Materials and Methods for Mass Spectrometric Protein Analysis

Abstract

The present invention describes systems, processes, and methods for characterizing proteins or polypeptides and / or identifying clip sites on proteins or polypeptides. In some embodiments, the systems, processes, and methods include generating reporter ions and subsequent tandem mass spectrometry triggered by the reporter ions.

Description

[Technical Field]

[0001] (Reference to electronically submitted sequence listing) This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII sequence listing with the filename "206389_0027_00WO_SequenceListing_ST25.txt", created on October 5, 2020, and having a size of 10 KB. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety.

[0002] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Patent Application No. 62 / 913,406, filed October 10, 2019, the disclosure of which is incorporated herein by reference in its entirety.

[0003] FIELD OF THE INVENTION The present invention relates to polypeptides and methods for identifying and characterizing clip sites on proteins or polypeptides. In one aspect, the present invention relates to a method for identifying clip sites on proteins or polypeptides through the generation of reporter ions and subsequent tandem mass spectrometry triggered by the reporter ions. [Background technology]

[0004] Recent advances in protein engineering technology have enabled novel therapeutic modalities that are unique in their structural and conformational diversity (see AlDeghaither et al., J. Clin. Pharmacol., 2015, 55, Suppl 3:S4-20). These novel drugs also tend to suffer from disadvantages, including rapid proteolysis, which can compromise efficacy and safety profiles. Protein clipping is ubiquitous and can occur during culture or process development, but is most often due to host cell-derived proteases that are site-specific or have broad substrate specificity (Dorai et al., Biotechnology and Bioengineering, 2009, 103:162-176; Dorai et al., Biotechnology Progress, 2011, 27:220-231). Clipping can also occur via non-enzymatic mechanisms, where a significant number of sites prone to clipping are influenced by factors such as side chain type, changes in local flexibility due to secondary, tertiary, and quaternary structures, and pre-analytical variables (e.g., pH, temperature, metals, and radicals) often used in assessing their developmental potential (Vlasak & Ionescu, mAbs, 2011, 3:253-263; Jarasch, J. Pharm. Sci., 2015, 104:1885-1898). Analytical methods used to monitor the level of clipped species as well as purity and integrity are some of the important quality attributes of the product or process (Torkashvand et al., Iranian Biomedical Journal, 2017, 21:131-141; Duval et al., Biotechnology Progress, 2012, 28:608-622).

[0005] Identifying the N-terminal sequence of an intact or cleaved protein is important for its biochemical and structural characterization. Conventional shotgun proteomics analysis makes it difficult to identify the protein N-terminus due to the frequent detection of protein N-terminal peptides. Characterizing the clipped site of a protein therapeutic by shotgun mass spectrometry is challenging due to the sequencing of the most abundant peptides present in complex protein digests. The relatively low stoichiometry of clipped species can potentially result in the failure to detect peptides with neo-N-termini. Furthermore, in-solution and in-source fragmentation artifacts can potentially cause false-positive identification of neo-N-terminal peptides sequenced by mass spectrometry. Summary of the Invention [Problem to be solved by the invention]

[0006] There remains a need in the art for the development of methods for identifying and characterizing polypeptides and clip sites on polypeptides. The present invention addresses these needs. [Means for solving the problem]

[0007] In one aspect, the present invention relates to a method for characterizing a polypeptide, the method comprising: (i) labeling a polypeptide with an N-terminal labeling reagent to obtain a labeled polypeptide; (ii) digesting the labeled polypeptide to produce a mixture comprising one or more unlabeled peptides and one or more labeled peptides; (iii) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (iv) subjecting the eluate to electron induced dissociation mass spectrometry, such as electron transfer dissociation (ETD) or electron capture dissociation (ECD) mass spectrometry, to obtain a mass spectrum, such as an ETD or ECD mass spectrum, of each of the one or more labeled peptides; (v) identifying each of the one or more labeled peptides by detecting a reporter ion in a mass spectrum, such as an ETD or ECD mass spectrum, of each of the one or more labeled peptides; (vi) subjecting each of the identified labeled peptides to a second mass analysis, thereby generating a second mass spectrum for each of the labeled peptides; (vii) characterizing the polypeptide by analyzing the ETD or ECD mass spectrum and a second mass spectrum of each of the labeled peptides; Includes.

[0008] In some embodiments, the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In further embodiments, the labeled polypeptide is a TMPP-labeled polypeptide and the labeled peptide is a TMPP-labeled peptide.

[0009] In some embodiments, the reporter ion is a TMPP reporter ion. In further embodiments, the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0010] In some embodiments, the second mass spectrometry is collision-induced dissociation (CID) mass spectrometry, high-energy collisional dissociation (HCD) mass spectrometry, or ultraviolet photodissociation (UVPD) mass spectrometry, hi further embodiments, the second mass spectrum of the labeled peptide is a CID, HCD, or UVPD mass spectrum of the TMPP-labeled peptide.

[0011] In identifying embodiments, the TMPP reporter ion triggers CID mass spectrometry, HCD mass spectrometry, or UVPD mass spectrometry.

[0012] In another general aspect, the invention relates to a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more clip polypeptides with an N-terminal labeling reagent to obtain one or more labeled clip polypeptides; (iii) digesting the labeled clip polypeptide to produce a mixture containing unlabeled and labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to electron induced dissociation tandem mass spectrometry, such as electron transfer dissociation (ETD) or electron capture dissociation (ECD) mass spectrometry, to obtain a mass spectrum, such as an ETD or ECD mass spectrum, of each of the labeled peptides; (vi) identifying the labeled peptides by detecting a reporter ion in a mass spectrum, such as an ETD or ECD mass spectrum, for each of the labeled peptides; (vii) subjecting the identified labeled peptides to a second mass analysis, thereby generating a second mass spectrum for each of the labeled peptides; (viii) characterizing the polypeptides by analyzing the ETD or ECD mass spectrum and a second mass spectrum for each of the labeled peptides; Includes:

[0013] In some embodiments, the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In further embodiments, the labeled polypeptide is a TMPP-labeled polypeptide and the labeled peptide is a TMPP-labeled peptide.

[0014] In some embodiments, the reporter ion is a TMPP reporter ion. In further embodiments, the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0015] In some embodiments, the second mass spectrometry is collision-induced dissociation (CID) mass spectrometry, high-energy collisional dissociation (HCD) mass spectrometry, or ultraviolet photodissociation (UVPD) mass spectrometry, hi further embodiments, the second mass spectrum of the labeled peptide is a CID, HCD, or UVPD mass spectrum of the TMPP-labeled peptide.

[0016] In an identifying embodiment, the TMPP reporter ion triggers CID mass spectrometry, HCD mass spectrometry, or UVPD mass spectrometry.

[0017] In another general aspect, the invention relates to a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting one or more TMPP-labeled clip polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to electron induced dissociation mass spectrometry, such as electron transfer dissociation (ETD) or electron capture dissociation (ECD) mass spectrometry, to obtain a mass spectrum, such as an ETD or ECD mass spectrum, of each of the TMPP-labeled peptides; (vi) for each of the TMPP-labeled peptides, detecting or isolating a TMPP reporter ion in a mass spectrum, such as an ETD or ECD mass spectrum; (vii) upon detection or separation of the TMPP reporter ions, subjecting each of the TMPP-labeled peptides to collision-induced dissociation (CID) mass spectrometry or higher-energy collision-induced dissociation (HCD) mass spectrometry, thereby producing a CID or HCD mass spectrum, respectively, for each of the TMPP-labeled peptides; (viii) identifying clip sites on the protein by analyzing the ETD or ECD mass spectrum and the CID or HCD mass spectrum for each of the TMPP-labeled peptides; Includes:

[0018] In another aspect, the invention relates, in part, to a system for identifying a clip site on a polypeptide and / or for characterizing a polypeptide in a sample, hi various embodiments, the system includes a liquid chromatography (LC) device and a tandem mass spectrometer.

[0019] In one embodiment, the tandem mass spectrometer comprises: (i) a first ionizer; (ii) a first mass to charge ratio filter or mass to charge ratio mass analyser arranged and adapted in a first mode of operation to transmit ions having a mass to charge ratio within a first range; (iii) a first ion mobility spectrometer, detector, or separator; (iv) attenuation means for attenuating ions in an operational mode; (v) a controller configured to control operation of the attenuation means such that ions having a mass to charge ratio within the first range but having one or more undesirable first charge states are substantially attenuated; (vi) a second ionizer; (vii) a second ion mobility spectrometer, detector, or separator; (viii) a data system configured to acquire unmixed signals of fragment ions and to non-redundantly encode trigger ions, the non-redundant encoding being arranged to avoid or minimize repeated overlap of any two ion signals from different parent species over multiple repetitions of any individual gate time; and Includes:

[0020] In various embodiments, the clip site on the polypeptide or the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In one embodiment, the first ionizer produces TMPP reporter ions.

[0021] In one embodiment, the sample is subjected to an LC instrument to produce an eluate, hi one embodiment, the eluate is subjected to tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum.

[0022] In some embodiments, the first mass spectrum and the second mass spectrum are analyzed by comparison to information in a database or spectral library.

[0023] In one embodiment, the first ionization device is an electron induced dissociation device. In some embodiments, the electron induced dissociation device is an electron transfer dissociation (ETD) device or an electron capture dissociation (ECD) device.

[0024] In some embodiments, the second ionization device is a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, or an ultraviolet photodissociation (UVPD) device.

[0025] In some embodiments, the mass spectrometer further comprises a collision device, a fragmentation device, or a reaction device.

[0026] In some embodiments, the attenuation means comprises an ion gate or an ion barrier. In one embodiment, the attenuation means is located downstream of the ion mobility spectrometer or separator.

[0027] In some embodiments the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged and adapted in a first mode of operation to attenuate ions having mass to charge ratios outside a first range, hi some embodiments the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged upstream or downstream of the ion mobility spectrometer or separator.

[0028] In some embodiments, the first undesired charge state is selected from one or more of the following: (i) monovalent, (ii) divalent, (iii) trivalent, (iv) tetravalent, (v) pentavalent, and (vi) multivalent.

[0029] In some embodiments, the system further comprises an ion guide, ion trap or ion trapping region positioned upstream of said ion mobility spectrometer or separator, said ion guide, ion trap or ion trapping region arranged to trap, store or accumulate ions and then periodically pulse ions into or towards said ion mobility spectrometer or separator.

[0030] In another aspect, the invention relates, in part, to a reporter ion for identifying a clip site on a polypeptide. In one embodiment, the clip site is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In one embodiment, TMPP is ionized to generate a reporter ion.

[0031] In another aspect, the invention relates, in part, to reporter ions for characterizing polypeptides. In one embodiment, the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In one embodiment, the TMPP is ionized to generate a reporter ion.

[0032] In one embodiment, the TMPP is ionized by a mass spectrometer to generate reporter ions. In one embodiment, the mass spectrometer is a tandem mass spectrometer. In various embodiments, the tandem mass spectrometer comprises an electron transfer dissociation (ETD) instrument, an electron capture dissociation (ECD) instrument, a collision-induced dissociation (CID) instrument, a high-energy collisional dissociation (HCD) instrument, an ultraviolet photodissociation (UVPD) instrument, or any combination thereof.

[0033] In some embodiments, the reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0034] In some embodiments, the reporter ion is

[0035] [ka]

[0036] [ka] It is a compound having the structure:

[0037] In another aspect, the invention relates, in part, to compositions for identifying clip sites on polypeptides.

[0038] In another aspect, the invention pertains, in part, to compositions for characterizing polypeptides.

[0039] In various embodiments, the composition comprises at least one reporter ion and a polypeptide of the invention.

[0040] In another aspect, the invention also relates, in part, to a kit for identifying a clip site on a polypeptide or characterizing a polypeptide in a sample, the kit including N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling a clip site on a polypeptide or N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling a polypeptide, and an instructional material.

[0041] Other aspects, features, and advantages of the present invention will become apparent from the following disclosure, including the detailed description of the invention and its preferred embodiments, and the appended claims. [Brief explanation of the drawings]

[0042] The following detailed description of various embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there is shown in the drawings exemplary embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown. [Figure 1] A representative ETD product ion spectrum of the TMPP-labeled peptide DIQMTQSPSTL (SEQ ID NO: 1), corresponding to the N-terminal sequence of the light chain of the NIST antibody, is shown. The mass spectrum consisted primarily of the diagnostic TMPP reporter ion (m / z = 533 Da) and a c-type backbone product ion (m / z = 590 Da) consisting of the N-terminal TMPP tag. [Figure 2A] Schematic diagrams of the structures of the 533 Da, 590 Da, and 573 Da reporter ions are shown with their exact masses. Figure 2A shows a schematic diagram of the structure of the 533 Da (TMPP) reporter ion, Figure 2B shows a schematic diagram of the structure of the 590 Da (TMPP-Ac-NH) reporter ion, and Figure 2C shows a schematic diagram of the structure of the 573 Da (TMPP-Ac) reporter ion. [Figure 2B] Schematic diagrams of the structures of the 533 Da, 590 Da, and 573 Da reporter ions are shown with their exact masses. Figure 2A shows a schematic diagram of the structure of the 533 Da (TMPP) reporter ion, Figure 2B shows a schematic diagram of the structure of the 590 Da (TMPP-Ac-NH) reporter ion, and Figure 2C shows a schematic diagram of the structure of the 573 Da (TMPP-Ac) reporter ion. [Figure 2C] Schematic diagrams of the structures of the 533 Da, 590 Da, and 573 Da reporter ions are shown with their exact masses. Figure 2A shows a schematic diagram of the structure of the 533 Da (TMPP) reporter ion, Figure 2B shows a schematic diagram of the structure of the 590 Da (TMPP-Ac-NH) reporter ion, and Figure 2C shows a schematic diagram of the structure of the 573 Da (TMPP-Ac) reporter ion. [Figure 2D] The formulas for calculating the reporter ion generation efficiency in ETD (FIG. 2D) and HCD (FIG. 2E) are shown. [Figure 2E] The formulas for calculating the reporter ion generation efficiency in ETD (FIG. 2D) and HCD (FIG. 2E) are shown. [Figure 3] A representative reverse-phase chromatography buffer gradient and the corresponding total ion chromatogram of the NIST digest after TMPP labeling are shown. Most unlabeled peptides eluted at 2-30% organic in a shallow gradient of 10 min length. A surrogate peptide corresponding to the TMPP-labeled N-terminus of the NIST antibody light chain eluted at 12 min. [Figure 4A] HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4B]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4C]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4D]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4E]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4F]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4G]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4H]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4I]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4J]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4K]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4L]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4M]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4N]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4O]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4P]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4Q]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4R]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4S]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4T]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4U]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4V]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4W]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4X]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4Y]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 4Z]HK (Figures 4A and 4B), ADYEK (SEQ ID NO: 12) (Figure 4C), VYACEVTHQGLSSPVTK (SEQ ID NO: 13) (Figure 4D), SFNR (SEQ ID NO: 14) (Figure 4E), EAK (Figure 4F), VQWK (SEQ ID NO: 15) (Figure 4G), DTLMISR (SEQ ID NO: 16) (Figure 4H), FNWYVDGVEVHNAK (SEQ ID NO: 17) (Figure 4I), TKPR (SEQ ID NO: 18) (Figure 4J), EEQYNSTYR (SEQ ID NO: 19) (Figure 4K), VVSVLTVLHQDWLNGK (SEQ ID NO: 20) (Figure 4L), EYK (Figure 4M), CK (Figure 4N), GQPR (SEQ ID NO: 2 Representative ETD-MS2 and triggered CID-MS2 spectra of 20 TMPP-labeled synthetic peptides are shown, including EPQVYTLPPSR (SEQ ID NO: 22) (Figures 4P-4R), STSGGTAALGCLVKD (SEQ ID NO: 23) (Figures 4S-4U), STSGGTAALGCLVKDYFPEPVTVSWN (SEQ ID NO: 24) (Figure 4V), VVSLTVLHQDWLNGKE (SEQ ID NO: 25) (Figures 4W-4X), VVSLTVLHQDWLNGK (SEQ ID NO: 26) (Figure 4Y), and VSLTVLHQDWLNGK (SEQ ID NO: 27) (Figure 4Z). [Figure 5A] Representative results showing peptide intensity as a function of observed retention time overlaid with an AcCN gradient are shown. [Figure 5B] Representative results showing peptide intensity as a function of observed retention time overlaid with an AcCN gradient are shown. [Figure 6A] Representative results demonstrating TMPP+ETD efficiency as a function of mass are shown. [Figure 6B] Representative results demonstrating TMPP+ETD efficiency as a function of sequence length are shown. [Figure 6C] Representative results demonstrating TMPP+ETD efficiency as a function of charge are shown. [Figure 6D]Representative results are shown demonstrating the difference in intensity as a function of efficiency for the diagnostic ions 533 and 590, which are TMPP+ and TMPP-Ac-NH2+, respectively. For the same peptide, a smaller efficiency value (single digit) was observed for the diagnostic ion 590 compared to the diagnostic ion 533 (two digits). Thus, the intensity of the diagnostic ion 533 was significantly higher than that of the diagnostic ion 590. [Figure 7A] Representative results are shown demonstrating that the overall ETD efficiency, considering all product ions, exhibits a slight charge-state-dependent decrease. ETD efficiency can be defined by the equation shown in Figure 2D. The original %ETD efficiency, defined by Gunawardena et al. (Gunawardena HP et al., 2005, Journal of the American Chemical Society, 127:12627-12639), is Equation 5, which was an estimate of the overall ETD efficiency for all backbone fragments of a polypeptide (reported as a percentage). Equations 1–3 were derived as estimates of the reporter ion-specific ETD efficiency. Equation 4 was an estimate of the ETD efficiency for the reporter ion as well as the backbone fragments of the polypeptide. In contrast, Figures 7C and 7D show representative results demonstrating a charge-state-dependent increase in the overall backbone efficiency estimated by Equation 5, in which the TMPP+ reporter ion was ignored. Figure 7A shows representative results demonstrating the overall ETD efficiency as a function of mass using Equation 4. Figure 7B shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 4. Figure 7C shows representative results demonstrating overall ETD efficiency as a function of mass using Equation 5. Figure 7D shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 5. [Figure 7B]Representative results are shown demonstrating that the overall ETD efficiency, considering all product ions, exhibits a slight charge-state-dependent decrease. ETD efficiency can be defined by the equation shown in Figure 2D. The original %ETD efficiency, defined by Gunawardena et al. (Gunawardena HP et al., 2005, Journal of the American Chemical Society, 127:12627-12639), is Equation 5, which was an estimate of the overall ETD efficiency for all backbone fragments of a polypeptide (reported as a percentage). Equations 1–3 were derived as estimates of the reporter ion-specific ETD efficiency. Equation 4 was an estimate of the ETD efficiency for the reporter ion as well as the backbone fragments of the polypeptide. In contrast, Figures 7C and 7D show representative results demonstrating a charge-state-dependent increase in the overall backbone efficiency estimated by Equation 5, in which the TMPP+ reporter ion was ignored. Figure 7A shows representative results demonstrating the overall ETD efficiency as a function of mass using Equation 4. Figure 7B shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 4. Figure 7C shows representative results demonstrating overall ETD efficiency as a function of mass using Equation 5. Figure 7D shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 5. [Figure 7C]Representative results are shown demonstrating that the overall ETD efficiency, considering all product ions, exhibits a slight charge-state-dependent decrease. ETD efficiency can be defined by the equation shown in Figure 2D. The original %ETD efficiency, defined by Gunawardena et al. (Gunawardena HP et al., 2005, Journal of the American Chemical Society, 127:12627-12639), is Equation 5, which was an estimate of the overall ETD efficiency for all backbone fragments of a polypeptide (reported as a percentage). Equations 1–3 were derived as estimates of the reporter ion-specific ETD efficiency. Equation 4 was an estimate of the ETD efficiency for the reporter ion as well as the backbone fragments of the polypeptide. In contrast, Figures 7C and 7D show representative results demonstrating a charge-state-dependent increase in the overall backbone efficiency estimated by Equation 5, in which the TMPP+ reporter ion was ignored. Figure 7A shows representative results demonstrating the overall ETD efficiency as a function of mass using Equation 4. Figure 7B shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 4. Figure 7C shows representative results demonstrating overall ETD efficiency as a function of mass using Equation 5. Figure 7D shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 5. [Figure 7D]Representative results are shown demonstrating that the overall ETD efficiency, considering all product ions, exhibits a slight charge-state-dependent decrease. ETD efficiency can be defined by the equation shown in Figure 2D. The original %ETD efficiency, defined by Gunawardena et al. (Gunawardena HP et al., 2005, Journal of the American Chemical Society, 127:12627-12639), is Equation 5, which was an estimate of the overall ETD efficiency for all backbone fragments of a polypeptide (reported as a percentage). Equations 1–3 were derived as estimates of the reporter ion-specific ETD efficiency. Equation 4 was an estimate of the ETD efficiency for the reporter ion as well as the backbone fragments of the polypeptide. In contrast, Figures 7C and 7D show representative results demonstrating a charge-state-dependent increase in the overall backbone efficiency estimated by Equation 5, in which the TMPP+ reporter ion was ignored. Figure 7A shows representative results demonstrating the overall ETD efficiency as a function of mass using Equation 4. Figure 7B shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 4. Figure 7C shows representative results demonstrating overall ETD efficiency as a function of mass using Equation 5. Figure 7D shows representative results demonstrating overall ETD efficiency as a function of charge using Equation 5. [Figure 7E] Figure 7F shows representative results demonstrating TMPP+HCD efficiency as a function of mass using Equation 6. Figure 7F shows representative results demonstrating TMPP+HCD efficiency as a function of charge using Equation 6. [Figure 7F] Representative results are shown demonstrating TMPP+HCD efficiency as a function of charge using Equation 6. [Figure 8] Representative results are shown demonstrating the relationship between ETD efficiency and labeled peptides grouped by the number of tyrosine and lysine residues per peptide. [Figure 9A] Representative results are shown demonstrating the overall distribution of peptide reaction or TMPP labeling efficiency. [Figure 9B]Representative results are shown demonstrating the overall distribution of peptide reactivity or TMPP labeling efficiency as predicted by Equation 7. [Figure 10A-1] Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 10A-2] Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 10B-1]Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 10B-2] Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 10C-1]Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 10C-2] Representative results are shown demonstrating the generation of true positives (TP), false positives (FP), true negatives (TN), and false negatives (FN) for labeled and unlabeled peptides. As shown in Figure 10A, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated no reporter ions, which were true negatives (TN). As shown in Figure 10B, the labeled peptide generated a characteristic TMPP reporter ion that was a true positive (TP), while the unlabeled peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). As shown in Figure 10C, the modified peptide generated no diagnostic ions that were false negatives (FN), while the unmodified peptide counterpart generated an interfering ion similar in mass to the TMPP reporter ion that was a false positive (FP). [Figure 11A] Representative results are shown demonstrating the area under the curve (AUC) of the ROC curve for each diagnostic ion and elution time. Figure 11A shows representative predictive power for the TMPP+ (533) diagnostic ion. Figure 11B shows representative predictive power for TMPP peptide retention times. Figure 11C shows representative predictive power for the TMPP-Ac+ (573) diagnostic ion. Figure 11D shows representative predictive power for the TMPP-Ac-NH2+ (591) diagnostic ion. [Figure 11B] Representative results are shown demonstrating the area under the curve (AUC) of the ROC curve for each diagnostic ion and elution time. Figure 11A shows representative predictive power for the TMPP+ (533) diagnostic ion. Figure 11B shows representative predictive power for TMPP peptide retention times. Figure 11C shows representative predictive power for the TMPP-Ac+ (573) diagnostic ion. Figure 11D shows representative predictive power for the TMPP-Ac-NH2+ (591) diagnostic ion. [Figure 11C] Representative results are shown demonstrating the area under the curve (AUC) of the ROC curve for each diagnostic ion and elution time. Figure 11A shows representative predictive power for the TMPP+ (533) diagnostic ion. Figure 11B shows representative predictive power for TMPP peptide retention times. Figure 11C shows representative predictive power for the TMPP-Ac+ (573) diagnostic ion. Figure 11D shows representative predictive power for the TMPP-Ac-NH2+ (591) diagnostic ion. [Figure 11D] Representative results are shown demonstrating the area under the curve (AUC) of the ROC curve for each diagnostic ion and elution time. Figure 11A shows representative predictive power for the TMPP+ (533) diagnostic ion. Figure 11B shows representative predictive power for TMPP peptide retention times. Figure 11C shows representative predictive power for the TMPP-Ac+ (573) diagnostic ion. Figure 11D shows representative predictive power for the TMPP-Ac-NH2+ (591) diagnostic ion. [Figure 12A] Representative MS / MS spectra are shown showing evidence for the neo-N-terminus of the IAWLVK sequence (SEQ ID NO: 5) generated due to protease activity. Figure 12A shows a representative product ion spectrum obtained from ETD-MS2 of a doubly charged ion that generated the characteristic diagnostic ion TMPP+ (m / z = 533 Da). Figure 12B shows a CID-MS2 spectrum triggered by a representative diagnostic reporter ion (m / z = 533) of the doubly charged ion. Figure 12C shows a representative ETD spectrum of the unconjugated peptide in the absence of the diagnostic ion. [Figure 12B]Representative MS / MS spectra are shown showing evidence for the neo-N-terminus of the IAWLVK sequence (SEQ ID NO: 5) generated due to protease activity. Figure 12A shows a representative product ion spectrum obtained from ETD-MS2 of a doubly charged ion that generated the characteristic diagnostic ion TMPP+ (m / z = 533 Da). Figure 12B shows a CID-MS2 spectrum triggered by a representative diagnostic reporter ion (m / z = 533) of the doubly charged ion. Figure 12C shows a representative ETD spectrum of the unconjugated peptide in the absence of the diagnostic ion. [Figure 12C] Representative MS / MS spectra are shown showing evidence for the neo-N-terminus of the IAWLVK sequence (SEQ ID NO: 5) generated due to protease activity. Figure 12A shows a representative product ion spectrum obtained from ETD-MS2 of a doubly charged ion that generated the characteristic diagnostic ion TMPP+ (m / z = 533 Da). Figure 12B shows a CID-MS2 spectrum triggered by a representative diagnostic reporter ion (m / z = 533) of the doubly charged ion. Figure 12C shows a representative ETD spectrum of the unconjugated peptide in the absence of the diagnostic ion. [Figure 13A] Representative results are shown demonstrating ETD-MS2 and diagnostic ion-induced CID-MS2 product ion spectral evidence of surrogate peptides corresponding to sequential clipping of the GLP1 sequence. Figure 13A shows a representative result demonstrating the surrogate peptide of AWLVK (SEQ ID NO: 6) resulting from the I / A clip. Figure 13B shows a representative result demonstrating the surrogate peptide of WLVK (SEQ ID NO: 7) resulting from the A / W clip. Figure 13C shows a representative result demonstrating the surrogate peptide of LVK resulting from the W / L clip. [Figure 13B]Representative results are shown demonstrating ETD-MS2 and diagnostic ion-induced CID-MS2 product ion spectral evidence of surrogate peptides corresponding to sequential clipping of the GLP1 sequence. Figure 13A shows a representative result demonstrating the surrogate peptide of AWLVK (SEQ ID NO: 6) resulting from the I / A clip. Figure 13B shows a representative result demonstrating the surrogate peptide of WLVK (SEQ ID NO: 7) resulting from the A / W clip. Figure 13C shows a representative result demonstrating the surrogate peptide of LVK resulting from the W / L clip. [Figure 13C] Representative results are shown demonstrating ETD-MS2 and diagnostic ion-induced CID-MS2 product ion spectral evidence of surrogate peptides corresponding to sequential clipping of the GLP1 sequence. Figure 13A shows a representative result demonstrating the surrogate peptide of AWLVK (SEQ ID NO: 6) resulting from the I / A clip. Figure 13B shows a representative result demonstrating the surrogate peptide of WLVK (SEQ ID NO: 7) resulting from the A / W clip. Figure 13C shows a representative result demonstrating the surrogate peptide of LVK resulting from the W / L clip. [Figure 14] A schematic diagram of the clipping site of dulaglutide (sequence number 2) to generate surrogate peptides of EFIAWLVK (sequence number 3), FIAWLVK (sequence number 4), IAWLVK (sequence number 5), AWLVK (sequence number 6), WLVK (sequence number 7) and LVK is shown. [Figure 15A] Representative extracted ion chromatograms (XICs) of dulaglutide surrogate peptides are shown. Figure 15A shows representative extracted ion chromatograms of TMPP-unlabeled surrogate peptides, including peptides SEQ ID NOS: 4-7 and LVK. Figure 15B shows representative extracted ion chromatograms of labeled surrogate peptides, including TMPP-FIAWLVK (SEQ ID NOS: 8), TMPP-IAWLVK (SEQ ID NOS: 9), TMPP-AWLVK (SEQ ID NOS: 10), TMPP-WLVK (SEQ ID NOS: 11), and TMPP-LVK. The XICs demonstrated that each TMPP-labeled peptide eluted during two rapid gradients between 10 and 13 minutes. [Figure 15B]Representative extracted ion chromatograms (XICs) of dulaglutide surrogate peptides are shown. Figure 15A shows representative extracted ion chromatograms of TMPP-unlabeled surrogate peptides, including peptides SEQ ID NOS: 4-7 and LVK. Figure 15B shows representative extracted ion chromatograms of labeled surrogate peptides, including TMPP-FIAWLVK (SEQ ID NOS: 8), TMPP-IAWLVK (SEQ ID NOS: 9), TMPP-AWLVK (SEQ ID NOS: 10), TMPP-WLVK (SEQ ID NOS: 11), and TMPP-LVK. The XICs demonstrated that each TMPP-labeled peptide eluted during two rapid gradients between 10 and 13 minutes. [Figure 16A] Representative results are shown demonstrating the generation of characteristic reporter ions via different dissociation modes on the LVK peptide sequence. Figure 16A shows representative results demonstrating the generation of characteristic reporter ions via high-energy collisional dissociation (HCD) on the LVK peptide sequence. Figure 16B shows representative results demonstrating the generation of characteristic reporter ions via ultraviolet photodissociation (UVPD) on the LVK peptide sequence. Figure 16C shows representative results demonstrating the generation of characteristic reporter ions via electron transfer dissociation (ETD) on the LVK peptide sequence. [Figure 16B] Representative results are shown demonstrating the generation of characteristic reporter ions via different dissociation modes on the LVK peptide sequence. Figure 16A shows representative results demonstrating the generation of characteristic reporter ions via high-energy collisional dissociation (HCD) on the LVK peptide sequence. Figure 16B shows representative results demonstrating the generation of characteristic reporter ions via ultraviolet photodissociation (UVPD) on the LVK peptide sequence. Figure 16C shows representative results demonstrating the generation of characteristic reporter ions via electron transfer dissociation (ETD) on the LVK peptide sequence. [Figure 16C]Representative results are shown demonstrating the generation of characteristic reporter ions via different dissociation modes on the LVK peptide sequence. Figure 16A shows representative results demonstrating the generation of characteristic reporter ions via high-energy collisional dissociation (HCD) on the LVK peptide sequence. Figure 16B shows representative results demonstrating the generation of characteristic reporter ions via ultraviolet photodissociation (UVPD) on the LVK peptide sequence. Figure 16C shows representative results demonstrating the generation of characteristic reporter ions via electron transfer dissociation (ETD) on the LVK peptide sequence. [Figure 17A] Representative results demonstrating GLP1 peptide clipping with FI are shown in the presence and absence of cathepsin D, as well as in buffer solutions of different pH and buffer composition used for TMPP derivatization. Cathepsin D-treated samples are labeled +Cathepsin D, and TMPP-derivatized samples are labeled +TMPP. The x-axis shows sample and reaction conditions: 100 mM MES pH 6, 100 mM HEPES pH 7, 100 mM sodium phosphate pH 8; TMPP reagent premixed with DMF; and unreacted control in PBS pH 7. The y-axis shows peak area. Figure 17A shows a representative panel of extracted ion chromatograms (XICs) for the precursor peptide FIAWLVK (SEQ ID NO: 4) and the corresponding clipped product IAWLVK (SEQ ID NO: 5). The y-axis shows peak area, and the x-axis shows retention time. Figure 17B shows representative peak areas of the XIC for the precursor peptide FIAWLVK (SEQ ID NO: 4) and the corresponding clipped product IAWLVK (SEQ ID NO: 5). [Figure 17B]Representative results demonstrating GLP1 peptide clipping with FI are shown in the presence and absence of cathepsin D, as well as in buffer solutions of different pH and buffer composition used for TMPP derivatization. Cathepsin D-treated samples are labeled +Cathepsin D, and TMPP-derivatized samples are labeled +TMPP. The x-axis shows sample and reaction conditions: 100 mM MES pH 6, 100 mM HEPES pH 7, 100 mM sodium phosphate pH 8; TMPP reagent premixed with DMF; and unreacted control in PBS pH 7. The y-axis shows peak area. Figure 17A shows a representative panel of extracted ion chromatograms (XICs) for the precursor peptide FIAWLVK (SEQ ID NO: 4) and the corresponding clipped product IAWLVK (SEQ ID NO: 5). The y-axis shows peak area, and the x-axis shows retention time. Figure 17B shows representative peak areas of the XIC for the precursor peptide FIAWLVK (SEQ ID NO: 4) and the corresponding clipped product IAWLVK (SEQ ID NO: 5). DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention is based in part on the discovery that the use of TMPP labeling in combination with electron transfer dissociation (ETD) mass spectrometry revealed a facile TMPP labeling technique that was most powerful for small tryptic peptides. + Furthermore, the present invention is based, in part, on the unexpected discovery that a reporter ion was generated by collision-induced dissociation (CID) MS. 2 The spectra complement the ETD identification and MS 2 It is based on the unexpected discovery that it provides a real-time in silco filtering mechanism that triggers a scan and ensures that a CID scan is performed only if a reporter ion is observed.

[0044] Thus, the present invention relates, in part, to novel systems, processes, and methods for characterizing proteins or polypeptides and / or identifying clip sites on proteins or polypeptides. In various embodiments, the systems, processes, and methods include high-throughput LC-MS for facile generation of reporter ions in ETD. In some embodiments, reporter generation facilitates subsequent MS / MS analysis. In some embodiments, MS / MS analysis includes complementary ion activation modes, such as CID, higher-energy collisional dissociation (HCD), and / or ultraviolet photodissociation (UVPD), via intensity- and m / z-dependent trigger events, to further sequence the protein or polypeptide. For example, in one embodiment, the present invention focuses on a method for identifying clipped polypeptides that includes ETD-MS2, where a reporter ion derived from TMPP triggers MS2 analysis to autonomously filter out clipped polypeptides.

[0045] definition As used herein, each of the following terms has the meaning associated with it in this section. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to practice or test the present invention, preferred methods and materials are described.

[0046] All patents, published patent applications, and publications cited herein are incorporated by reference as if set forth in their entirety. The discussion of documents, operations, materials, devices, articles and the like which has been included in the present specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these items constitute part of the prior art to any invention disclosed or claimed.

[0047] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the object article. By way of example, "an element" means one element or more than one element.

[0048] Unless otherwise specified, any numerical values, such as concentrations or concentration ranges, described herein should be understood in all cases to be modified by the word "about." Thus, numerical values ​​typically include ±10% of the stated value. For example, an amount of about 50 ppm or less includes 45 ppm or less to 55 ppm or less. As used herein, the use of numerical ranges expressly includes all possible subranges, including integers and fractions of values ​​within the range, and all individual numerical values ​​within the range, unless the context clearly indicates otherwise.

[0049] The term "about" will be understood by those of skill in the art and will vary to some extent depending on the context in which the term is used. When referring to measurable values ​​such as amounts and durations, the term "about" as used herein is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, where such variations are appropriate for practicing the methods of the present disclosure.

[0050] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to mean the inclusion of a specified integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the word "comprise" can be replaced with the words "containing" or "including," or, as sometimes used herein, can also be replaced with the word "having."

[0051] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. When used herein in connection with aspects or embodiments of the present invention, any of the above terms "comprising," "containing," "including," and "having" can be substituted with the terms "consisting of" or "essentially consisting of" to vary the scope of the disclosure.

[0052] As used herein, the connective term "and / or" between multiple listed elements is understood to encompass both individual and combined alternatives. For example, when two elements are connected by "and / or," the first alternative refers to the first element being applicable without the second element. The second alternative refers to the second element being applicable without the first element. The third alternative refers to the first and second elements being applicable together. Any one of these alternatives is understood to be within the meaning and therefore meets the requirements of the term "and / or" as used herein. The simultaneous applicability of two or more of the alternatives is also understood to be within the meaning and therefore meets the requirements of the term "and / or."

[0053] As used herein, "MS / MS" or "MS 2" refers to tandem mass spectrometry. Tandem mass spectrometry is a technique in instrumental analysis in which two or more mass analyzers are linked together using additional reaction steps to increase the ability to analyze a sample. Tandem use of mass spectrometry can occur when the reaction steps are separated in space (tandem in space) and / or when the reaction steps are separated in time (tandem in time). A common use of tandem mass spectrometry is the analysis of biomolecules such as proteins, peptides, organic and inorganic molecules, lipids, metabolites, and oligonucleotides.

[0054] As used herein, a "reporter ion" or "diagnostic ion" refers to a characteristic product ion of a labeled peptide or polypeptide containing an N-terminal tag or label, observed in an ETD mass spectrum. Typically, it is the most predominant product ion in the mass spectrum and is used to trigger a subsequent MS / MS event to further sequence the labeled peptide or polypeptide.

[0055] As used herein, the term "label" refers to a detectable compound or composition that is directly or indirectly conjugated to a probe to produce a "labeled" probe. The label may itself be detectable (e.g., a small molecule or charge label).

[0056] The term "amplification" refers to a process that increases the number of copies of a target reporter ion present in a sample.

[0057] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and is not limited to the maximum number of amino acids that a protein or peptide sequence can contain. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, e.g., commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, which are commonly referred to in the art as proteins, of which there are many forms. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins, among others. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or any combination thereof.

[0058] As used herein, the terms "amino acid," "amino acid monomer," or "amino acid residue" refer to any of the 20 naturally occurring amino acids or synthetic amino acids with unnatural side chains, and include both the D and L optical isomers.

[0059] As used herein, the terms "natural amino acid," "naturally encoded amino acid," "naturally occurring amino acid," and "genetically encoded amino acid" refer to an amino acid that is one of the 20 common amino acids or pyrrolysine or selenocysteine. The term "natural amino acid" includes, but is not limited to, proteinogenic amino acids.

[0060] "Unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "non-naturally encoded amino acid," "unnatural amino acid," "non-naturally occurring amino acid," "non-genetically encoded amino acid," and various hyphenated and non-hyphenated versions thereof. The term "unnatural amino acid" includes, but is not limited to, amino acids that occur in nature by modification of naturally encoded amino acids (including, but not limited to, the common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into growing polypeptide chains by the translation complex. Examples of naturally occurring non-naturally encoded amino acids include, but are not limited to, N-acetylglucosaminoyl-L-serine, N-acetylglucosaminoyl-L-threonine, and O-phosphotyrosine. Furthermore, the term "unnatural amino acid" includes, but is not limited to, non-proteinogenic amino acids and amino acids that do not occur in nature and may be obtained synthetically (e.g., Q-proline-based amino acids) or by modification of unnatural amino acids.

[0061] "Isolated" means altered or removed from the natural state. For example, a protein or peptide that is naturally present in a living animal is not "isolated," but the same protein or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated peptide or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0062] As used herein, the term "identical" refers to two or more sequences or subsequences that are the same.

[0063] Furthermore, as used herein, the term "substantially identical" refers to two or more sequences that have a percentage of contiguous units that are the same when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be "substantially identical" if the contiguous units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over the designated region. Such percentages describe the "percent identity" of two or more sequences. Sequence identity can exist over a region that is at least about 75-100 contiguous units in length, over a region that is about 50 contiguous units in length, or, if not specified, over the entire sequence. This definition also refers to the complement of a test sequence.

[0064] As used herein, the term "instructional material" includes publications, records, drawings, or any other media that can be used to communicate the usefulness of reporter ions, systems, and / or methods of the present invention in kits for identifying clip sites on polypeptides or characterizing polypeptides. Optionally, or alternatively, the instructional material may describe one or more methods for labeling a polypeptide or a clip site on a polypeptide with TMPP. Optionally, or alternatively, the instructional material may describe one or more methods for analyzing a TMPP-labeled clip site on a polypeptide or a TMPP-labeled polypeptide using a system or method of the present invention. Kit instructional material may, for example, be affixed to a container containing one or more components of the present invention or shipped together with a container containing one or more components of the present invention. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient use the instructional material and the components in conjunction.

[0065] Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0066] explanation The present invention relates, in part, to novel systems, processes, and methods for characterizing proteins or polypeptides and / or identifying clip sites on proteins or polypeptides. In various aspects of the invention, the systems, processes, and methods include high-throughput LC-MS for facile generation of reporter ions in ETD. In some embodiments, reporter generation facilitates subsequent MS / MS analysis (i.e., tandem mass spectrometry). In some embodiments, MS / MS analysis includes complementary ion activation modes, such as CID, higher-energy collisional dissociation (HCD), and / or ultraviolet photodissociation (UVPD), via intensity- and m / z-dependent trigger events, to further sequence the protein or polypeptide. For example, in one embodiment, the invention focuses on a method for identifying clipped polypeptides, including ETD-MS2, in which a reporter ion derived from TMPP triggers MS2 analysis to autonomously filter for clipped polypeptides.

[0067] Identification of the clip site Mass spectrometry is an important emerging method for the identification and characterization of clip sites. Depending on the performance and mass range of available mass spectrometers, two approaches are used to characterize proteins, including the "top-down" strategy and the "bottom-up" strategy.

[0068] In "top-down" strategies for protein analysis, intact proteins are ionized by either electrospray ionization (ESI) or matrix-assisted laser desorption / ionization (MALDI) and then introduced into a mass analyzer. However, intact MS is only useful for detecting degradation products that are within the instrument's limit of detection (LOD), while low-level fragments are often not observed in intact MS analyses that require peptide-level analysis.

[0069] In "bottom-up" proteomics, the presence of proteins is identified at the peptide level. The general procedure for this strategy involves using one or more proteolytic enzymes (such as trypsin, pepsin, or chymotrypsin) to obtain a mass of individual peptides derived from a protein. These peptides are then introduced into a mass spectrometer and identified by peptide mass fingerprinting or tandem mass spectrometry. The masses are then compared to a database, such as a sequence database or spectral library, and a probability-based scoring system is used to determine the closest protein match. This approach therefore uses peptide-level identification to infer the presence of clipped peptides. Smaller, more uniform fragments are easier to analyze than intact proteins and can also be determined with higher accuracy; therefore, this "bottom-up" approach is the preferred method for research in proteomics and protein characterization.

[0070] However, characterization of protein clip sites using bottom-up approaches is challenging due to the sequencing of the most abundant peptides present in complex protein digests. The relatively low stoichiometry of clipped peptides may result in peptides with neo-N-termini not being detected. Furthermore, in-solution and in-source fragmentation artifacts can potentially cause false-positive identification of neo-N-terminal peptides sequenced by mass spectrometry.

[0071] To increase the reliability of protein N-terminal identification, chemical derivatization of N-terminal amine groups with (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) or dimethyl labeling followed by mass spectrometry is commonly performed. Using this approach, the N-terminus of a protein of interest is labeled with TMPP or dimethyl before trypsin digestion and LC-MS analysis. Therefore, the N-terminus of a protein can be easily identified because only the N-terminal tryptic peptide contains the label. Peptides with N-terminal derivatization, such as TMPP, improve peptide ionization and retention during chromatography and generate unique fragment ions during tandem mass spectrometry, significantly facilitating the sequencing of these peptides.

[0072] One method based on chemical labeling is the demethylation of the N-terminus and ε-amino group of lysines with aqueous formaldehyde via reductive methylation. In MS / MS analysis, this labeling strategy provides signal enhancement of the a1 and yn-1 ions, which are undetectable from most underivatized fragments. Due to its simplicity and low cost, dimethyl labeling is a promising strategy for protein N-terminal identification.

[0073] Alternatively, the TMPP labeling approach is simple and has been successfully applied to different proteins. Two features of this labeling reagent enhance the sensitivity of the method: (i) the TMPP label introduces a permanent positive charge, resulting in enhanced ionization efficiency and therefore better detection of low-abundance peptides. (ii) The hydrophobic TMPP group shifts the retention time of TMPP-derivatized peptides in reversed-phase chromatography to a less complex part of the chromatogram, increasing detection sensitivity, especially for short N-terminal peptides that would otherwise not be retained on the column. Furthermore, TMPP is fully compatible with all standard detergents, chaotropes, and reducing conditions used for protein extraction in proteomics, making TMPP labeling a commonly used method for protein N-terminal sequencing.

[0074] The TMPP labeling approach has been demonstrated to detect protein clipping and proteogenomic mapping of N-terminal heterogeneity from proteins excised from SDS-PAGE gels. Furthermore, this labeling reagent increases the hydrophobicity of N-terminal peptides, improving their ionization ability and modifying their fragmentation patterns due to the introduced positive charge.

[0075] Reporter ion-triggered tandem mass spectrometry Site-specific localization of the TMPP tag allows for unambiguous determination of the mature or neo-N-terminus. In addition to the backbone product ion, the 273 Da TMPP reporter ion formed via collision-induced dissociation (CID) can be diagnostic for the presence of the processed N-terminus. However, reporter ions generated via CID may be less informative due to their low abundance.

[0076] This application describes a novel, high-throughput LC-MS method for the facile generation of TMPP reporter ions in electron transfer dissociation (ETD) tandem mass spectrometry. The abundant generation of these reporter ions enables subsequent MS / MS events using complementary ion activation modes, such as CID, HCD, or UVPD, via intensity- and m / z-dependent trigger events for further sequenced peptides. The reporter ions generated via ETD are novel, and triggering of these reporters facilitates filtering of spectra containing TMPP-labeled peptides, aiding in database searches or rapid manual verification of spectra.

[0077] In one general aspect, the present application relates to a method for characterizing a polypeptide, the method comprising: (i) labeling a polypeptide with an N-terminal labeling reagent to obtain a labeled polypeptide; (ii) digesting the labeled polypeptide to produce a mixture comprising one or more unlabeled peptides and one or more labeled peptides; (iii) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (iv) subjecting the eluate to electron transfer dissociation (ETD) mass spectrometry; (v) identifying the one or more labeled peptides by detecting reporter ions in the ETD mass spectrum of the one or more labeled peptides; (vi) subjecting the identified labeled peptide(s) to a second mass analysis, thereby generating a second mass spectrum for each of the labeled peptides; (vii) characterizing the polypeptide by analyzing the ETD mass spectrum and a second mass spectrum of each of the labeled peptides; Includes.

[0078] According to embodiments of the present application, the polypeptide to be characterized may be a fragment resulting from clipping of a protein or peptide (clipped polypeptide), including, but not limited to, an enzyme, an antibody (e.g., a monoclonal antibody, a diabody, a triabody, a tetrabody) or an antigen-binding fragment thereof, a biomolecule antigen, a fusion protein, a fusion peptide, a scaffold protein or peptide, a protein or peptide drug conjugate, or any other polypeptide or peptide useful as a therapeutic or diagnostic modality. The polypeptide itself may have one or more clipping sites and thus may be clipped to generate a clipped peptide.

[0079] N-terminal labeling reagents can react with terminal amine groups, including any primary amine-reactive reagent. Examples of reagents include, but are not limited to, Sanger's reagent, dansyl derivatives, phenylisothiocyanate (PITC), dimethoxypyrimidine-2-isothiocyanate (DMPITC), N-hydroxysuccinimide (NHS) reagent, (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP), dimethyl labeling reagent, tandem mass tag (TMT), and isobaric tag for relative and absolute quantification (iTRAQ).

[0080] The N-terminal labeling reagent suitable for the present invention is a fixed charge derivatization reagent. Examples of these reagents include, but are not limited to, TMPP, TMT, or iTRAQ. These reagents can add a tag with a fixed positive charge to a peptide. The fixed charge tag results in better ionization, and the hydrophobicity of the tag results in greater retention in reversed-phase chromatography. Furthermore, subsequent fragmentation of the fixed charge peptide in mass spectrometry produces various backbone fragments and charged tag fragments, including reporter ions, which are essentially diagnostic and can facilitate peptide identification. In particular, reporter ions are generated from charge loss during fragmentation.

[0081] Some of these reagents are also characterized by steric bulkiness that improves reaction specificity towards the free N-terminus and not towards any other free amines such as lysine.

[0082] In some embodiments, the N-terminal labeling reagent is TMPP. In further embodiments, the labeled polypeptide is a TMPP-labeled polypeptide and the labeled peptide is a TMPP-labeled peptide.

[0083] In some embodiments, the TMPP labeling of the peptide is primarily at the N-terminus, with additional unlabeled lysine or tyrosine residues being labeled with diagnostic TMPP. + Does not affect ions.

[0084] In some embodiments, the enzyme used to digest the peptide comprises any proteolytic enzyme known in the art.

[0085] In certain embodiments, TMPP-labeled polypeptides are digested to produce a mixture containing unlabeled and TMPP-labeled peptides. When this mixture is subjected to liquid chromatography (LC) to produce an LC eluate, this method allows for rapid separation of the TMPP-labeled peptides from the unlabeled peptides in the mixture because the TMPP label is hydrophobic and elutes later in the reverse-phase gradient. This allows for predictable retention times for the TMPP-labeled peptides, further improving specificity and reducing false-positive peptide identifications.

[0086] In some embodiments, the liquid chromatography (LC) separation step iii) can be omitted so that there is no liquid chromatography (LC) for separation. Thus, in the subsequent step iv), the mixture containing one or more unlabeled peptides and one or more labeled peptides is directly subjected to tandem mass spectrometry via direct infusion or flow injection. For example, in one embodiment, the tandem mass spectrometry includes electron transfer dissociation (ETD). Generally, ETD is a type of electron-induced dissociation method, so other types of electron-based dissociation methods, such as electron capture dissociation (ECD), can also be used herein as an alternative to ETD. In some embodiments, other dissociation methods, such as higher-energy collisional dissociation (HCD), can also be used herein as an alternative to ETD.

[0087] According to embodiments of the present application, the derivatized precursor ions subjected to ETD are independent of the level of derivatization of the peptide. For example, a peptide derivatized at 100% can have the same ETD efficiency as the same peptide derivatized at 1%. This is important when considering these reactions in the context of protein clip site identification; the derivatization efficiency of the protein does not affect the ETD efficiency of the surrogate peptide.

[0088] In some embodiments, the reporter ion is a TMPP reporter ion. In further embodiments, the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da. The nominal mass of an element is the mass number of its most abundant naturally occurring stable isotope; for an ion or molecule, the nominal mass is the sum of the nominal masses of the constituent atoms. The accuracy of the nominal mass is often better than 10 ppm.

[0089] Preferably, the method of the present invention comprises obtaining a tetrahydrofuran having an m / z of about 533 Da (TMPP) upon electron transfer dissociation (ETD). + ), and in some cases, approximately 590 Da (TMPP-Ac-NH2 + ) or upon higher energy collisional dissociation (HCD) at m / z approximately 573 Da (TMPP-Ac + This allows for the easy generation of TMPP reporter ions of approximately 533 Da. Typically, the reporter ion at approximately 533 Da is the most predominant product ion. The intense product ion can be used to trigger subsequent MS / MS events to further sequence the peptide. Thresholds can be used to trigger subsequent MS / MS events, including mass m / z thresholds and intensity thresholds. For example, filters (mass tolerance) are often used to trigger specific specificities, such as m / z thresholds. In certain embodiments, the filter is set to a base peak of 533.1935 or 590.2150 Da / charge or the exact mass-to-charge in Thomson, with a mass tolerance ranging from 1 to 20 ppm for triggering. The mass tolerance may be 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, or any number therebetween, preferably 5 ppm.

[0090] Alternatively, the intensity of the reporter ion can be used as a threshold, which can be set using the instrumentation software. For example, the intensity can be set to any particular number, such as 10% of the base peak, and then any reporter ion with an intensity at or greater than 10% of the base peak can trigger a subsequent MS / MS event.

[0091] In a preferred embodiment, the method involves identifying the TMPP-labeled peptide by detecting or isolating a TMPP reporter ion in an ETD mass spectrum of the labeled peptide.

[0092] In accordance with embodiments of the present application, generally, TMPP is + The tendency to generate reporter ions favors doubly charged precursor ions over triply charged precursor ions for peptides with similar mass or the same number of amino acids. Therefore, the diagnostic utility of TMPP+ ions generated by ETD is entirely appropriate for tryptic peptides, most of which are doubly charged.

[0093] In some embodiments, the second mass spectrometry is collision-induced dissociation (CID) mass spectrometry, high-energy collisional dissociation (HCD) mass spectrometry, or ultraviolet photodissociation (UVPD) mass spectrometry. Thus, in some embodiments, the tandem mass spectrometry is collision-induced dissociation (CID) mass spectrometry (CID-MS). 2 ), high-energy collisional dissociation tandem mass spectrometry (HCD-MS 2 ), or ultraviolet photodissociation tandem mass spectrometry (UVPD-MS 2 )

[0094] In some embodiments, the second mass spectrum is a CID, HCD, or UVPD mass spectrum of the TMPP-labeled peptide.

[0095] In some embodiments, the TMPP reporter ions trigger CID, HCD, or UVPD mass spectrometry. Triggered mass spectrometry techniques include MS 2Trigger MS to confirm the presence of reporter ions generated from the first ETD mass spectrometry portion 2 The low occurrence of makes the filtering of data suitable for manual inspection. This therefore eliminates the need for an in-situ approach or manual inspection of ETD mass spectra with reporter ion peaks.

[0096] In some embodiments, the TMPP reporter ion is generated from charge loss.

[0097] In some embodiments, the LC is a high performance liquid chromatogram (HPLC) or an ultra performance liquid chromatogram (UPLC).

[0098] In some embodiments, the method is high-throughput.

[0099] In some embodiments, the ETD mass spectrum (i.e., the first mass spectrum) and the second mass spectrum, such as a CID, HCD, or UVPD mass spectrum, are analyzed by comparison to information in a sequence database or spectral library.

[0100] In a preferred embodiment, the identified TMPP-labeled peptides are subjected to CID mass spectrometry.

[0101] According to embodiments of the present application, TMPP labeling can also occur on lysine and tyrosine residues of peptides, and when subjected to ETD, peptides with these TMPP modifications can also generate diagnostic reporter ions, thus enabling CID-MS analysis. 2 These CID spectra are false positive identifications of the reporter. Nevertheless, subsequent examination of the sequence ions in ETD and diagnostic ion-triggered CID spectra can site-specifically localize TMPP on the sequence and help eliminate false positives. For example, subsequent triggered CID-MS 2The spectrum can provide backbone ion information that can be used to determine whether the TMPP moiety is assigned to the N-terminus or side chain of lysine or tyrosine. 2 and trigger CID-MS 2 The complementary nature of the TMPP-clip assay can be useful for rapid screening of potential clipped species, regardless of the amino acid sequence of the surrogate proteolytic peptide containing the TMPP moiety. 2 for TMPP + The ability to generate ions offers complete interrogation of sequences for precise localization of TMPP moieties or confirmation of sequence with high confidence.

[0102] In a preferred embodiment, the present application relates to a method for characterizing N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptides in a sample, the method comprising: (i) subjecting the sample to tandem mass spectrometry including electron transfer dissociation (ETD); (ii) identifying the TMPP-labeled peptide by detecting or isolating the TMPP reporter ion in the ETD mass spectrum of the TMPP-labeled peptide; (iii) subjecting the identified TMPP-labeled peptide to a second mass analysis to generate a second mass spectrum of the TMPP-labeled peptide; (iv) characterizing the TMPP-labeled peptide by analyzing the ETD mass spectrum and a second mass spectrum; Includes:

[0103] It should be noted that the N-terminal labeling reagent, preferably TMPP, can also be attached to amine groups in the side chains of lysines and tyrosines. When TMPP-labeled peptides are subjected to ETD, reporter ions are also generated, and therefore CID-MS 2If a second mass spectrometry analysis is triggered, such as a reporter ion, these second mass spectra will result in false positive identification of the reporter ion. However, careful inspection of the sequence ions in the ETD spectrum and reporter ion-triggered CID spectrum can site-specifically localize TMPP on the sequence and help eliminate false positives.

[0104] In another general aspect, the present application also relates to a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more polypeptides with an N-terminal labeling reagent to obtain one or more labeled polypeptides; (iii) digesting the labeled clip polypeptide to produce a mixture containing unlabeled and labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to tandem mass spectrometry including electron transfer dissociation (ETD); (vi) identifying the labeled peptides by detecting a reporter ion in the ETD mass spectrum for each of the labeled peptides; (vii) subjecting the identified labeled peptides to a second mass analysis, thereby generating a second mass spectrum for each of the labeled peptides; (viii) characterizing the polypeptide by analyzing the ETD mass spectrum and a second mass spectrum for each of the labeled peptides; Includes.

[0105] As used herein, the term "protein" encompasses natural proteins, synthetic proteins, recombinant proteins, or peptides thereof. Examples of proteins that can be analyzed by the methods of the present invention include, but are not limited to, enzymes, antibodies (e.g., monoclonal antibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies) or antigen-binding fragments thereof, biomolecule antigens, fusion proteins, fusion peptides, scaffold proteins or peptides, protein or peptide drug conjugates, or any other polypeptides or peptides useful as therapeutic or diagnostic modalities.

[0106] According to embodiments of the present application, the clipped polypeptides to be characterized may be fragments resulting from the clipping of proteins such as enzymes, antibodies, and biomolecular antigens. The polypeptides themselves may have one or more clipping sites and thus may be clipped to generate clipped peptides.

[0107] In some embodiments, the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP). In further embodiments, the labeled polypeptide is a TMPP-labeled polypeptide and the labeled peptide is a TMPP-labeled peptide.

[0108] In some embodiments, the N-terminal labeling reagent is TMPP. In further embodiments, the labeled polypeptide is a TMPP-labeled polypeptide and the labeled peptide is a TMPP-labeled peptide.

[0109] In some embodiments, step iv) may be absent, such that there is no liquid chromatography (LC) for separation. Thus, in the subsequent step v), the mixture containing unlabeled and labeled peptides is directly subjected to electron transfer dissociation (ETD) or electron capture dissociation (ECD) or other electron induced dissociation tandem mass spectrometry by direct infusion or flow injection.

[0110] In some embodiments, the reporter ion is a TMPP reporter ion. In further embodiments, the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0111] In a preferred embodiment, the method involves identifying the TMPP-labeled peptide by detecting or isolating a TMPP reporter ion in an ETD mass spectrum of the labeled peptide.

[0112] In some embodiments, the second mass spectrometry is collision-induced dissociation (CID) mass spectrometry, high-energy collisional dissociation (HCD) mass spectrometry, or ultraviolet photodissociation (UVPD) mass spectrometry. Thus, in some embodiments, the tandem mass spectrometry is collision-induced dissociation (CID) mass spectrometry (CID-MS). 2 ), high-energy collisional dissociation tandem mass spectrometry (HCD-MS 2 ), or ultraviolet photodissociation tandem mass spectrometry (UVPD-MS 2 )

[0113] In some embodiments, the second mass spectrum is a CID, HCD, or UVPD mass spectrum of the TMPP-labeled peptide.

[0114] In some embodiments, the TMPP reporter ion triggers CID, HCD, or UVPD mass spectrometry.

[0115] In some embodiments, the TMPP reporter ion is generated from charge loss.

[0116] In some embodiments, the LC is a high performance liquid chromatogram (HPLC) or an ultra performance liquid chromatogram (UPLC).

[0117] In one embodiment, the protein is a therapeutic protein. In one embodiment, the protein is a non-therapeutic protein.

[0118] In some embodiments, the method is high-throughput.

[0119] In some embodiments, the ETD mass spectrum and a second mass spectrum, such as a CID, HCD, or UVPD mass spectrum, are analyzed by comparison with information in a database or spectral library, such as Uniprot, NIST, or SpectraST, However, these databases or public libraries do not have the 533 or 590 reporter ions annotated as a basis for identification and are therefore used for identification of non-reporter ions.

[0120] In a preferred embodiment, the identified TMPP-labeled peptides are subjected to CID mass spectrometry.

[0121] In another general aspect, the invention relates to a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting one or more TMPP-labeled clip polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to tandem mass spectrometry including electron transfer dissociation (ETD), thereby generating an ETD mass spectrum for each of the TMPP-labeled peptides; (vi) detecting or isolating the TMPP reporter ions in the ETD mass spectrum; and (vii) upon detection or separation of the TMPP reporter ions, subjecting each of the TMPP-labeled peptides to collision-induced dissociation (CID) mass spectrometry or high-energy collisional dissociation (HCD) mass spectrometry or ultraviolet photodissociation (UVPD) mass spectrometry, thereby producing a CID or HCD or UVPD mass spectrum, respectively, for each of the TMPP-labeled peptides; (viii) Identifying clip sites on the protein by analyzing the ETD mass spectrum and the CID or HCD or UPVD mass spectrum for each of the TMPP-labeled peptides; Includes.

[0122] In some embodiments, the clipped polypeptides to be characterized may be fragments resulting from clipping of proteins such as enzymes, antibodies, and biomolecular antigens. The polypeptides themselves may have one or more clipping sites and thus may be clipped to generate clipped peptides.

[0123] In some embodiments, step iv) may be absent, such that there is no liquid chromatography (LC) for separation. Thus, in the subsequent step v), the mixture containing unlabeled and labeled peptides is directly subjected to tandem mass spectrometry including electron transfer dissociation (ETD) via direct infusion or flow injection.

[0124] In some embodiments, the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0125] In some embodiments, the ETD mass spectrum and the CID or HCD or UPVD mass spectrum are analyzed by comparison to information in a database or spectral library.

[0126] In light of the present disclosure, any suitable mass spectrometer can be used with the inventions described herein. For example, tandem mass spectrometry including ETD can be performed using any instrument capable of performing ETD reactions. There are several models and instrument vendors capable of performing this type of dissociation, which are known in the art. For example, electron capture dissociation (ECD) has similarities to ETD and can therefore perform dissociation and produce the same reporter ions. Therefore, instruments capable of performing ECD reactions can also be used as an alternative to ETD tandem mass spectrometry. Similarly, CID tandem mass spectrometry can be performed using any instrument capable of performing CID reactions, including low-energy CID and high-energy CID.

[0127] System for identifying clip sites on polypeptides and / or for characterizing polypeptides The present invention also relates, in part, to a system for identifying a clip site on a polypeptide or for characterizing a polypeptide in a sample. In various aspects of the invention, the system includes a liquid chromatography (LC) device and a tandem mass spectrometer.

[0128] In one embodiment, the LC device is a high performance liquid chromatography (HPLC) device.

[0129] In some embodiments, the tandem mass spectrometer comprises: (i) a first ionizer; (ii) a first mass to charge ratio filter or mass to charge ratio mass analyser arranged and adapted in a first mode of operation to transmit ions having a mass to charge ratio within a first range; (iii) a first ion mobility spectrometer, detector, or separator; (iv) attenuation means for attenuating ions in an operational mode; (v) a controller configured to control operation of the attenuation means such that ions having a mass to charge ratio within the first range but having one or more undesirable first charge states are substantially attenuated; (vi) a second ionizer; (vii) a second ion mobility spectrometer, detector, or separator; (viii) a data system configured to acquire unmixed signals of fragment ions and non-redundantly encode trigger ions, the non-redundant encoding being arranged to avoid or minimize repeated overlap of any two ion signals from different parent species over multiple repetitions of any individual gate time; and Includes:

[0130] In one embodiment, the sample is subjected to an LC instrument to produce an eluate.

[0131] In one embodiment, the eluate is subjected to tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum.

[0132] In some embodiments, the clip site on the polypeptide or the polypeptide is labeled with an N-terminal labeling reagent. For example, in one embodiment, the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0133] In one embodiment, the first ionizer generates TMPP reporter ions. In various embodiments, the TMPP reporter ions have a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da. In one embodiment, the TMPP reporter ions trigger a second mass analysis.

[0134] In one embodiment, the first ionization device is an electron induced dissociation device. In some embodiments, the electron induced dissociation device is an electron transfer dissociation (ETD) device or an electron capture dissociation (ECD) device. Thus, in various embodiments, the first mass spectrum is an ETD or ECD mass spectrum.

[0135] In some embodiments, the second ionization device is a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, or an ultraviolet photodissociation (UVPD) device. Thus, in various embodiments, the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

[0136] In some embodiments, the first mass spectrum and the second mass spectrum are analyzed by comparison to information in a database or spectral library.

[0137] In some embodiments, the mass spectrometer further comprises a collision device, a fragmentation device, or a reaction device.

[0138] In some embodiments, the attenuation means comprises an ion gate or an ion barrier. In some embodiments, the attenuation means is located downstream of the ion mobility spectrometer or separator.

[0139] In some embodiments the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged and adapted in a first mode of operation to attenuate ions having mass to charge ratios outside a first range, hi some embodiments the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged upstream or downstream of the ion mobility spectrometer or separator.

[0140] In some embodiments, the first undesired charge state is selected from one or more of the following: (i) monovalent, (ii) divalent, (iii) trivalent, (iv) tetravalent, (v) pentavalent, and (vi) multivalent.

[0141] In some embodiments, the mass spectrometer further comprises an ion guide, ion trap or ion trapping region positioned upstream of said ion mobility spectrometer or separator, said ion guide, ion trap or ion trapping region arranged to trap, store or accumulate ions and then periodically pulse ions into or towards said ion mobility spectrometer or separator.

[0142] Reporter ions for identifying clip sites on the polypeptide and / or for characterizing the polypeptide. In another aspect, the present invention also relates, in part, to a reporter ion for identifying a clip site on a polypeptide and / or for characterizing a polypeptide. In one embodiment, the clip site is labeled with an N-terminal labeling reagent. In one embodiment, the polypeptide is labeled with an N-terminal labeling reagent. For example, in one embodiment, the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0143] In one embodiment, the N-terminal labeling reagent is ionized to produce a reporter ion, for example, in one embodiment, TMPP is ionized to produce a reporter ion.

[0144] In one embodiment, the N-terminal labeling reagent is ionized by a mass spectrometer to produce a reporter ion, for example, in one embodiment, TMPP is ionized by a mass spectrometer to produce a reporter ion.

[0145] In one embodiment, the mass spectrometer is a tandem mass spectrometer. In various embodiments, the tandem mass spectrometer is any tandem mass spectrometer described herein. For example, in some embodiments, the tandem mass spectrometer comprises an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, an ultraviolet photodissociation (UVPD) device, or any combination thereof.

[0146] In one embodiment, the N-terminal labeling reagent is ionized by a mass spectrometry technique to produce a reporter ion, for example, in one embodiment, TMPP is ionized by a mass spectrometry technique to produce a reporter ion.

[0147] In one embodiment, the mass spectrometry technique is a tandem mass spectrometry technique. In various embodiments, the tandem mass spectrometry technique is any tandem mass spectrometry technique described herein. For example, in some embodiments, the tandem mass spectrometry technique includes electron transfer dissociation (ETD), electron capture dissociation (ECD), collision-induced dissociation (CID), high-energy collisional dissociation (HCD), ultraviolet photodissociation (UVPD), or any combination thereof.

[0148] In some embodiments, the reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0149] In some embodiments, the reporter ion is

[0150] [ka]

[0151] [ka] It is a compound having the structure:

[0152] In another aspect, the invention also relates, in part, to compositions for identifying clip sites on polypeptides.

[0153] In another aspect, the invention pertains, in part, to compositions for characterizing polypeptides.

[0154] In various embodiments, the composition comprises at least one reporter ion and a polypeptide of the invention.

[0155] kit The present invention also relates to kits useful in the methods of the present invention. Such kits include various combinations of components useful in any of the methods described elsewhere herein, including, for example, materials for identifying clip sites on a polypeptide and / or materials for characterizing the polypeptide, as well as instructional materials. For example, in one embodiment, the kit includes components useful for identifying clip sites on a polypeptide in a sample. In one embodiment, the components useful for identifying clip sites on a polypeptide in a sample include TMPP. In another embodiment, the kit includes components useful for characterizing a polypeptide in a sample. In one embodiment, the components useful for characterizing a polypeptide in a sample include TMPP.

[0156] In one embodiment, the instructional material describes steps for labeling a polypeptide with TMPP. In one embodiment, the instructional material describes steps for labeling a clip site on a polypeptide with TMPP. In some embodiments, the instructional material describes one or more methods for analyzing a TMPP-labeled clip site on a polypeptide or a TMPP-labeled polypeptide using a system of the invention. In some embodiments, the instructional material describes one or more methods for analyzing a TMPP-labeled clip site on a polypeptide or a TMPP-labeled polypeptide using a system of the invention.

[0157] Embodiments of the present invention The present invention also provides the following non-limiting embodiments.

[0158] Embodiment 1 is a method for characterizing N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptides in a sample, the method comprising: (i) subjecting the sample to electron transfer dissociation (ETD) or electron capture dissociation (ECD) or other electron-based dissociation tandem mass spectrometry or higher energy collision dissociation (HCD) tandem mass spectrometry; (ii) identifying the TMPP-labeled peptide by detecting or isolating the TMPP reporter ion in an ETD or ECD, other electron-induced dissociation mass spectrum, or HCD mass spectrum of the TMPP-labeled peptide; and (iii) subjecting the identified TMPP-labeled peptide to a second mass analysis to generate a second mass spectrum of the TMPP-labeled peptide; (iv) characterizing the TMPP-labeled peptide by analyzing an ETD or ECD or other electron-induced dissociation mass spectrum or an HCD mass spectrum, and a second mass spectrum; Includes.

[0159] Embodiment 1a is the method of embodiment 1, wherein an ETD is used in the method.

[0160] Embodiment 1b is the method of embodiment 1, wherein an ECD is used in the method.

[0161] Embodiment 1c is a further embodiment in which the second mass analysis is CID tandem mass spectrometry (CID-MS 2 ) and the second mass spectrum is a CID mass spectrum.

[0162] Embodiment 1d is a further embodiment in which the second mass analysis is HCD tandem mass spectrometry (HCD-MS 2 ) and the second mass spectrum is an HCD mass spectrum.

[0163] Embodiment 1d is a further embodiment in which the second mass analysis is UVPD tandem mass spectrometry (UVPD-MS 2 ) and the second mass spectrum is a UVPD mass spectrum.

[0164] Embodiment 2 is the method of any one of embodiments 1-1d, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da or about 573 Da or about 590 Da.

[0165] Embodiment 2a is the method of embodiment 2, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da.

[0166] Embodiment 2b is the method of embodiment 2a, wherein the TMPP reporter ion has an exact mass-to-charge (m / z) of 533.1935.

[0167] Embodiment 2c is the method of embodiment 2a or 2b, wherein the TMPP-labeled peptide is identified by detecting a second TMPP reporter ion in an ETD or ECD or other electron-induced dissociation mass spectrum of the TMPP-labeled peptide, the second TMPP reporter ion having a nominal mass-to-charge (m / z) of about 590 Da.

[0168] Embodiment 2d is the method of embodiment 2c, wherein the second TMPP reporter ion has an exact mass-to-charge (m / z) of 590.2150.

[0169] Embodiment 2e is the method of any one of embodiments 2a-2d, wherein the TMPP-labeled peptide is identified by detecting a second or third TMPP reporter ion in an ETD or ECD or HCD or other electron-induced dissociation mass spectrum of the TMPP-labeled peptide, the second or third TMPP reporter ion having a nominal mass-to-charge (m / z) of about 573 Da.

[0170] Embodiment 2f is the method of embodiment 2e, wherein the second or third TMPP reporter ion has an exact mass-to-charge (m / z) of 573.1884.

[0171] Embodiment 3 is the method of any one of embodiments 1-2f, wherein the TMPP reporter ion is generated from charge loss.

[0172] Embodiment 3a is the method of embodiment 3, wherein the TMPP reporter ion is the predominant product ion in an ETD or ECD or other electron induced dissociation mass spectrum.

[0173] Embodiment 3b is the method of embodiment 3a, wherein the TMPP reporter ion is the predominant product ion in the ETD mass spectrum.

[0174] Embodiment 3c is the method of embodiment 3a, wherein the TMPP reporter ion is the predominant product ion in the ECD mass spectrum.

[0175] Embodiment 3d is the method of any one of embodiments 1-3c, wherein the TMPP reporter ion is generated from a doubly charged peptide.

[0176] Embodiment 3e is an embodiment in which the TMPP reporter ion is TMPP + The method of any one of embodiments 1 to 3c, wherein:

[0177] Embodiment 3f is a method for preparing a TMPP reporter ion comprising the steps of: + The method of any one of embodiments 1 to 3c, wherein:

[0178] Embodiment 3f is an embodiment in which the TMPP reporter ion is TMPP-Ac + The method of any one of embodiments 1 to 3c, wherein:

[0179] Embodiment 4 is the method of any one of embodiments 1-3f, wherein the TMPP reporter ion triggers a second mass analysis.

[0180] Embodiment 4a is the method of any one of embodiments 1-4, wherein the TMPP reporter ion triggers the second mass analysis via an intensity threshold or m / z threshold.

[0181] Embodiment 4b is the method of embodiment 4 or 4a, wherein a filter (mass tolerance) is used to trigger the second mass analysis.

[0182] Embodiment 4c is the method of embodiment 4b, wherein the filter is set to a base peak of exact mass of 533.1935, 573.1884, or 590.2150 Da with a mass tolerance in the range of 1 to 20 ppm for triggering, such as 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, or any number therebetween, preferably a mass tolerance of 5 ppm.

[0183] Embodiment 4d is the method of embodiment 4 or 4a, wherein the intensity of the reporter ion is used as a threshold for triggering the second mass analysis.

[0184] Embodiment 4e is the method of embodiment 4d, wherein the intensity is set to 10% or greater than the intensity of the base peak.

[0185] Embodiment 4f is a method for determining whether the TMPP reporter ions are amplified by CID tandem mass spectrometry (CID-MS 2 4e. The method of any one of embodiments 1-4e, wherein the method triggers collision-induced dissociation (CID) mass spectrometry to provide:

[0186] Embodiment 4g is a method for determining whether the TMPP reporter ions are detected by HCD tandem mass spectrometry (HCD-MS 2 4e. The method of any one of embodiments 1-4e, wherein the method triggers a higher-energy collisional dissociation (HCD) mass spectrometry analysis resulting in:

[0187] Embodiment 4h is a method for determining whether the TMPP reporter ions are ionized by UVPD tandem mass spectrometry (UVPD-MS 2 4e. The method of any one of embodiments 1-4e, wherein the method triggers ultraviolet photodissociation (UVPD) mass spectrometry to provide:

[0188] Embodiment 5 is a method for characterizing a polypeptide, the method comprising: (i) labeling a polypeptide with an N-terminal labeling reagent to obtain a labeled polypeptide; (ii) digesting the labeled polypeptide to produce a mixture comprising one or more unlabeled peptides and one or more labeled peptides; (iii) optionally subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (iv) subjecting the eluate from step iii) or the mixture from step ii) to electron transfer dissociation (ETD) or electron capture dissociation (ECD) or other electron induced dissociation tandem mass spectrometry; (v) identifying the labeled peptide by detecting a reporter ion in an ETD or ECD or other electron-induced dissociation mass spectrum of the labeled peptide; (vi) subjecting the identified labeled peptide to a second mass analysis, thereby generating a second mass spectrum of the labeled peptide; (vii) characterizing the polypeptide by analyzing the ETD or ECD or other electron induced dissociation mass spectrum and a second mass spectrum; Includes:

[0189] Embodiment 6 is the method of embodiment 5, wherein the polypeptide is a fragment polypeptide or a clip polypeptide.

[0190] Embodiment 6a is the method of embodiment 6, wherein the fragment polypeptide or clip polypeptide results from clipping of a protein.

[0191] Embodiment 6b is the method of embodiment 6 or 6a, wherein the polypeptide comprises one or more clip sites.

[0192] Embodiment 6c is the method of any one of Embodiments 6-6b, wherein the protein is an enzyme, an antibody (e.g., a monoclonal antibody, a bispecific antibody, a trispecific antibody, a tetraspecific antibody) or an antigen-binding fragment thereof, a biomolecule antigen, a fusion protein, a fusion peptide, a scaffold protein or peptide, a protein or peptide drug conjugate, or any other polypeptide or peptide useful as a therapeutic or diagnostic modality.

[0193] Embodiment 6d is the method of embodiment 6a, wherein the protein is a therapeutic protein.

[0194] Embodiment 6e is the method of embodiment 6a, wherein the protein is a non-therapeutic protein.

[0195] Embodiment 6f is the method of any one of embodiments 5-6e, wherein an ETD is used in the method.

[0196] Embodiment 6g is the method of any one of embodiments 5-6e, wherein an ECD is used in the method.

[0197] Embodiment 7 is a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more polypeptides with an N-terminal labeling reagent to obtain one or more labeled polypeptides; (iii) digesting the labeled clip polypeptide to produce a mixture containing unlabeled and labeled peptides; (iv) optionally subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate from step iv) or the mixture from step iii) to electron transfer dissociation (ETD) or electron capture dissociation (ECD) or other electron induced dissociation tandem mass spectrometry; (vi) for each of the labeled peptides, identifying the labeled peptide by detecting a reporter ion in an ETD or ECD or other electron induced dissociation mass spectrum; (vii) subjecting the identified labeled peptides to a second mass analysis, thereby generating a second mass spectrum for each of the labeled peptides; (viii) characterizing the polypeptide by analyzing the ETD or ECD or other electron induced dissociation mass spectrum and a second mass spectrum; Includes.

[0198] Embodiment 8 is the method of embodiment 7, wherein the protein is an enzyme, an antibody (e.g., a monoclonal antibody, a bispecific antibody, a trispecific antibody, a tetraspecific antibody) or an antigen-binding fragment thereof, a biomolecule antigen, a fusion protein, a fusion peptide, a scaffold protein or peptide, a protein or peptide drug conjugate, or any other polypeptide or peptide useful as a therapeutic or diagnostic modality.

[0199] Embodiment 8a is the method of embodiment 7, wherein the protein is a therapeutic protein.

[0200] Embodiment 8b is the method of embodiment 7, wherein the protein is a non-therapeutic protein.

[0201] Embodiment 8c is the method of any one of embodiments 7 to 8b, wherein the clip polypeptide of the protein has one or more clip sites.

[0202] Embodiment 8d is the method of any one of embodiments 7-8c, wherein an ETD is used in the method.

[0203] Embodiment 8e is the method of any one of embodiments 7 to 8d, wherein an ECD is used in the method.

[0204] Embodiment 9 is the method of any one of embodiments 5 to 8e, wherein the N-terminal labeling reagent is a fixed charge derivatization reagent.

[0205] Embodiment 9a is the method of embodiment 9, wherein the N-terminal labeling reagent adds a positive charge to the polypeptide.

[0206] Embodiment 9b is the method of embodiment 9 or 9a, wherein the N-terminal labeling reagent results in a reporter ion generated from charge loss.

[0207] Embodiment 9c is the method of embodiment 9, wherein the N-terminal labeling reagent is TMPP.

[0208] Embodiment 9d is the method of any one of embodiments 5 to 9c, wherein the efficiency of N-terminal labeling is 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any number therebetween.

[0209] Embodiment 10 is the method of any one of embodiments 5 to 9d, wherein the labeled polypeptide is a TMPP-labeled polypeptide.

[0210] Embodiment 10a is the method of embodiment 10, wherein the labeled peptide is a TMPP-labeled peptide.

[0211] Embodiment 11 is the method of any one of embodiments 5 to 10a, wherein the LC is a high performance liquid chromatogram (HPLC) or an ultra performance liquid chromatogram (UPLC), preferably an HPLC.

[0212] Embodiment 11a is the method of embodiment 11, wherein the TMPP-labeled peptide elutes later in the reverse-phase gradient than the corresponding unlabeled peptide.

[0213] Embodiment 11b is the method of any one of embodiments 5 to 10a, wherein the mixture from step iii) is subjected to tandem mass spectrometry involving electron capture dissociation (ECD).

[0214] Embodiment 11c is the method of embodiment 11b, wherein the mixture is subjected to tandem mass spectrometry involving ETD via direct or flow injection.

[0215] Embodiment 11d is the method of any one of embodiments 5 to 10a, wherein the mixture from step iii) is subjected to tandem mass spectrometry including electron transfer dissociation (ETD).

[0216] Embodiment 11e is the method of embodiment 11d, wherein the mixture is subjected to tandem mass spectrometry involving ECD via direct or flow injection.

[0217] Embodiment 12 is the method of any one of embodiments 5 to 11e, wherein the reporter ion is a TMPP reporter ion.

[0218] Embodiment 12a is the method of embodiment 12, wherein the TMPP reporter ions have a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0219] Embodiment 12b is the method of embodiment 12a, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da.

[0220] Embodiment 12c is the method of embodiment 12b, wherein the TMPP reporter ion has an exact mass-to-charge (m / z) of about 533.1935.

[0221] Embodiment 12d is the method of embodiment 12b or 12c, wherein the labeled peptide is identified by detecting a second TMPP reporter ion in an ETD or ECD or other electron-induced dissociation mass spectrum of the TMPP-labeled peptide, the second TMPP reporter ion having a nominal mass-to-charge (m / z) of about 590 Da.

[0222] Embodiment 12e is the method of embodiment 12d, wherein the second TMPP reporter ion has an exact mass-to-charge (m / z) of 590.2150.

[0223] Embodiment 12f is the method of any one of embodiments 12a-12e, wherein the labeled peptide is identified by detecting a second or third TMPP reporter ion in an ETD or ECD or HCD or other electron-induced dissociation mass spectrum of the TMPP-labeled peptide, wherein the second or third TMPP reporter ion has a nominal mass-to-charge (m / z) of about 573 Da.

[0224] Embodiment 12g is the method of embodiment 12f, wherein the second or third TMPP reporter ion has an exact mass-to-charge (m / z) of 573.1884.

[0225] Embodiment 12h is the method of any one of embodiments 12-12g, wherein the TMPP reporter ion is generated from charge loss.

[0226] Embodiment 12i is the method of any one of embodiments 12-12h, wherein the TMPP reporter ion is the predominant product ion in an ETD or ECD or other electron induced dissociation mass spectrum.

[0227] Embodiment 12j is the method of any one of embodiments 5-12i, wherein the TMPP reporter ion is generated from a doubly charged peptide.

[0228] Embodiment 12k is an embodiment wherein the TMPP reporter ion is TMPP + 13. The method of embodiment 12, wherein

[0229] Embodiment 121 is an embodiment in which the TMPP reporter ion is TMPP-Ac-NH + 13. The method of embodiment 12, wherein

[0230] Embodiment 12m is an embodiment in which the TMPP reporter ion is TMPP-Ac+ 13. The method of embodiment 12, wherein

[0231] Embodiment 13 is a method for determining whether the second mass spectrometry is a CID tandem mass spectrometry (CID-MS 2 12m), wherein the second mass spectrum is a CID mass spectrum.

[0232] Embodiment 13a is a further embodiment in which the second mass analysis is HCD tandem mass spectrometry (HCD-MS 2 12m), and the second mass spectrum is a HCD mass spectrum.

[0233] Embodiment 13b is a method in which the second mass analysis is UVPD tandem mass spectrometry (UVPD-MS 2 12m), and the second mass spectrum is a UVPD mass spectrum.

[0234] Embodiment 14 is the method of any one of embodiments 5 to 13b, wherein the TMPP reporter ion triggers a second mass analysis.

[0235] Embodiment 14a is the method of any one of embodiments 5-14, wherein the TMPP reporter ion triggers the second mass analysis via intensity and m / z.

[0236] Embodiment 14b is the method of embodiment 14 or 14a, wherein a filter (mass tolerance) is used to trigger the second mass analysis.

[0237] Embodiment 14c is the method of embodiment 14b, wherein the filter is set to a base peak of exact mass of 533.1935 or 590.2150 Da with a mass tolerance in the range of 1 to 20 ppm for triggering, such as 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, or any number therebetween, preferably a mass tolerance of 5 ppm.

[0238] Embodiment 14d is the method of embodiment 14 or 14a, wherein the intensity of the reporter ion is used as a threshold for triggering the second mass analysis.

[0239] Embodiment 14e is the method of embodiment 14d, wherein the intensity is set to 10% or greater than the intensity of the base peak.

[0240] Embodiment 14f is a method for determining whether the TMPP reporter ions are amplified by CID tandem mass spectrometry (CID-MS 2 14e. The method of any one of embodiments 5-14e, wherein the method triggers collision-induced dissociation (CID) mass spectrometry to provide:

[0241] Embodiment 14g is a method for determining whether the TMPP reporter ions are isolated by HCD tandem mass spectrometry (HCD-MS 2 14e. The method of any one of embodiments 5-14e, wherein the method triggers a high-energy collisional dissociation (HCD) mass spectrometry analysis resulting in:

[0242] Embodiment 14h is a method for determining whether the TMPP reporter ions are present in a tandem mass spectrometer (UVPD-MS). 2 14e. The method of any one of embodiments 5-14e, wherein the method triggers ultraviolet photodissociation (UVPD) mass spectrometry to provide:

[0243] Embodiment 15 is the method of any one of embodiments 5 to 14h, wherein the method is high throughput.

[0244] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the ETD or ECD or other electron induced dissociation mass spectrum and the second mass spectrum are analyzed by comparison with information in a database or spectral library.

[0245] Embodiment 16a is the method of embodiment 16, wherein an ETD mass spectrum and a second mass spectrum are analyzed.

[0246] Embodiment 16b is the method of embodiment 16, wherein an ECD mass spectrum and a second mass spectrum are analyzed.

[0247] Embodiment 16c is the method of any one of embodiments 16-16b, wherein the second mass spectrum is a CID mass spectrum.

[0248] Embodiment 16d is the method of any one of embodiments 16-16b, wherein the second mass spectrum is an HCD mass spectrum.

[0249] Embodiment 16e is the method of any one of embodiments 16-16b, wherein the second mass spectrum is a UVPD mass spectrum.

[0250] Embodiment 17 is the method of any one of embodiments 5 to 16e, wherein the method eliminates false positive identification of clip sites.

[0251] Embodiment 17a is the method of embodiment 17, wherein the false positive identification is caused by an unlabeled polypeptide or peptide.

[0252] Embodiment 17b is the method of embodiment 17, wherein the false positive identification is caused by labeling at a lysine residue.

[0253] Embodiment 17c is the method of embodiment 17, wherein the false positive identification is caused by labeling at a tyrosine residue.

[0254] Embodiment 18 is a method for identifying a clip site on a protein, the method comprising: (i) obtaining a sample containing one or more clip polypeptides of a protein; (ii) labeling one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting one or more TMPP-labeled clip polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) optionally subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate from step iv) or the mixture from step iii) to electron transfer dissociation (ETD) tandem mass spectrometry, thereby generating an ETD or ECD or other electron induced dissociation mass spectrum for each of the TMPP-labeled peptides; (vi) detecting or isolating the TMPP reporter ions in an ETD or ECD or other electron induced dissociation mass spectrum; (vii) upon detection or separation of the TMPP reporter ions, subjecting each of the TMPP-labeled peptides to collision-induced dissociation (CID) mass spectrometry or high-energy collisional dissociation (HCD) mass spectrometry or ultraviolet photodissociation (UVPD) mass spectrometry, thereby generating a CID or HCD or UPVD mass spectrum, respectively, for each of the TMPP-labeled peptides; (viii) identifying clip sites on the protein by analyzing ETD or ECD or other electron-induced dissociation mass spectra and CID or HCD or UPVD mass spectra; Includes.

[0255] Embodiment 19 is the method of embodiment 18, wherein the protein is an enzyme, an antibody (e.g., a monoclonal antibody, a bispecific antibody, a trispecific antibody, a tetraspecific antibody) or an antigen-binding fragment thereof, a biomolecule antigen, a fusion protein, a fusion peptide, a scaffold protein or peptide, a protein or peptide drug conjugate, or any other polypeptide or peptide useful as a therapeutic or diagnostic modality.

[0256] Embodiment 19a is the method of embodiment 19, wherein the protein is a therapeutic protein.

[0257] Embodiment 19b is the method of embodiment 19, wherein the protein is a non-therapeutic protein.

[0258] Embodiment 19c is the method of any one of embodiments 18 to 19b, wherein the clip polypeptide of the protein has one or more clip sites.

[0259] Embodiment 19d is the method of any one of embodiments 18-19c, wherein an ETD is used in the method.

[0260] Embodiment 19e is the method of any one of embodiments 18-19d, wherein an ECD is used in the method.

[0261] Embodiment 19f is the method of any one of embodiments 18-19e, wherein the efficiency of TMPP labeling is 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any number therebetween.

[0262] Embodiment 20 is the method of any one of embodiments 18 to 19f, wherein the LC is a high performance liquid chromatogram (HPLC) or an ultra performance liquid chromatogram (UPLC), preferably an HPLC.

[0263] Embodiment 20a is the method of embodiment 20, wherein the TMPP-labeled peptide elutes later in the reverse-phase gradient compared to the corresponding unlabeled peptide.

[0264] Embodiment 20b is the method of any one of embodiments 18-19b, wherein the mixture from step iii) is subjected to tandem mass spectrometry involving electron transfer dissociation (ETD).

[0265] Embodiment 20c is the method of embodiment 20b, wherein the mixture is subjected to tandem mass spectrometry with ETD via direct or flow injection.

[0266] Embodiment 20d is the method of any one of embodiments 18-19a, wherein the mixture from step iii) is subjected to tandem mass spectrometry involving electron capture dissociation (ECD).

[0267] Embodiment 20e is the method of embodiment 20c, wherein the mixture is subjected to tandem mass spectrometry involving ECD via direct or flow injection.

[0268] Embodiment 21 is the method of any one of embodiments 18-20c, wherein the TMPP reporter ions have a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0269] Embodiment 21a is the method of embodiment 21, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da.

[0270] Embodiment 21b is the method of embodiment 21a, wherein the TMPP reporter ion has an exact mass-to-charge (m / z) of 533.1935.

[0271] Embodiment 21c is the method of embodiment 21a or 21b, further comprising detecting a second TMPP reporter ion in an ETD or ECD or other electron-induced dissociation mass spectrum, wherein the second TMPP reporter ion has a nominal mass-to-charge (m / z) of about 590 Da.

[0272] Embodiment 21d is the method of embodiment 21c, wherein the second TMPP reporter ion has an exact mass to charge (m / z) of 590.2150.

[0273] Embodiment 21e is the method of any one of embodiments 21a-21d, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 573 Da.

[0274] Embodiment 21f is the method of embodiment 21e, wherein the TMPP reporter ion has an exact mass-to-charge (m / z) of 573.1884.

[0275] Embodiment 21g is the method of any one of embodiments 21-21f, wherein the TMPP reporter ion is generated from charge loss.

[0276] Embodiment 21h is the method of any one of embodiments 21-21g, wherein the TMPP reporter ion is the predominant product ion in an ETD or ECD or other electron induced dissociation mass spectrum.

[0277] Embodiment 21i is the method of any one of embodiments 21-21h, wherein the TMPP reporter ion is generated from a doubly charged peptide.

[0278] Embodiment 21j is an embodiment in which the TMPP reporter ion is TMPP + 22. The method of embodiment 21, wherein

[0279] Embodiment 21k is an embodiment in which the TMPP reporter ion is TMPP-Ac-NH + 22. The method of embodiment 21, wherein

[0280] Embodiment 21l is an embodiment in which the TMPP reporter ion is TMPP-Ac + 22. The method of embodiment 21, wherein

[0281] Embodiment 22 is a method for determining whether a TMPP reporter ion is a nucleotide sequence or a nucleotide sequence, wherein the TMPP reporter ion is a nucleotide sequence or a nucleotide sequence, and the TMPP reporter ion is a nucleotide sequence or a nucleotide sequence, wherein the TMPP reporter ion is a nucleotide sequence or a nucleotide sequence, ... 2 ) by collision-induced dissociation (CID) mass spectrometry, or HCD tandem mass spectrometry (HCD-MS 2 ) by high-energy collision dissociation (HCD) mass spectrometry, or UVPD tandem mass spectrometry (UVPD-MS 2 21l. The method of any one of embodiments 18-21l, wherein ultraviolet photodissociation (UVPD) mass spectrometry is triggered to provide:

[0282] Embodiment 22a is a method for determining whether TMPP reporter ions are identified via intensity and m / z by CID-MS. 2 CID mass spectrometry, or HCD-MS, which results in 2 HCD mass spectrometry, or UVPD-MS, which results in 2 23. The method of embodiment 22, wherein the UVPD mass spectrometry analysis results in:

[0283] Embodiment 22b is the method of embodiment 22 or 22a, wherein a filter (mass tolerance) is used to trigger the second mass analysis.

[0284] Embodiment 22c is the method of embodiment 22b, wherein the filter is set to a base peak of exact mass of 533.1935 or 590.2150 Da with a mass tolerance in the range of 1 to 20 ppm for triggering, such as 1 ppm, 2 ppm, 3 ppm, 4 ppm, 5 ppm, 6 ppm, 7 ppm, 8 ppm, 9 ppm, 10 ppm, 11 ppm, 12 ppm, 13 ppm, 14 ppm, 15 ppm, 16 ppm, 17 ppm, 18 ppm, 19 ppm, 20 ppm, or any number therebetween, preferably a mass tolerance of 5 ppm.

[0285] Embodiment 22d is the method of embodiment 22 or 22a, wherein the intensity of the reporter ion is used as a threshold for triggering the second mass analysis.

[0286] Embodiment 22e is the method of embodiment 22d, wherein the intensity is set to 10% or greater than the intensity of the base peak.

[0287] Embodiment 23 is the method of any one of embodiments 18 to 22e, wherein the method is high throughput.

[0288] Embodiment 24 is the method of any one of embodiments 18 to 23, wherein the ETD or ECD or other electron induced dissociation mass spectrum and the CID mass spectrum are analyzed by comparison with information in a database or spectral library.

[0289] Embodiment 24a is the method of any one of embodiments 18-23, wherein the ETD or ECD or other electron induced dissociation mass spectrum and the HCD mass spectrum are analyzed by comparison to information in a database or spectral library.

[0290] Embodiment 25 is the method of any one of embodiments 18 to 24a, wherein the method eliminates false positive identification of clip sites.

[0291] Embodiment 25a is the method of embodiment 25, wherein the false positive identification is caused by an unlabeled polypeptide or peptide.

[0292] Embodiment 25b is the method of embodiment 25, wherein the false positive identification is caused by labeling at a lysine residue.

[0293] Embodiment 25c is the method of embodiment 25, wherein the false positive identification is caused by labeling at a tyrosine residue.

[0294] Embodiment 26 is a system for identifying a clip site on a polypeptide or for characterizing a polypeptide in a sample.

[0295] Embodiment 26a is the system of embodiment 26, wherein the system comprises a liquid chromatography (LC) instrument and a tandem mass spectrometer.

[0296] Embodiment 26b is an embodiment wherein the tandem mass spectrometer comprises: (i) a first ionizer; (ii) a first mass to charge ratio filter or mass to charge ratio mass analyser arranged and adapted in a first mode of operation to transmit ions having a mass to charge ratio within a first range; (iii) a first ion mobility spectrometer, detector, or separator; (iv) attenuation means for attenuating ions in an operational mode; (v) a controller configured to control operation of the attenuation means such that ions having a mass to charge ratio within the first range but having one or more undesirable first charge states are substantially attenuated; (vi) a second ionizer; (vii) a second ion mobility spectrometer, detector, or separator; (viii) a data system configured to acquire unmixed signals of the fragment ions and to non-redundantly encode the trigger ions, the non-redundant encoding being arranged to avoid or minimize repetitive overlap of any two ion signals from different parent species over multiple repetitions of any individual gate time.

[0297] Embodiment 27 is a system according to any one of embodiments 26 to 26b, wherein the LC device is a high performance liquid chromatography (HPLC) device.

[0298] Embodiment 28 is the system of any one of embodiments 26-27, wherein the sample is subjected to an LC device to generate an eluate.

[0299] Embodiment 28a is the system of embodiment 28, wherein the eluate is subjected to tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum.

[0300] Embodiment 29 is the system according to any one of embodiments 26 to 28, wherein the clip site on the polypeptide or the polypeptide is labeled with an N-terminal labeling reagent.

[0301] Embodiment 29a is the system of embodiment 29, wherein the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0302] Embodiment 29b is the system of embodiment 29a, wherein the first ionizer produces TMPP reporter ions.

[0303] Embodiment 29c is the system of embodiment 29b, wherein the TMPP reporter ions have a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0304] Embodiment 29d is the system of any one of embodiments 29a-29c, wherein the TMPP reporter ion triggers a second mass analysis.

[0305] Embodiment 30 is the system of any one of embodiments 26-29, wherein the first ionization device is an electron induced dissociation device.

[0306] Embodiment 30a is the system of embodiment 30, wherein the electron induced dissociation device is an electron transfer dissociation (ETD) device or an electron capture dissociation (ECD) device.

[0307] Embodiment 30b is the system of embodiment 30a, wherein the first mass spectrum is an ETD or ECD mass spectrum.

[0308] Embodiment 31 is the system of any one of embodiments 26 to 30, wherein the second ionization device is a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, or an ultraviolet photodissociation (UVPD) device.

[0309] Embodiment 31a is the system of embodiment 31, wherein the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

[0310] Embodiment 32 is the system of embodiment 30b or 31a, wherein the first mass spectrum and the second mass spectrum are analyzed by comparison to information in a database or spectral library.

[0311] Embodiment 33 is the system of any one of embodiments 26 to 32, wherein the mass spectrometer further comprises a collision device, a fragmentation device, or a reaction device.

[0312] Embodiment 34 is the system of any one of embodiments 26 to 33, wherein the attenuation means comprises an ion gate or an ion barrier.

[0313] Embodiment 34a is the system of any one of embodiments 26 to 34, wherein the attenuation means is located downstream of the ion mobility spectrometer or separator.

[0314] Embodiment 35 is a system described in any one of embodiments 26 to 34a, wherein the first mass to charge ratio filter or mass to charge ratio mass analyzer is configured and adapted in a first mode of operation to attenuate ions having mass to charge ratios outside a first range.

[0315] Embodiment 35a is the system of any one of embodiments 26 to 35, wherein a first mass to charge ratio filter or mass to charge ratio mass analyzer is located upstream or downstream of the ion mobility spectrometer or separator.

[0316] Embodiment 36 is the system of any one of embodiments 26-35a, wherein the first undesired charge state is selected from one or more of the following: (i) monovalent, (ii) divalent, (iii) trivalent, (iv) tetravalent, (v) pentavalent, and (vi) multivalent.

[0317] Embodiment 37 is a system described in any one of embodiments 26 to 36, wherein the mass spectrometer further includes an ion guide, ion trap, or ion trapping region positioned upstream of the ion mobility spectrometer or separator, the ion guide, ion trap, or ion trapping region being configured to trap, store, or accumulate ions and then periodically pulse the ions into or towards the ion mobility spectrometer or separator.

[0318] Embodiment 38 is a reporter ion for identifying a clip site on a polypeptide and / or for characterizing a polypeptide.

[0319] Embodiment 38a is the reporter ion of embodiment 38, wherein the clip site is labeled with an N-terminal labeling reagent.

[0320] Embodiment 38b is the reporter ion of embodiment 38, wherein the polypeptide is labeled with an N-terminal labeling reagent.

[0321] Embodiment 38c is the reporter ion of embodiment 38a or 38b, wherein the N-terminal labeling reagent is ionized to produce the reporter ion.

[0322] Embodiment 38d is the reporter ion of embodiment 38a or 38b, wherein the N-terminal labeling reagent is N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0323] Embodiment 38e is the reporter ion of embodiment 38d, wherein TMPP is ionized to produce the reporter ion.

[0324] Embodiment 39 is the reporter ion of any one of embodiments 38a-38c, wherein the N-terminal labeling reagent is ionized by a mass spectrometer to produce the reporter ion.

[0325] Embodiment 39a is the reporter ion of any one of embodiments 38a-38c, wherein the N-terminal labeling reagent is ionized by a mass spectrometry technique to produce the reporter ion.

[0326] Embodiment 40 is the reporter ion of embodiment 38d or 38e, wherein TMPP is ionized by a mass spectrometer to produce the reporter ion.

[0327] Embodiment 40a is the reporter ion of embodiment 38d or 38e, wherein TMPP is ionized by a mass spectrometry technique to produce the reporter ion.

[0328] Embodiment 41 is the reporter ion of embodiment 39 or 40, wherein the mass spectrometer is a tandem mass spectrometer.

[0329] Embodiment 41a is the reporter ion of embodiment 41, wherein the tandem mass spectrometer comprises an electron transfer dissociation (ETD) instrument, an electron capture dissociation (ECD) instrument, a collision-induced dissociation (CID) instrument, a high-energy collisional dissociation (HCD) instrument, an ultraviolet photodissociation (UVPD) instrument, or any combination thereof.

[0330] Embodiment 42 is the reporter ion of embodiment 39a or 40a, wherein the mass spectrometry technique is a tandem mass spectrometry technique.

[0331] Embodiment 42a is the reporter ion of embodiment 42, wherein the tandem mass spectrometry technique is any tandem mass spectrometry technique described herein. For example, in some embodiments, the tandem mass spectrometry technique comprises electron transfer dissociation (ETD), electron capture dissociation (ECD), collision-induced dissociation (CID), high-energy collisional dissociation (HCD), ultraviolet photodissociation (UVPD), or any combination thereof.

[0332] Embodiment 42b is the reporter ion of embodiment 42a, wherein the reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[0333] Embodiment 42c is an embodiment wherein the reporter ion is:

[0334] [ka] The reporter ion of embodiment 42a is a compound comprising the structure:

[0335] Embodiment 43 is a composition for identifying a clip site on a polypeptide, the composition comprising at least one reporter ion and a polypeptide described herein.

[0336] Embodiment 44 is a composition for characterizing a polypeptide, the composition comprising at least one reporter ion and a polypeptide as described herein.

[0337] Embodiment 45 is a kit for identifying a clip site on a polypeptide in a sample, the kit comprising a reporter ion for identifying the clip site on the polypeptide.

[0338] Embodiment 45a is the kit of embodiment 45, wherein the clip site is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0339] Embodiment 45b is a kit according to embodiment 45a, wherein TMPP is ionized to produce a reporter ion.

[0340] Embodiment 46 is a kit for characterizing a polypeptide in a sample, the kit comprising a reporter ion for characterizing the polypeptide.

[0341] Embodiment 46a is the kit of embodiment 46, wherein the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[0342] Embodiment 46b is a kit according to embodiment 46a, wherein TMPP is ionized to produce a reporter ion.

[0343] The following examples are presented to further illustrate the principles of the present invention, but it should be understood that the following examples do not limit the invention, the scope of which is defined by the appended claims.

[0344] Experimental Example The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should not be construed as being limited to the following examples in any way, but rather as embracing any and all variations that become evident as a result of the teachings provided herein.

[0345] Without further description, it is believed that one of ordinary skill in the art can, using the foregoing description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following examples, therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0346] Example 1: N-Terminal TMPP Labeling and Detection of Diagnostic Ions by Electron Transfer Dissociation (ETD) The diagnostic utility of the TMPP reporter ion was investigated by labeling a NIST antibody standard with TMPP and by data-dependent ETD-MS / MS tryptic peptide mapping. The NIST antibody possessed mature N-termini from both the light and heavy chains. One of the two surrogate peptides corresponded to the N-terminus of the NIST antibody light chain, which had a free primary amine, while the N-terminus of the heavy chain consisted of a secondary amine resulting from cyclization of glutamine to form an N-terminal pyroglutamic acid residue. Any neo-N-termini on the NIST antibody were potential degradation or clipping products of the molecule during storage.

[0347] Figure 1 shows the ETD product ion spectrum of a peptide corresponding to the N-terminal sequence of the light chain of the NIST antibody. The mass spectrum consisted primarily of a diagnostic TMPP reporter ion (m / z = 533 Da) and a c-type backbone product ion consisting of the N-terminal TMPP tag. It is interesting to note that this peptide sequence was generated due to in-source fragmentation of a larger tryptic peptide that generated reporter ions to a much lesser extent during ETD, likely due to charge segregation on the C-terminal arginine residue (Xia et al., Journal of the American Chemical Society, 2007, 129:12232-12243). Nevertheless, the approach employed included electron transfer dissociation to generate a dominant reporter ion peak, localization of the TMPP tag on the N-terminus using high sequence coverage, and reporter ion triggering of a complementary activation event to confirm the presence of the mature N-terminus of the NIST light chain. The absence of additional low-level clipped species due to degradation of the NIST antibody was also confirmed by examining and filtering ETD-MS / MS spectra with the diagnostic reporter ions at 533 and 590 Da across the data set. See Figures 2A-B for the structures of the reporter ions at 533 and 590 Da.

[0348] The triggered CID approach made data filtering suitable for manual inspection due to the low occurrence of triggered MS2 scans that confirmed the presence of reporter ions generated from ETD-MS / MS events across the data set, thereby eliminating the need for an in silico approach or manual inspection of ETD-MS / MS scans with reporter ion peaks.

[0349] Example 2: Rapid separation and predictable retention times of TMPP-labeled peptides Next, we evaluated rapid separation conditions for high-throughput detection of TMPP-labeled peptides. Complete reversed-phase separation, cleanup, and re-equilibration of peptides were achieved in a total run time of 20 minutes. Figure 3 shows the reversed-phase chromatography buffer gradient and the corresponding total ion chromatogram of the NIST digest after TMPP labeling. It is important to note that most unlabeled peptides eluted between 2 and 30% organic in a shallow gradient of 10 minutes. A surrogate peptide corresponded to the N-terminus of the NIST antibody light chain, eluting at 12 minutes. TMPP labeling increased the hydrophobicity of N-terminal peptides, and TMPP-labeled peptides were mostly observed between two steep gradients: 2 and 85% organic in 1 minute. The ability to separate TMPP-labeled peptides and improve retention time predictability further improved peptide specificity and reduced false-positive identifications.

[0350] It was also important to note that the corresponding unlabeled NIST mAb N-terminal peptide counterpart at 3.5 min was also observed. Complex samples with multiple clipped species likely consisted of both TMPP-labeled and unlabeled peptide counterparts (due to less than 100% labeling efficiency), and these unlabeled peptides eluted earlier in the gradient, were usually of lower intensity, and in some cases were below the LOD compared to the labeled peptides.

[0351] We then evaluated the possibility of observing unlabeled peptides and assessed the retention time predictability of TMPP-labeled peptides by derivatizing 15 synthetic peptide standards from the NIST sequence. A 20-minute HPLC gradient injection of these peptide mixtures was monitored by recording the retention times and intensities of the unlabeled and TMPP-labeled peptides. The reaction efficiency of TMPP labeling was assessed by obtaining the peak area ratio of the TMPP-labeled peptide normalized to the total intensity observed for that peptide. In the analysis, 15 TMPP peptides eluted between 12 and 14 minutes. The labeling efficiency of these peptides ranged from 8 to 100%, with 8 of the 15 peptides reacting with 100% efficiency. It was noteworthy that all TMPP-labeled peptides were identified by MS / MS despite the varying reaction efficiencies. Their unlabeled counterparts eluted in a much broader retention time window of 4 to 12 minutes. Due to the high reaction efficiency, only 7 of the 15 unlabeled peptides were identified by MS / MS. The remaining 8 unlabeled peptides were detected in MS1 ​​but did not trigger MS2-ETD due to their low LOD. Peptide labeling enabled improved identification and retention time predictability. The overall gradient LC run time can be shortened for rapid identification of clip sites for less complex samples or extended for more complex mixtures.

[0352] Example 3: Diagnostic ions of TMPP-labeled synthetic peptide standards Several synthetic peptide standards corresponding to NIST mAbs were derivatized with TMPP labels and analyzed by ETD-MS. 2 and diagnostic reporter ion-triggered MS 2These peptides were subjected to LC-MS using CID events. Their propensity to generate diagnostic TMPP reporter ions (m / z = 533 Da, 590 Da) in ETD spectra was then assessed using these synthetic peptide standards with different lengths, amino acid compositions, and sites for TMPP labeling. Table 1 shows that most peptides triggered a subsequent MS2-CID scan upon detection of the TMPP reporter ion. The triggered scans were used to measure reporter ion intensities, i.e., TMPP. + Some efficiency estimates for the ETD spectra of NIST peptides were based on previously reported % efficiency calculations for fragmentation of peptide backbone bonds, and for TMPP-derivatized peptides. + and TMPP-Ac + (Gunawardena et al., Journal of the American Chemical Society, 2005, 127:12627-12639) for the reporter ions. See Figure 2D for the calculation of ETD efficiency estimates for these two reporter ions.

[0353] Due to differences in reporter ion abundance from synthetic NIST peptides, TMPP + Efficiency is TMPP-Ac + It was observed that the reporter ion intensity was significantly higher than that of TMPP. + It was also interesting to note that the reporter ion contribution significantly contributed to the overall ETD efficiency of the backbone bond.

[0354] [Table 1]

[0355] Related ETD-MS 2 and TMPP + Triggered CID-MS 2 See Figures 4A-4Z for spectra.

[0356] In addition, subsequent mass-triggered MS 2 It should also be noted that the fidelity of generating scans is likely influenced by data-dependent criteria on which subsequent scan decisions are made relative to the overall intensity of the reporter ions and the AGC settings of the precursor ions (Bowers et al., Scientific Reports, 2018, 8:10399). For example, both N-terminally labeled peptides FNWYVDGVEVHNAK (SEQ ID NO: 17) and VVSLTVLHQDWLNGK (SEQ ID NO: 26) exhibited lower intensity TMPP. + However, a mass trigger was only observed for FNWYVDGVEVHNAK (SEQ ID NO: 17, FIG. 4I), while the VVSLTVLHQDWLNGK (SEQ ID NO: 26) peptide produced diagnostic ions. 2 The trigger was absent (Figure 4L). Subsequent MS1 intensity examination revealed that the peptide VVSLTVLHQDWLNGK (SEQ ID NO: 26) was below the MS / MS threshold. Some interesting dissociation behavior was also observed based on the sequence composition of the derivatized peptide. The single residue extension or the location of the TMPP label led to the formation of TMPP as a charged species. +These results demonstrate a significant effect on the gas-phase dissociation of TMPP. For example, TMPP derivatized at both the N-terminus and lysine side chain of the peptide VVSLTVLHQDWLNGK (SEQ ID NO: 26, Figure 4Y) generated predominant reporter ions during ETD and triggered MS2-CID spectra. The peptide VVSLTVLHQDWLNGKE (SEQ ID NO: 25, Figure 4W), which contains a glutamic acid residue appended to the C-terminus with TMPP at the N-terminus, was also observed to generate abundant diagnostic reporter ions and triggered MS2-CID spectra. Without being bound by any particular theory, it is hypothesized that the propensity to generate reporter ions via ETD likely depends on a number of different factors, including the position of the TMPP label, the amino acid composition, and the charge state of the peptide. The synthetic peptide data suggested that TMPP labeling occurred predominantly at the N-terminus, with derivatization of tyrosine and lysine residues occurring to a lesser extent (i.e., 14 of 15 N-termini, 1 of 6 had a tyrosine residue, and 1 of 10 lysine residues were TMPP-labeled).

[0357] It was expected that the solvent accessibility of polar residues in intact proteins may result in unwanted labeling of lysine and tyrosine residues, as previously reported (Abello et al., Journal of Proteome Research, 2007, 6:4770-4776). However, triggered MS 2 This approach effectively facilitated localization of the TMPP modification and helped unambiguously determine the clip site. TMPP-labeled lysine-containing peptides were analyzed by ETD-MS. 2 was clearly localized exclusively to the N-terminus by diagnostic ion-triggered CID-MS 2 The data complemented the ETD spectra and improved reliable localization.

[0358] Most TMPP-labeled tyrosine-containing peptides were analyzed by ion-triggered CID-MS, which improved reliable localization. 2 ETD-MS with spectra 2However, when the sequence ions were insufficient to localize the TMPP modification site, ETD-MS was used. 2 Figure 4D shows an annotated ETD-MS analysis of a single TMPP-labeled peptide, VYACEVTHQGLSSPVTK (SEQ ID NO: 13), where a search engine incorrectly assigned a TMPP modification to the N-terminus. 2 The spectra are shown. However, based on the c-type and z-type ETD ions, the TMPP moiety cannot be unambiguously assigned to either the N-terminus or a side chain derivative of tyrosine. The utility of subsequent triggered MS2-CID scans produced a convincing y16 ion that unambiguously localized TMPP on the tyrosine residue. Figure 4P shows the ETD-MS analysis of a single TMPP-labeled peptide, EPQVYTLPPSR (SEQ ID NO: 22), which produced only diagnostic ions with no backbone sequence ions. 2 The spectra are shown. Sequence identification was based on the m / z of the precursor ion in the MS1 spectrum, and the reporter ion indicated that the peptide likely possessed a TMPP moiety. The triggered MS2-CID spectrum produced several backbone ions lacking the TMPP modification: y7, y8, and y10, along with ions b1–b4 with the TMPP moiety, localizing TMPP to the N-terminus. After examining all MS2-CID spectra, it was important to note that only a few spectra exhibited a diagnostic ion at 573, which has limited diagnostic utility.

[0359] ETD-MS 2 and triggered CID-MS 2 The complementary nature of how TMPP was used in these experiments helped to rapidly screen potential clipped species regardless of the amino acid sequence of the surrogate proteolytic peptide containing the TMPP moiety. The tandem MS approach described herein allows for ETD-MS of peptides with various lengths and charge states. 2 The ability of the TMPP moiety to generate a predominant diagnostic immonium ion makes it suitable for ETD-MS to trigger subsequent CID scans.2 This was important for the reporter ions generated. 2 was shown to produce TMPP diagnostic ions quite consistently, while fragment ions of both the ETD and CID backbones localized the TMPP modification to a single residue.

[0360] Example 4: Diagnostic ions of TMPP-labeled compounds derived from cell lysates Next, we investigated the efficiency of generating reporter ions for a large pool of tryptic peptides and their TMPP derivatives derived from K562 cell lysates to determine the TMPP fragments generated using ETD and HCD-type fragmentation. + The diagnostic utility of the reporter ions was investigated. The complexity of the peptides required a change in the overall LC separation time; therefore, the peptides were subjected to two single-shot, six-fold longer run times (120 min) and analyzed by ETD-MS. 2 and HCD-MS 2 Dissociation of peptides was performed separately for each run. Figure 5A shows peptide intensity as a function of observed retention time, overlaid with the AcCN gradient. Unlabeled peptides eluted up to approximately 30% AcCN as expected, while TMPP-labeled peptides eluted adjacent to the two rapid organic gradients (0 to 85% AcCN) as in the shorter gradient runs. The observation of TMPP-labeled synthetic standard peptides with higher overall intensity distributions indicated increased peptide hydrophobicity and improved ionization of TMPP-labeled peptides. Figure 5B shows the observed time distribution of a subset of TMPP-labeled peptides with their corresponding unlabeled peptides. The observed time difference (delta time) between the labeled peptides and the corresponding unlabeled peptides is then shown as a function of observed retention time, overlaid with the AcCN gradient. TMPP-labeled sequences eluted later for almost all peptides, as indicated by large positive values. TMPP-labeled peptides were observed at higher retention times and higher delta times in the two rapid AcCN gradients of the gradient.

[0361] The tendency to generate diagnostic TMPP reporter ions (m / z = 533 Da, 590 Da) in ETD spectra and TMPP reporter m / z 573 Da in HCD spectra was carefully evaluated for peptides with different lengths, amino acid compositions, charge states, and TMPP-labeled sites. The efficiency of generating diagnostic ions for each TMPP-labeled peptide was estimated using several different methods. Reporter ion intensities were compared with the TMPP reporter ions derived from both types of ETD. + and TMPP-AC-NH2 + The product ion intensities of various types were normalized as shown by the relationship in Equations 1 to 3 (see Figure 2D) for TMPP. + Reporter ion abundance is TMPP-Ac-NH2 + was more dominant (Figures 6A to 6C).

[0362]

number

[0363] Furthermore, we investigated the overall ETD efficiency results for all backbone reporters and C, Z-type product ions (Gunawardena et al., Journal of the American Chemical Society, 2005, 127:12627-12639) as shown in Equation 4, as well as all backbone fragments except for the reporter ions shown in Equation 5. Similar to the ETD reporter ion estimation in Equation 1, the HCD-derived reporter ions were normalized to the product ions as shown in Equation 6 (see Figure 2E). As shown in Figures 5A-5B, the TMPP + (533 Da) Efficiency was reported as a function of peptide precursor mass or peptide length, grouped by precursor charge state. + It was important to note the charge state dependence of the reporter ion ETD efficiency. + The efficiency was highest for doubly charged precursor ions and was observed to decrease linearly with peptide mass.+ The efficiency is TMPP-Ac-NH2 + (590 Da). + The tendency to generate reporter ions favored doubly charged precursor ions over triply charged precursor ions for peptides with similar mass or the same number of amino acids. This observation was intriguing because backbone c and z fragment ions of these same peptides showed increasing efficiency with increasing charge state (e.g., Figures 7A-7D).

[0364]

number

[0365] The overall backbone ETD efficiency of the TMPP-labeled peptide was then evaluated, considering all backbone fragment ions and all backbone fragment ions excluding the reporter ion. As shown in Figure 7A, the overall ETD efficiency, considering all product ions, showed a slight charge-state-dependent decrease. In contrast, Figure 7B shows that the overall ETD efficiency of the TMPP-labeled peptide showed a slight charge-state-dependent decrease. + For the overall backbone efficiency estimated by eq. 5, ignoring reporter ions, we show that the ETD efficiency exhibited a charge-state-dependent increase, which was generally observed for unmodified peptides as reported elsewhere. Taking all these observations into account, the effect of TMPP on the overall efficiency estimates, especially for doubly charged ions, is unclear. + The contribution of reporter ion intensity was significant. TMPP generated by ETD + The diagnostic utility of the ions was perfectly suited to tryptic peptides, most of which are doubly charged. As shown in Figure 7C, the HCD-derived TMPP-Ac + (573 Da) Efficiency was reported as a function of peptide precursor mass, grouped by precursor charge state. Overall, HCD generated TMPP-Ac. + The reporter ion efficiency is related to the ETD-generated TMPP. + The TMPP-Ac of most peptides was significantly lower than that of the reporter ion. +Reporter ion efficiencies were less than 1%, with a significant proportion having no diagnostic reporter ion or zero efficiency. There were several peptides with extreme efficiencies as high as approximately 30% (two outliers approaching 60% efficiency were false-positive assignments where the precursor m / z was the same as the reporter ion m / z). No charge-state dependence on HCD efficiency of the TMPP-Ac+ reporter ion was observed.

[0366] Next, we investigated the possibility of off-labeling TMPP to lysine and tyrosine residues. Twelve PSMs of TMPP labeled with tyrosine residues were identified from a total of 520 tyrosine-containing peptides against sequence matches (PSMs). Of these 520 tyrosine-containing PSMs, 262 PSMs had TMPP-derivatized N-termini, while the remaining 246 PSMs were unmodified. No PSMs of TMPP labeled with lysine residues were identified from a total of 1,441 lysine-containing PSMs. Of the 1,441 lysine-containing PSMs, 771 PSMs had TMPP-derivatized N-termini, 11 PSMs had TMPP-derivatized tyrosine, and the remaining 659 PSMs were unmodified. Figure 8 shows that ETD efficiency does not affect labeled peptides grouped by the number of tyrosine and lysine residues per peptide. These data indicate that the TMPP labeling of the peptides was predominantly at the N-terminus under the reaction conditions described, with additional unlabeled lysine or tyrosine residues present in the diagnostic TMPP + This suggests that the derivatization efficiency of the TMPP ions does not affect the ETD efficiency of the surrogate peptides. Figure 9 shows the overall distribution of the peptide reaction or TMPP labeling efficiency as estimated by Equation 7. The derivatization precursor ions subjected to ETD did not affect the level of derivatization. In other words, a peptide derivatized at 100% has the same ETD efficiency as the same peptide derivatized at 1%. This is important when considering these reactions in the context of protein clip site identification; the derivatization efficiency of the protein did not affect the ETD efficiency of the surrogate peptides.

[0367] Finally, we investigated the diagnostic utility of each reporter ion generated by ETD and HCD, as well as the LC retention time. TMPP-derivatized peptides and their unmodified counterparts were confidently identified through searches against human protein sequences, and sequence ions confidently localized the TMPP moiety to the N-terminus of the majority of peptides. Search results were used to determine the class of TMPP-labeled peptides and separate them from unlabeled peptides. The dissociation efficiency of the diagnostic ions (ETD or HCD) for each spectrum was then subjected to logistic regression and random forest models to determine the sensitivity and specificity of each diagnostic ion, generating receiver operating characteristic (ROC) curves for both ETD and HCD diagnostic ions. Similarly, the retention times of both labeled and unlabeled peptides were used to determine the sensitivity and specificity of elution time. We also demonstrated how search results and diagnostic ion abundances could be used to classify or misclassify spectra. As shown in Figure 10A, labeled peptides contained characteristic TMPP ions that were true positives (TPs). + As shown in Figure 10B, the labeled peptide produced a characteristic TMPP ion, which was a true positive (TP), whereas the unlabeled peptide counterpart did not produce a reporter ion, which was a true negative (TN). + TMPP generated reporter ions, but the unlabeled peptide counterpart was a false positive (FP). + As shown in Figure 10C, the modified peptide did not produce any diagnostic ions that were false negatives (FNs), whereas the unmodified peptide counterpart produced false positives (FPs). + Interfering ions with similar masses to the reporter ions were generated. Figures 11A and 11B show the area under the curve (AUC) of the ROC curve for each diagnostic ion and elution time. Among the reporter ions, TMPP + The reporter ion was the most diagnostic, with the highest AUC of 98%, and TMPP-Ac-NH2 + AUC was 85%, TMPP-Ac + The AUC was 84% ​​and the ETD-generated TMPP +The ROC curve for the 99% observed retention time AUC was the most diagnostic of all assays, suggesting that the diagnostic ion was the most accurate and specific compared to other reporter ions.

[0368] Example 5: Application of TMPP labeling and ETD reporter ion-triggered CID to detect therapeutic protein degradation Finally, TMPP labeling was applied to investigate the clipping site of dulaglutide, a commercially available therapeutic GLP1 agonist known to undergo protease-induced clipping of the GLP1 peptide. Dulaglutide, a GLP1-Fc fusion protein treated with cathepsin D, was used to analyze the clipping site using ETD-MS. 2 and diagnostic ion-triggered CID-MS 2 was used to study the putative clip site on the GLP1 peptide.

[0369] Previous studies have reported that cathepsin D induces cleavage of GLP1 at W25 / L26 (Dorai et al., Biotechnology Progress, 2011, 27:220-231; Deacon et al., Diabetes, 2004, 53:2181-2189; Manandhar et al., Journal of Medicinal Chemistry, 2015, 58:1020-1037). Figures 12A-12C show the MS analysis. 2 The spectra demonstrated evidence of neo-N-termini generated due to protease activity. 2 The resulting product ion spectrum (Figure 12A) contains the characteristic diagnostic ion TMPP + (m / z = 533 Da). Furthermore, c-type ions were primarily observed at the TMPP site localization. The site localization of TMPP on the N-terminus indicated a neo-N-terminus due to the FI clip, whereas a second TMPP on the C-terminal lysine residue can be inferred by the KG clip, since TMPP conjugated to the lysine residue was resistant to trypsinization of the sample after conjugation.

[0370] TMPP labeling also occurs on lysine and tyrosine residues, and when peptides with TMPP modifications are subjected to ETD, diagnostic reporter ions are also generated, thus allowing for CID-MS analysis. 2 It was important to note that these CID spectra were false positive identifications of the reporter. Nevertheless, careful inspection of the sequence ions in the ETD and diagnostic ion-triggered CID spectra revealed that TMPP was site-specifically localized on the sequence, helping to eliminate the false positives. Figure 12B shows the diagnostic reporter ion (m / z = 533)-triggered CID-MS of a doubly charged ion. 2 The spectra are shown. The b- and y-type ions supported TMPP site localization of both the N- and C-terminal lysines. It was important to note that the peptide sequence did not produce a characteristic CID-induced reporter ion (m / z=573).

[0371] An ETD spectrum of the unconjugated peptide (Figure 12C), in which no diagnostic ions were present, was also generated. The product ion distribution of the unconjugated peptide resulted in a mixture of both c- and z-type ions.

[0372] Full analysis of the dulaglutide peptide identified additional clippings of GLP1. Figures 13A-13C show ETD-MS of surrogate peptides corresponding to sequential clippings of the GLP1 sequence. 2 and diagnostic ion-triggered CID-MS 2 Evidence from product ion spectra is shown. The surrogate peptide resulting from the I / A clip produced a diagnostic ion exclusively at 533 Da, whereas those resulting from the A / W and W / L clips produced diagnostic ions at 533 Da and 590 Da during ETD. The predominant diagnostic ion at 533 Da was observed for each peptide by CID-MS. 2 Events were triggered and CID product ion spectra were generated. 2 Spectra complement ETD identification and triggered MS 2Scans confirmed the presence of reporter ions seamlessly generated from ETD-MS / MS to unambiguously identify the neo-N-terminus across the data set.

[0373] Figure 14 shows the clipping site of dulaglutide to generate a surrogate peptide from the neo-N-terminus. Figure 15A shows the extracted ion chromatogram (XIC) of the surrogate peptide without TMPP labeling, and Figure 15B shows the chromatogram of the labeled surrogate peptide.

[0374] To generate reporter ions for the TMPP-labeled neo-N-terminal peptides formed by clipping, we further investigated the utility of other dissociation modes, such as HCD (Michalski et al., Journal of Proteome Research, 2012, 11:5479-5491), which is known to generate more internal fragments, and UVPD, in addition to CID. Table 2 summarizes the results obtained for the clipped sites of the surrogate peptides IAWLVK (SEQ ID NO: 5), AWLVK (SEQ ID NO: 6), WLVK (SEQ ID NO: 7), and LVK of the GLP1 sequence. While all of these peptides generated a characteristic 533 Da diagnostic ion via ETD, only the LVK peptide sequence exhibited diagnostic ions in the HCD and UVPD dissociation modes. As shown in Figures 16A and 16B, HCD produced a characteristic diagnostic ion at 573 Da due to amide bond dissociation (Sadagopan et al., Journal of the American Society for Mass Spectrometry, 2000, 11:107-119; He et al., Journal of the American Society for Mass Spectrometry, 2012, 23:1182-1190), and UVPD produced a diagnostic ion at 181 Da, likely due to further dissociation and rearrangement (Huang et al., Analytical Chemistry, 1997, 69:137-144). In addition to not detecting diagnostic ions for all peptides, significantly lower relative peak intensities were observed, in contrast to the ETD-generated diagnostic ions (Figure 16C), making triggering of these ions less informative.

[0375] Table 2. Dissociation methods and identified clip sites of GLP1 based on reporter ion TMPP-specific sequence ions (HCD, CID, and UVPD diagnostic ions at 573 Da, 181 Da; ETD diagnostic ion at 533 Da).

[0376] [Table 2]

[0377] XIC demonstrated that each TMPP-labeled peptide eluted during two rapid gradients between 10 and 13 minutes. The short LVK peptides observed primarily in the endogenous sample exhibited low retention and were likely difficult to detect in peptide mapping experiments. However, the same peptides after TMPP labeling exhibited significant column retention during reversed-phase chromatography due to the peptide's overall enhanced hydrophobicity. The fact that unlabeled peptides were also observed suggested that TMPP labeling was not complete. The extent to which these TMPP derivatization reactions could be achieved was investigated, and optimal conditions were used for TMPP labeling (Figure 17A-B). Despite the presence of unlabeled counterparts with different retention times from their precursors, the identity of the TMPP label at the neo-N-terminus, resulting from clipping, was further verified.

[0378] In conclusion, the easy diagnostic ion TMPP + and TMPP-Ac-NH2 + The use of TMPP labeling in combination with electron transfer dissociation mass spectrometry has been reported as a means of generating reporter ions. Among reporter ions, ETD is the most intense and readily identified TMPP for small tryptic peptides. +This observation was atypical of typical backbone dissociation efficiencies, which typically increased with precursor ion charge for peptides of similar length. The ETD efficiency of doubly charged ions was lower than that of triply or quadruply charged ions due to the generation of neutral product ions as a result of a single cleavage (Xia et al., Journal of the American Chemical Society, 2007, 129:12232-12243; Gunawardena et al., Journal of the American Chemical Society, 2005, 127:12627-12639). Thus, the data indicated that the fixed charge groups on the TMPP moiety facilitate efficient electron recombination to generate favorable fragments that retain charge (Gunawardena et al., Molecular & Cellular Proteomics, 2016, 15:740-751). Using synthetic standard peptides of the NIST monoclonal antibody against TMPP, + The various factors that influence reporter ion generation were also demonstrated by generating a large pool of peptides from K562 cell lysates with varying lengths, charge states, and sequence compositions. + The reporter ion efficiency was highest for small, doubly charged peptides. In contrast, HCD showed no charge-state dependence for TMPP-Ac. + Reporter ions were generated. ETD generated TMPP + The diagnostic utility of the ions was evaluated using ROC analysis of HCD-generated TMPP-AC + The AUC was determined by 98% compared to 85% for the ion. + The ability to generate ions indicated a complete interrogation of the sequence or confirmation of the sequence with a high degree of confidence for precise location of the TMPP moiety when ETD failed to generate sufficient backbone fragments for doubly charged ions. 2High fidelity was demonstrated for a panel of TMPP-derivatized NIST-synthetic peptides and tryptic peptides generated from a TMPP-derivatized GLP1-Fc fusion protein. N-terminal labeling established the clip site prior to digestion and mass spectrometry analysis, both of which are known to generate spurious fragments that can be mistaken for clip sites.

[0379] Finally, TMPP to investigate the cathepsin-induced clip site of GLP1 + Diagnostic Reporter Ion-Triggered MS 2 The usefulness of ETD-MS of surrogate peptides corresponding to sequential clipping of the GLP1 sequence was demonstrated. 2 and diagnostic ion-triggered MS2-CID product ion spectra were obtained. + Diagnostic ions and subsequent reporter ion triggered CID-MS 2 This was reliably confirmed via CID-MS. 2 Spectra complement ETD identification and trigger MS 2 Scans were performed only when a CID scan was detected, providing a real-time in silico filtering mechanism. Reporter ion triggering was performed to ensure high-confidence identification and seamless assembly of the N-terminus down the entire data set. This analysis mode reduced ambiguity in clip site detection when labeling was not performed and eliminated the need to evaluate spurious artifacts caused by sample digestion and mass spectrometry conditions.

[0380] In summary, protein therapeutics were susceptible to clipping via enzymatic and nonenzymatic mechanisms, resulting in degraded protein neo-N-termini. Determination of the neo-N-termini of therapeutics was typically performed by chemical derivatization of the N-terminal amine group with TMPP, followed by proteolysis and mass spectrometry. Identification of TMPP-labeled peptides was possible by mapping peptide sequences bearing TMPP modifications to product ion spectra derived from collisional activation. Site-specific localization of the TMPP tag allowed unambiguous determination of the mature or neo-N-terminus. In addition to the backbone product ions, a 273 Da TMPP reporter ion formed via CID could be diagnostic for the presence of the processed N-terminus. However, reporter ions generated via CID were less informative due to their low abundance. Herein, a novel high-throughput LC-MS method was demonstrated to easily generate TMPP reporter ions at m / z 533 Da and, in some cases, 590 Da during ETD. The abundant generation of these reporters enabled subsequent MS / MS events using complementary ion activation modes such as CID, HCD, or UVPD via intensity- and m / z-dependent trigger events to further sequence peptides.

[0381] More specifically, we demonstrated the utility of TMPP-derived reporter ions for identifying clipped peptides via ETD-MS2 and diagnostic ion-triggered MS2 events for autonomously filtering clipped peptides. The approach described herein for efficient generation of reporter ions of TMPP-labeled standard peptides was demonstrated to represent both N-terminally clipped species and undesired TMPP labeling at lysine and tyrosine residues. Using a large pool of TMPP-labeled peptides with various sequence compositions, lengths, and charge states, we also demonstrated the diagnostic utility of reporter ions generated via ETD-MS2 on HCD-MS2. Finally, we applied the approach described herein to investigate sequential clipping of the GLP1 peptide of dulaglutide, a commercially available GLP1 agonist, following treatment with cathepsin D. Comparing the utility of the TMPP reporter ion for complementary dissociation modes: ETD, HCD, CID, and UVPD, suggested that the facile charge-loss peak at m / z = 533 Da of the TMPP+ ion generated via ETD was most diagnostic for TMPP-labeled peptides with neo-N-termini. The rapid separation method enabled efficient separation of TMPP-labeled peptides from unlabeled peptides in complex samples, improved retention time predictability of TMPP-labeled peptides, further improved specificity of clipped peptides, and reduced false-positive identifications.

[0382] The materials and methods used in the above experimental examples are described below.

[0383] Chemicals and Reagents (N-Succinimidyloxycarbonyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP), 4-morpholineethanesulfonic acid monohydrate (MES), N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), trimethylammonium bicarbonate (TMAB), dibasic sodium phosphate (NaHPO), monobasic sodium phosphate (NaHPO), dimethylformamide (DMF), bovine spleen cathepsin D, 1,4-dithiothreitol (DTT), iodoacetamide (IAA), and NIST-IgG1-K1 monoclonal antibody were all purchased from Sigma (St. Louis, MO).

[0384] Peptide standards from NIST monoclonal antibodies were synthesized to 99.99% purity by Biomatik Corporation (Ontario, Canada). Human K562 pre-digested cell extract, and sequencing-grade trypsin and endoproteinase Lys-C were purchased from Promega (Madison, Wisconsin). GLP1-Fc fusion protein was purchased from Myoderm (Norristown, Pennsylvania). Optimal LC-MS-grade acetonitrile, water, and formic acid, hydroxylamine, and Gybco PBS buffer were all purchased from Thermofisher Scientific (Waltham, Massachusetts).

[0385] N-terminal labeling and protein digestion Synthetic peptides, peptides derived from K562 predigests, NIST monoclonal antibodies, and GLP1-Fc fusion proteins were all derivatized with TMPP. Derivatization was performed in the following buffers: 100 mM MES pH 6, HEPES pH 7, and sodium phosphate pH 8, respectively. A fresh 100 mM TMPP solution was prepared by dissolving 100 mg of TMPP in 1.3 mL of DMF. TMPP labeling was performed by adapting a derivatization protocol published elsewhere (Deng et al., Methods in Molecular Biology, 2015, 1295:249-258).

[0386] Briefly, 10 μl of TMPP solution was added to 50 μg of peptide and protein, mixed briefly, followed by the addition of 40 μL of buffer, and the resulting mixture was then incubated for 1 hour. The reaction was quenched with 1 μL of hydroxylamine and then lyophilized to dryness. The dried peptide was reconstituted in 0.1% FA water for MS, and the dried protein was reconstituted in TMAB for trypsinization.

[0387] Proteins were digested using a protocol described elsewhere (Gunawardena et al., Molecular & Cellular Proteomics, 2016, 15:740-751). Briefly, proteins were reduced with DTT and subsequently alkylated with iodoacetamide. Proteins were then subjected to proteolysis with endoproteinase Lys-C for 1 h at 37 °C, followed by a 4-fold dilution of 25 mM TMAB, pH 8.0, 1 mM CaCl2, followed by trypsin digestion for 4 h at 37 °C. Digestion was stopped by adding formic acid to 0.1%. The peptide solution was desalted on a Sep-Pak Light C18 cartridge (Waters, Milford, MA) and collected for mass spectrometry analysis.

[0388] device All analyses were performed using an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA) coupled to an OrbitrapLumos (Thermo Scientific, San Jose, CA) Tribrid mass spectrometer equipped with an electrospray ion source using Tune application software 2.1.1565.18 and Xcalibur 4.0.27.13.

[0389] LC-MS / MS analysis All samples submitted for LC-MS / MS analysis were separated on an Agilent Infinity 1290 UHPLC (Agilent Technologies, Santa Clara, CA) using an AdvanceBio Peptide Map Micro Bore Rapid Resolution Column (1 × 150 mm, 2.7 μm) at 65 °C. The following 20-min fast LC gradient program was used: 0 min, 2% B; 10 min, 30% B; 10.5 min, 2% B; 11.5 min, 85% B; 12 min, 2% B; 13 min, 85% B; 13.5 min, 2% B; followed by a 14–18 min wash step at 85% B and a 2 min re-equilibration at 2% B. A 120-min LC gradient method was used, utilizing water containing 0.1% formic acid as mobile phase A and acetonitrile as mobile phase B: 0 min, 2% B; 60 min, 30% B; 60.5 min, 2% B; 61.5 min, 85% B; 62 min, 2% B; 63 min, 85% B; 63.5 min, 2% B; followed by a wash step from 64.5 to 80 min, 85% B, and then a 20-min re-equilibration at 2% B. The flow rate for all gradients was set at 0.2 mL / min, and the selected injection volume was 2 μL. The mass spectrometer was configured for data-dependent MS analysis. 2 The ETD, CID, HCD, and UVPD techniques were operated in positive ionization mode. The interface conditions were as follows: emitter voltage, -2600 V; vaporizer temperature, 325 °C; ion transfer tube, 325 °C; sheath gas, 55 arb; aux gas, 10 arb; and sweep gas, 1 arb.

[0390] Setting the method Unless otherwise stated, the internal mass analyzer settings utilized for MS scans were as follows: RF lens, 60%; AGC (automatic gain control) objective, 4e5; maximum injection time, 50 ms; and 1 μ scan in profile mode at 50K resolution on an Orbitrap mass analyzer. The method was then adapted for any HCD MS. 2A series of filters were included sequentially before the events. A monoisotopic peak selection filter was included and set to peptide for all methods. A 1e5 intensity filter was utilized for all methods unless otherwise noted. An optional charge state filter was included for some methods to select precursor charge states 2-6. An optional dynamic exclusion (DE) filter was included for some methods with either a 12-second or 3-second exclusion window, with common parameters: exclude n = 1; + / - 3 ppm; excluded isotope; and a single charge state per precursor. APEX detection was included in one method and set to an expected peak width of 6 seconds; and a desired apex window of 30%. Five ddMS runs were performed with the following settings unless otherwise noted. 2 OT-ETD scans were performed using the following: quadrupole isolation, 2 m / z isolation window; 50 ms response time; detector type: Orbitrap; automated m / z normal scan range, 15K resolution, 100 m / z first mass; AGC target: 2e5, ion injection for all available parallelizable time, maximum injection time: 50 ms; 1 μscan, profile. Target mass trigger (TMT) was used for ddMS. 2 Following IT-CID, ions 533.193, 690.214 were included; + / - 5 ppm error tolerance; detection of either two or one ion from the specified list; only ions within the top 10 most intense for all mass triggers were included. 2 OT-CID conditions were as follows unless otherwise stated: MS n Level, 2; Quadrupole isolation, 1.6 m / z isolation window; CID collision energy, 30; Activation Q, 0.25; Detector type, Orbitrap, automated m / z normal scan range, 15K resolution; AGC target, 5e4, ion injection for all available parallelizable time, maximum injection time 22ms; 1µ scan, profile. ddMS 2 OT-ETD and ddMS 2 The number of dependent scans between IT-CID was set to 1. Unless otherwise stated, five ddMS scans were performed with the following settings: 2OT-HCD scans were performed: quadrupole isolation, 1.6 m / z isolation window; HCD collision energy, 40%, stepwise 5%; detector type, Orbitrap, automated m / z normal scan range, 15K resolution; 100 m / z first mass; AGC target, 5e4, ion injection for all available parallelizable time, maximum injection time 35 ms; 1 μ scan, profile.

[0391] Data analysis Data analysis was performed using Xcalibur visualization software from Thermo Scientific (San Jose, CA), Byos3.9 chromatography and mass spectrometry data analysis software from Protein Metrics (Cupertino, CA), and R3.6 statistical programming software (Vienna, Austria).

[0392] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that modifications to the above-described embodiments may be made without departing from the broad inventive concept thereof. It is therefore understood that the invention is not limited to the particular embodiments disclosed, but that it is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

[0393] Those skilled in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein relates to all such variations and modifications to such elements and methods known to those skilled in the art.

[0394] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations. The invention described herein may include the following aspects. [1] 1. A method for characterizing N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptides in a sample, comprising: (i) subjecting the sample to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP-labeled peptide; (ii) identifying the TMPP-labeled peptide by detecting or isolating a TMPP reporter ion in a first mass spectrum of the TMPP-labeled peptide; (iii) subjecting the identified TMPP-labeled peptide to a second mass analysis to generate a second mass spectrum of the TMPP-labeled peptide; (iv) characterizing the TMPP-labeled peptide by analyzing the first mass spectrum and the second mass spectrum; The method comprising: [2] 1. A method for characterizing a polypeptide, comprising: (i) labeling the polypeptide with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) to obtain a TMPP-labeled polypeptide; (ii) digesting the TMPP-labeled polypeptide to produce a mixture comprising one or more unlabeled peptides and one or more TMPP-labeled peptides; (iii) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (iv) subjecting the eluate to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of each of the one or more TMPP-labeled peptides; (v) identifying the one or more TMPP-labeled peptides by detecting and isolating a TMPP reporter ion in the first mass spectrum of each of the one or more TMPP-labeled peptides; (vi) subjecting the identified one or more TMPP-labeled peptides to a second mass spectrometry analysis to generate a second mass spectrum for each of the one or more TMPP-labeled peptides; (vii) characterizing the polypeptide by analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP-labeled peptides; The method comprising: [3] 1. A method for identifying a clip site on a protein, comprising: (i) obtaining a sample containing one or more clip polypeptides of said protein; (ii) labeling the one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting the one or more TMPP-labeled clipped polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of each of the TMPP-labeled peptides; (vi) identifying each of the TMPP-labeled peptides by detecting and isolating a TMPP reporter ion in the first mass spectrum for each of the TMPP-labeled peptides; (vii) subjecting each of the identified TMPP-labeled peptides to a second mass spectrometry analysis to generate a second mass spectrum for each of the TMPP-labeled peptides; (viii) identifying the clip site on the protein by analyzing the first mass spectrum and the second mass spectrum for each of the TMPP-labeled peptides; The method comprising: [4] The method according to any one of the above [1] to [3], wherein the electron-induced dissociation is electron transfer dissociation (ETD) or electron capture dissociation (ECD). [5] The method according to any one of [1] to [4] above, wherein the first mass spectrum is an ETD or ECD mass spectrum. [6] The method according to any one of [1] to [5] above, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da. [7] The method according to any one of [1] to [6] above, wherein the TMPP reporter ion triggers the second mass analysis. [8] The method according to any one of [1] to [7] above, wherein the second mass spectrometry comprises collision-induced dissociation (CID), high-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD). [9] The method according to any one of [1] to [8] above, wherein the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

[10] The method according to [8] or [9] above, wherein the TMPP reporter ion triggers the CID, HCD, or UVPD mass spectrometry.

[11] The method according to any one of [1] to

[10] above, wherein the TMPP reporter ion is generated by charge loss.

[12] The method according to any one of [2] to

[11] above, wherein the LC is high performance liquid chromatogram (HPLC).

[13] The method according to any one of [3] to

[12] above, wherein the protein is a therapeutic protein.

[14] The method according to any one of [3] to

[12] above, wherein the protein is a non-therapeutic protein.

[15] The method according to any one of [1] to

[14] above, wherein the method is high-throughput.

[16] The method according to any one of [1] to

[15] above, wherein the first mass spectrum and the second mass spectrum are analyzed by comparing them with information in a database or a spectral library.

[17] The method according to any one of [1] to

[16] above, wherein the second mass spectrometry is the CID mass spectrometry.

[18] 1. A method for identifying a clip site on a protein, comprising: (i) obtaining a sample containing one or more clip polypeptides of said protein; (ii) labeling the one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting the one or more TMPP-labeled clipped polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to tandem mass spectrometry, thereby generating a first electron transfer dissociation (ETD) mass spectrum for each of the TMPP-labeled peptides; (vi) detecting or isolating a TMPP reporter ion in the ETD mass spectrum for each of the TMPP-labeled peptides; (vii) upon detection or separation of the TMPP reporter ions, subjecting each of the TMPP-labeled peptides to a second mass analysis comprising collision-induced dissociation (CID), high-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD), thereby producing a CID, HCD, or UVPD mass spectrum, respectively, for each of the TMPP-labeled peptides; (viii) identifying the clip site on the protein by analyzing the ETD mass spectrum and the CID, HCD, or UVPD mass spectrum for each of the TMPP-labeled peptides; The method comprising:

[19] 19. The method of any one of claims 18, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[20] The method according to

[18] or

[19] above, wherein the TMPP reporter ion is generated by charge loss.

[21] The method according to any one of

[18] to

[20] above, wherein the LC is high performance liquid chromatogram (HPLC).

[22] The method according to any one of

[18] to

[21] above, wherein the protein is a therapeutic protein.

[23] The method according to any one of

[18] to

[21] above, wherein the protein is a non-therapeutic protein.

[24] The method according to any one of

[18] to

[23] above, wherein the method is high-throughput.

[25] The method according to any one of

[18] to

[24] above, wherein the ETD mass spectrum and the CID or HCD or UVPD mass spectrum are analyzed by comparing them with information in a database or spectral library.

[26] The method according to any one of

[18] to

[24] above, wherein each of the TMPP-labeled peptides is subjected to the CID mass spectrometry.

[27] A system for identifying a clip site on a polypeptide or for characterizing a polypeptide in a sample, comprising a liquid chromatography (LC) device and a tandem mass spectrometer, the tandem mass spectrometer comprising: (i) a first ionizer; (ii) a first mass to charge ratio filter or mass to charge ratio mass analyser arranged and adapted in a first mode of operation to transmit ions having a mass to charge ratio within a first range; (iii) a first ion mobility spectrometer, detector, or separator; (iv) attenuation means for attenuating ions in an operational mode; (v) a controller configured to control operation of the attenuation means such that ions having a mass to charge ratio within the first range but having one or more undesirable first charge states are substantially attenuated; (vi) a second ionizer; (vii) a second ion mobility spectrometer, detector, or separator; (viii) a data system configured to acquire unmixed signals of fragment ions and to non-redundantly encode trigger ions, the non-redundant encoding being arranged to avoid or minimize repeated overlap of any two ion signals from different parent species over multiple repetitions of any individual gate time.

[28]

[27] The system according to

[27] above, wherein the first ionization device is an electron induced dissociation device.

[29] The system according to

[28] above, wherein the electron induced dissociation device is an electron transfer dissociation (ETD) device or an electron capture dissociation (ECD) device.

[30] The system according to any one of

[27] to

[29] above, wherein the second ionization device is a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, or an ultraviolet photodissociation (UVPD) device.

[31] The system according to any one of the above

[27] to

[30] , wherein the mass spectrometer further comprises a collision device, a fragmentation device, or a reaction device.

[32] The system according to any one of the above

[27] to

[31] , wherein the attenuation means includes an ion gate or an ion barrier.

[33] The system according to any one of the above

[27] to

[32] , wherein the attenuation means is arranged downstream of the ion mobility spectrometer or separator.

[34] 33. A system according to any one of claims 27 to 33, wherein the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged and adapted in the first mode of operation to attenuate ions having a mass to charge ratio outside the first range.

[35] The system according to any one of

[27] to

[34] above, wherein the first mass to charge ratio filter or mass to charge ratio mass analyzer is positioned upstream or downstream of the ion mobility spectrometer or separator.

[36] The system according to any one of

[27] to

[35] above, wherein the first undesirable charge state is selected from one or more of the following: (i) monovalent, (ii) divalent, (iii) trivalent, (iv) tetravalent, (v) pentavalent, and (vi) multivalent.

[37] 36. The system of claim 25, further comprising an ion guide, ion trap, or ion trapping region arranged upstream of the ion mobility spectrometer or separator, the ion guide, ion trap, or ion trapping region arranged to trap, store, or accumulate ions and then periodically pulse the ions into or towards the ion mobility spectrometer or separator.

[38] The system according to any one of

[27] to

[37] above, wherein the sample is subjected to the LC device to generate an eluate.

[39] The system described in

[38] above, wherein the eluate is subjected to the tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum.

[40] 39. The system according to claim 39, wherein the first mass spectrum is an ETD or ECD mass spectrum.

[41] 39. The system of claim 39, wherein the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

[42] The system according to any one of

[27] to

[41] above, wherein the clip site on the polypeptide or the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

[43] The system described in

[42] above, wherein the first ionization device generates TMPP reporter ions.

[44] The system described in

[43] above, wherein the TMPP reporter ions have a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[45] The system according to any one of claims 43 to 44, wherein the TMPP reporter ion triggers the second mass analysis.

[46] The system according to any one of the above

[27] to

[45] , wherein the LC is a high performance liquid chromatogram (HPLC).

[47] The system according to any one of

[38] to

[46] above, wherein the first mass spectrum and the second mass spectrum are analyzed by comparing them with information in a database or spectral library.

[48] a reporter ion for identifying a clip site on a polypeptide, the clip site is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP); the TMPP is ionized to generate the reporter ion; the reporter ion.

[49] A reporter ion for characterizing a polypeptide, comprising: the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP); the TMPP is ionized to generate the reporter ion; the reporter ion.

[50] The reporter ion according to

[48] or

[49] above, wherein the TMPP is ionized by a mass spectrometer to generate the reporter ion.

[51] The reporter ion according to

[50] above, wherein the mass spectrometer is a tandem mass spectrometer.

[52] The reporter ion according to

[51] above, wherein the tandem mass spectrometer comprises an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, an ultraviolet photodissociation (UVPD) device, or any combination thereof.

[53] 52. The reporter ion according to claim 52, wherein the reporter ion has a nominal mass-to-charge (m / z) of about 533 Da, about 573 Da, or about 590 Da.

[54] The reporter ion is

change

change

[53] above, wherein the compound has a structure selected from the group consisting of:

[55] A composition for identifying a clip site on a polypeptide, comprising at least one reporter ion and a polypeptide according to any one of

[48] and

[50] to

[54] above.

[56] A composition for characterizing a polypeptide, comprising at least one reporter ion according to any one of

[49] to

[54] above and a polypeptide.

[57] 1. A kit for identifying a clip site on a polypeptide or for characterizing a polypeptide in a sample, comprising: (i) N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling the clip site on the polypeptide or N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling the polypeptide; (ii) explanatory materials and The kit comprises:

Claims

1. 1. A method for characterizing N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) labeled peptides in a sample, comprising: (i) subjecting the sample to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of the TMPP-labeled peptide; (ii) identifying the TMPP-labeled peptide by detecting or isolating a TMPP reporter ion in a first mass spectrum of the TMPP-labeled peptide; (iii) subjecting the identified TMPP-labeled peptide to a second mass spectrometry analysis to generate a second mass spectrum of the TMPP-labeled peptide, wherein the second mass spectrometry analysis is selected from collision-induced dissociation (CID), high-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD); (iv) characterizing the TMPP-labeled peptide by analyzing the first mass spectrum and the second mass spectrum; and The method comprising:

2. 1. A method for characterizing a polypeptide, comprising: (i) labeling the polypeptide with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) to obtain a TMPP-labeled polypeptide; (ii) digesting the TMPP-labeled polypeptide to produce a mixture comprising one or more unlabeled peptides and one or more TMPP-labeled peptides; (iii) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (iv) subjecting the eluate to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of each of the one or more TMPP-labeled peptides; (v) identifying the one or more TMPP-labeled peptides by detecting or isolating a TMPP reporter ion in the first mass spectrum of each of the one or more TMPP-labeled peptides; (vi) subjecting the identified one or more TMPP-labeled peptides to a second mass spectrometry analysis to generate a second mass spectrum for each of the one or more TMPP-labeled peptides, wherein the second mass spectrometry analysis is selected from collision-induced dissociation (CID), high-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD); (vii) characterizing the polypeptide by analyzing the first mass spectrum and the second mass spectrum for each of the one or more TMPP-labeled peptides; The method comprising:

3. 1. A method for identifying a clip site on a protein, comprising: (i) obtaining a sample containing one or more clip polypeptides of said protein; (ii) labeling the one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting the one or more TMPP-labeled clipped polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to electron induced dissociation tandem mass spectrometry to obtain a first mass spectrum of each of the TMPP-labeled peptides; (vi) identifying each of the TMPP-labeled peptides by detecting or isolating a TMPP reporter ion in the first mass spectrum for each of the TMPP-labeled peptides; (vii) generating a second mass spectrum for each of the identified TMPP-labeled peptides by subjecting each of the identified TMPP-labeled peptides to a second mass analysis, wherein the second mass analysis is selected from collision-induced dissociation (CID), high-energy collisional dissociation (HCD), and ultraviolet photodissociation (UVPD); (viii) identifying the clip site on the protein by analyzing the first mass spectrum and the second mass spectrum for each of the TMPP-labeled peptides; The method comprising:

4. The method according to any one of claims 1 to 3, wherein the electron-induced dissociation is electron transfer dissociation (ETD) or electron capture dissociation (ECD).

5. The method of any one of claims 1 to 4, wherein the first mass spectrum is an ETD or ECD mass spectrum.

6. 6. The method of any one of claims 1 to 5, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of 533 Da, 573 Da, or 590 Da.

7. The method of any one of claims 1 to 6, wherein the TMPP reporter ion triggers the second mass analysis.

8. The method of any one of claims 1 to 7, wherein the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

9. 9. The method of claim 8, wherein the TMPP reporter ion triggers the CID, HCD, or UVPD mass spectrometry.

10. 10. The method of any one of claims 1 to 9, wherein the TMPP reporter ion is generated from charge loss.

11. The method of claim 2 or 3, wherein the LC is a high performance liquid chromatogram (HPLC).

12. The method of claim 3 , wherein the protein is a therapeutic protein.

13. The method of claim 3 , wherein the protein is a non-therapeutic protein.

14. The method of any one of claims 1 to 13, wherein the method is high throughput.

15. The method of any one of claims 1 to 14, wherein the first mass spectrum and the second mass spectrum are analyzed by comparing them with information in a database or spectral library.

16. The method according to any one of claims 1 to 7, wherein the second mass analysis is CID mass analysis.

17. 1. A method for identifying a clip site on a protein, comprising: (i) obtaining a sample containing one or more clip polypeptides of said protein; (ii) labeling the one or more clipped polypeptides with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP), thereby obtaining one or more TMPP-labeled clipped polypeptides; (iii) digesting the one or more TMPP-labeled clipped polypeptides to produce a mixture comprising unlabeled peptides and TMPP-labeled peptides; (iv) subjecting the mixture to liquid chromatography (LC) to produce an LC eluate; (v) subjecting the eluate to tandem mass spectrometry, thereby generating a first electron transfer dissociation (ETD) mass spectrum for each of the TMPP-labeled peptides; (vi) detecting or isolating a TMPP reporter ion in the ETD mass spectrum for each of the TMPP-labeled peptides; (vii) upon detection or separation of the TMPP reporter ions, subjecting each of the TMPP-labeled peptides to a second mass analysis comprising collision-induced dissociation (CID), high-energy collisional dissociation (HCD), or ultraviolet photodissociation (UVPD), thereby producing a CID, HCD, or UVPD mass spectrum for each of the TMPP-labeled peptides, respectively; (viii) identifying the clip site on the protein by analyzing the ETD mass spectrum and the CID, HCD, or UVPD mass spectrum for each of the TMPP-labeled peptides; The method comprising:

18. 18. The method of claim 17, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of 533 Da, 573 Da, or 590 Da.

19. 19. The method of claim 17 or 18, wherein the TMPP reporter ion is generated from charge loss.

20. The method according to any one of claims 17 to 19, wherein the LC is a high performance liquid chromatogram (HPLC).

21. The method of any one of claims 17 to 20, wherein the protein is a therapeutic protein.

22. The method of any one of claims 17 to 20, wherein the protein is a non-therapeutic protein.

23. The method of any one of claims 17 to 22, wherein the method is high throughput.

24. 24. The method of any one of claims 17 to 23, wherein the ETD mass spectrum and the CID or HCD or UVPD mass spectrum are analyzed by comparison with information in a database or spectral library.

25. The method of any one of claims 17 to 23, wherein each of the TMPP-labeled peptides is subjected to the CID mass spectrometry.

26. A system for identifying a clip site on a TMPP-labeled polypeptide or for characterizing a TMPP-labeled polypeptide in a sample, comprising a liquid chromatography (LC) device and a tandem mass spectrometer, the tandem mass spectrometer comprising: (i) a first ionizer, the first ionizer being an electron induced dissociation device; (ii) a first mass to charge ratio filter or mass to charge ratio mass analyser arranged and adapted in a first mode of operation to transmit ions having a mass to charge ratio within a first range; (iii) a first ion mobility spectrometer, detector, or separator; (iv) attenuation means for attenuating ions in the operational mode; (v) a controller configured to control operation of the attenuation means such that ions having a mass to charge ratio within the first range but having one or more undesirable first charge states are substantially attenuated; (vi) a second ionization device selected from a collision-induced dissociation (CID) device, a high-energy collisional dissociation (HCD) device, and an ultraviolet photodissociation (UVPD) device; (vii) a second ion mobility spectrometer, detector, or separator; (viii) a data system configured to acquire unmixed signals of fragment ions and to non-redundantly encode trigger ions, said non-redundant encoding being arranged to avoid or minimize repeated overlap of any two ion signals from different parent species in multiple repetitions of any individual gate time.

27. 27. The system of claim 26, wherein the electron induced dissociation device is an electron transfer dissociation (ETD) device or an electron capture dissociation (ECD) device.

28. 28. The system of claim 26 or 27, wherein the mass spectrometer further comprises a collision device, a fragmentation device, or a reaction device.

29. The system of any one of claims 26 to 28, wherein the attenuation means comprises an ion gate or an ion barrier.

30. A system according to any one of claims 26 to 29, wherein the attenuation means is located downstream of the ion mobility spectrometer or separator.

31. 31. A system according to any one of claims 26 to 30, wherein the first mass to charge ratio filter or mass to charge ratio mass analyser is arranged and adapted in the first mode of operation to attenuate ions having a mass to charge ratio outside the first range.

32. A system according to any one of claims 26 to 31, wherein the first mass to charge ratio filter or mass to charge ratio mass analyser is located upstream or downstream of the ion mobility spectrometer or separator.

33. The first undesired charge state is one of the following: (i) monovalent, (ii) divalent, (iii) trivalent (iv) tetravalent, (v) pentavalent, and (vi) multivalent; A system according to any one of claims 26 to 32.

34. 34. A system according to any one of claims 26 to 33, further comprising an ion guide, ion trap or ion trapping region arranged upstream of the ion mobility spectrometer or separator, the ion guide, ion trap or ion trapping region arranged to trap, store or accumulate ions and then periodically pulse ions into or towards the ion mobility spectrometer or separator.

35. The system of any one of claims 26 to 34, wherein the sample is subjected to the LC device to produce an eluate.

36. 36. The system of claim 35, wherein the eluate is subjected to the tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum.

37. 37. The system of claim 36, wherein the first mass spectrum is an ETD or ECD mass spectrum.

38. 37. The system of claim 36, wherein the second mass spectrum comprises a CID, HCD, or UVPD mass spectrum.

39. The system according to any one of claims 26 to 38, wherein the clip site on the polypeptide or the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP).

40. 40. The system of claim 39, wherein the first ionizer produces a TMPP reporter ion.

41. 41. The system of claim 40, wherein the TMPP reporter ion has a nominal mass-to-charge (m / z) of 533 Da, 573 Da, or 590 Da.

42. 42. The system of claim 40 or 41, wherein the TMPP reporter ions trigger mass analysis by the second ionization device.

43. The system according to any one of claims 26 to 42, wherein the LC is a high performance liquid chromatogram (HPLC).

44. A system described in any one of claims 35 to 43, wherein the eluate is subjected to tandem mass spectrometry to obtain a first mass spectrum and a second mass spectrum, and the first mass spectrum and the second mass spectrum are analyzed by comparing them with information in a database or spectral library.

45. a reporter ion for identifying a clip site on a polypeptide, the clip site is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP); the TMPP is ionized to produce the reporter ion; The reporter ion is generated by the method of any one of claims 1 to 25. the reporter ion.

46. A reporter ion for characterizing a polypeptide, comprising: the polypeptide is labeled with N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP); the TMPP is ionized to produce the reporter ion; The reporter ion is generated by the method of any one of claims 1 to 25. the reporter ion.

47. 47. The reporter ion of claim 45 or 46, wherein the TMPP is ionized by a mass spectrometer to produce the reporter ion.

48. 48. The reporter ion of claim 47, wherein the mass spectrometer is a tandem mass spectrometer.

49. 49. The reporter ion of claim 48, wherein the tandem mass spectrometer comprises an electron transfer dissociation (ETD) instrument, an electron capture dissociation (ECD) instrument, a collision-induced dissociation (CID) instrument, a high-energy collisional dissociation (HCD) instrument, an ultraviolet photodissociation (UVPD) instrument, or any combination thereof.

50. 50. The reporter ion of claim 49, wherein the reporter ion has a nominal mass-to-charge (m / z) of 533 Da, 573 Da, or 590 Da.

51. The reporter ion is 【Chemistry 1】 51. The reporter ion of claim 50, which is a compound having a structure selected from the group consisting of:

52. A composition for identifying a clip site on a polypeptide, said composition comprising at least one reporter ion and a polypeptide according to any one of claims 45 and 47 to 51.

53. A composition for characterizing a polypeptide, said composition comprising at least one reporter ion according to any one of claims 46 to 51 and a polypeptide.

54. A kit for identifying a clip site on a polypeptide or characterizing a polypeptide in a sample by the method of any one of claims 1 to 25, comprising: (i) N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling the clip site on the polypeptide or N-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl (TMPP) for labeling the polypeptide; (ii) explanatory materials; and The kit comprises:

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