System and method for assaying large molecules with improved sensitivity

Abstract

This invention relates to a system and method for assaying the presence of high molecular weight analytes such as proteins, antibodies, antigens, and receptors, using a targeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS / MS) system, optionally combined with affinity capture. [Solution] In some embodiments, the system is partially or fully automated. In some embodiments, the system may enable the detection of protein biomarkers (e.g., antibodies or antigens) from clinical or nonclinical biological tissue or fluid samples in the range of pg / mL to ng / mL.

Description

Technical Field

[0001] The present application relates to systems and methods for assaying the presence of macromolecular analytes such as proteins, e.g., antibodies, antigens, receptors, etc., optionally in combination with affinity capture, using a targeted two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS / MS) system. In some embodiments, the system is partially or fully automated. In some embodiments, the system may enable the detection of protein biomarkers (e.g., antibodies or antigens) from clinical or non-clinical biological tissue or fluid samples in the range of pg / mL to ng / mL.

Background Art

[0002] Historically, ligand binding assays have been the technology selected to assist in pharmacokinetic and biomarker evaluation for drug development (1). This technology is well established, relatively easy to perform, and has both high sensitivity and assay throughput. Therefore, it is widely used in bioanalytical applications. However, the main limitation of ligand binding assays is that their performance strongly depends on the availability and quality of critical reagents, typically antibodies or antibody pairs (1-3). Antibody development is difficult and costly and usually requires a lead time of 3 to 6 months (4, 5). Due to this limitation, reagent-independent bioanalytical strategies are highly desirable when high-quality antibody reagents are not available or when shorter assay development times are required.

[0003] Mass spectrometry can be a powerful alternative analytical tool for the evaluation of pharmacokinetics and biomarkers in drug development. Furthermore, structural information about the analyte can be obtained. The main advantage of mass spectrometry-based assays compared to ligand binding assays is that they are less dependent on high-quality binding reagents. However, an important limitation is that their sensitivity is relatively low.

[0004] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used as an alternative to ligand-binding assays and can quantify proteins in a reagent-independent manner (6-10). However, without enrichment with highly specific capture reagents such as anti-idiotype antibodies, the sensitivity of conventional LC-MS / MS is relatively low, typically in the range of 100 ng / mL to low μg / mL for monoclonal antibodies, for example (11-13). This may be insufficient for measuring antibody therapeutics at trough levels and may not be sufficiently sensitive to specific biomarkers.

[0005] To increase sensitivity compared to one-dimensional LC-MS / MS, approaches based on two-dimensional liquid chromatography (2D-LC) are used. An additional step of LC separation is included to enrich the analyte and reduce matrix interference from molecules in the sample. Keshishian, H. et al. successfully quantified low ng / mL range cardiovascular biomarkers in human plasma using immunodepletion, offline strong cation exchange (SCX) fractionation, and nanoLC-MS / MS approaches (14). More recently, Shi, T. et al. developed the PRISM-SRM method, which combines offline high-pH reversed-phase liquid chromatography (RPLC) fractionation with nanoLC-MS / MS, enabling the quantification of pg / mL range proteins in human plasma / serum (15). Compared to SCX, high-pH RPLC may offer higher resolution, higher recovery, and more reproducible separation of peptides, and a salt-free mobile phase may be better suited to MS detection (16). Limitations of the above methods are that they are labor-intensive and have relatively low throughput. To address these issues, several attempts have been made to automate 2D-LC-MS / MS methods based on high-pH and low-pH RPLC. For example, Shen, Y. et al. (17) and Zhang, G. et al. (18) implemented a method to control high-pH and low-pH RPLC using two independent HPLC systems with analytical flow, enabling the quantification of immunoglobulin A1 protease and myostatin with lower limits of quantification (LLOQ) of 50 ng / mL and 2.5 ng / mL, respectively. In another report, Zhang M. et al. described a capture microLC-MS strategy in which target peptides and all matrix molecules eluting before the target peptide in high-pH RPLC are selectively transferred to a high-volume trap column at high flow rate and then subjected to two-dimensional microflow LC-MS analysis operated at low flow rate. This method enabled 13-fold and 4-fold improvements in sensitivity compared to conventional analytical flow 1D-LC-MS / MS and microflow 1D-LC-MS / MS, respectively (19). This method used a relatively wide trap window.The data demonstrated that over 80% of the matrix peptides were removed from the trap column before microLC-MS analysis, but assay sensitivity may still be impaired by a significant amount of matrix molecules captured by the trap column that could co-elute the target peptide.

[0006] The methods and systems described herein utilize 2D-LC with mass spectrometry detection in some embodiments, enabling both complete automation and improved sensitivity compared to 1D-LC-MS / MS methods. [Overview of the Initiative]

[0007] This disclosure relates to a method for detecting an analyte in a sample. In some embodiments, the method includes (a) determining the peak apex (tapex) of the analyte by performing a high pH reversed-phase liquid chromatography (RPLC) survey scan on the analyte at a predetermined concentration in the presence of an internal standard, along with detection by mass spectrometry (MS); (b) obtaining an experimental sample containing the analyte; (c) performing high pH RPLC on the sample from (b); (d) collecting at least a portion of the eluate from the high pH RPLC in (c) corresponding to the average peak width of the Tapex+ / - analyte or internal standard in the survey scan from (a), wherein the at least portion is optionally collected in a trap column; (e) performing low pH RPLC on the collected portion of the eluate from the high pH RPLC in (d); and (f) detecting the analyte by MS. In some embodiments, the analyte is a peptide.

[0008] The Disclosure also relates to a method for detecting a peptide analyte in a sample, comprising: (a) obtaining an experimental sample and isolating proteins in the sample by performing affinity-based capture; (b) digesting the captured proteins with a protease to obtain a peptide analyte for detection; (c) determining the peak tapex of the analyte by performing a high pH reversed-phase liquid chromatography (RPLC) survey scan on the peptide analyte at a predetermined concentration in the presence of an internal standard peptide, along with detection by mass spectrometry (MS); (d) performing high pH RPLC on the digested sample from (b) containing the peptide analyte; (e) collecting at least a portion of the eluate from the high pH RPLC from (d) corresponding to the Tapex+ / - analyte of the analyte, the internal standard, or the average peak width of the peptide in the survey scan from (c), wherein the at least portion is optionally collected in a trap column; (f) performing low pH RPLC on the collected portion of the eluate from the high pH RPLC from (e); and (g) detecting the analyte by MS.

[0009] In any of the above methods, high pH RPLC and low pH RPLC can be performed at micro-flow rates. In any of the above methods, high pH RPLC and / or low pH RPLC can be performed at flow rates of 4-10 μL / min, 5-10 μL / min, 6-9 μL / min, 7-10 μL / min, 6-8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, high pH RPLC and / or low pH RPLC are performed on a C18 column. In some embodiments, both high pH RPLC and low pH RPLC are performed on a C18 column. In some embodiments, high pH RPLC and survey scans are performed at pH levels between 8 and 10, between 8 and 9, between 9 and 10, or at pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10, using mobile phase A containing ammonium formate and mobile phase B containing ammonium formate and acetonitrile (e.g., a 90% acetonitrile solution of ammonium formate). In some embodiments, high pH RPLC survey scans and high pH RPLC performed on a sample or digested sample are performed at substantially the same flow rate, pH, temperature, pressure, and / or column type (e.g., C8, C18, etc.). In any of the above methods, low pH RPLC can be performed at a pH between 2 and 4.5, between 2 and 4, between 2 and 3, between 2 and 2.5, between 2.5 and 3, between 3 and 4, or at pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, or pH 4.5, using mobile phase A containing formic acid and mobile phase B containing formic acid and acetonitrile (for example, a solution of formic acid in acetonitrile).

[0010] In any of the above methods, the sample may be a biological sample, but in some cases it is not a biological sample. In some cases, the sample is a biological fluid sample such as tears, saliva, lymph, urine, serum, cerebrospinal fluid, pleural fluid, ascites, or plasma. In some embodiments, the sample is a serum sample. In some cases, the sample is a tissue sample. In some cases, the sample is processed to concentrate or extract components containing the analyte before use in the method.

[0011] In any of the above methods, a portion of the eluate from the high-pH RPLC in which the low-pH RPLC is performed may correspond to the portion of the analyte with a Tapex of + / - 0.25 minutes at a flow rate of 8 μl / min in the C18 column. In some embodiments, a portion of the eluate from the high-pH RPLC in which the low-pH RPLC is performed may correspond to the portion of the analyte with a Tapex of + / - 0.3 minutes at a flow rate of 8 μl / min in the C18 column. In some embodiments, a portion of the eluate from the high-pH RPLC in which the low-pH RPLC is performed may correspond to the portion of the analyte with a Tapex of + / - 0.25 to 0.30 minutes at a flow rate of 8 μl / min in the C18 column.

[0012] In some embodiments, if the analyte is a peptide, the peptide or a peptide-containing protein is captured by attachment to particles coated with a protein binding partner such as a ligand or antigen. In some embodiments, the particles are coated with protein A, protein G, or a chimera of protein A and protein G. In some embodiments, the protein is an antibody. In some embodiments, the analyte is a portion of an antibody (e.g., an antigen-binding fragment such as Fab, Fv, scFv, or F(ab')2 fragment). In some cases, the analyte is an antigen or drug-binding target. In some embodiments, the protein analyte is cleaved into peptides, and the peptides are analyzed as analytes in the manner described herein. In some such cases, one or more proteases used for cleavage include one or more of the following: trypsin, endoproteinase LysC, endoproteinase ArgC, Staphylococcus aureus V8, endoproteinase GluC, chymotrypsin, or papain.

[0013] In some cases, the methods described herein detect more than one analyte in a sample. Thus, in some cases, the levels of two or more analytes are determined, and in some cases, the methods detect modified and unmodified forms of a molecule and / or determine the ratio of modified and unmodified forms of a molecule. In some cases where the methods detect modified and unmodified forms of a molecule, the modified and unmodified forms include modified and unmodified proteins, such as alkylated and non-alkylated proteins or glycosylated and non-glycosylated proteins, or molecules and metabolites of molecules.

[0014] In some methods described herein, high pH RPLC and low pH RPLC, as well as MS detection, are automated. In some cases, detecting the analyte by MS following low pH RPLC involves quantitatively determining the amount or concentration of the analyte in the sample. In some embodiments, the MS is tandem MS ("MS / MS") (e.g., multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem).

[0015] In some embodiments, at least a portion of the high-pH RPLC eluate is diluted with a low-pH mobile phase before performing the low-pH RPLC, and optionally, the dilution is further automatically controlled. In some embodiments, the method herein enables analyte detection sensitivity at least 30 times or 30 to 100 times higher than a one-dimensional HPLC-tandem MS (1D-LC-MS / MS) process at microflow rates, and / or the method enables analyte detection sensitivity at least 100 times higher than a one-dimensional HPLC-tandem MS (1D-LC-MS / MS) process at analytical flow rates.

[0016] In some embodiments, the method enables the detection of analytes in tissue samples at concentrations of less than 10 fmol / μg total protein, less than 1 fmol / μg total protein, 1 to 10 fmol / μg total protein, less than 0.1 fmol / μg total protein, 0.1 to 1 fmol / μg total protein, or 0.01 to 0.1 fmol / μg total protein; and / or enables the detection of analytes in fluid samples at concentrations of less than 10 ng / mL, less than 1 ng / mL, less than 100 pg / mL, or less than 10 pg / mL, for example, 1 to 10 ng / mL, 100 pg / mL to 1 ng / mL, or 10 to 100 pg / mL.

[0017] This disclosure also encompasses systems for carrying out the methods described herein. In some embodiments, the system comprises (a) an injection valve for injecting a sample containing an analyte into the system, connected to a high-performance liquid chromatography (HPLC) column and a first-dimensional pump, which controls the flow of the sample and mobile phase through the column; (b) a trap column for collecting at least a predetermined portion of the eluate from the first-dimensional HPLC column for analysis in a second-dimensional HPLC process; (c) a trap valve for controlling the flow path of the mobile phase from the first-dimensional HPLC process to the second-dimensional HPLC process; (d) a second-dimensional pump for controlling the flow of the mobile phase through the second-dimensional HPLC column; and (e) a mass spectrometer for analyzing the analyte after the second-dimensional HPLC process. In some systems, the mass spectrometer is a tandem mass spectrometer. In some cases, the positions of the injection valve and the trap valve may be automatically controlled during the second-dimensional liquid chromatography process, and the automatic control allows only a predetermined portion of the eluate from the first dimension to be separated in the second dimension. In some cases, a predetermined portion of the eluate from the first dimension corresponds to the portion containing Tapex + / - 0.25 minutes of the analyte at a flow rate of 8 μl / min in a C18 column. In some cases, a predetermined portion of the eluate from the first dimension corresponds to the portion containing Tapex + / - 0.3 minutes of the analyte at a flow rate of 8 μl / min in a C18 column. In some cases, a predetermined portion of the eluate from the first dimension corresponds to the portion consisting of Tapex + / - 0.25 to 0.30 minutes of the analyte at a flow rate of 8 μl / min in a C18 column.

[0018] In some cases, the system may further perform a survey scan to determine the tapex of the analyte in the first-dimensional HPLC column. In some cases, the mass spectrometer may perform multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem.

[0019] In some systems described herein, the analyte is a peptide. In some systems, the system can dilute a predetermined portion of the eluate from a one-dimensional process in a mobile phase buffer for a two-dimensional process, and optionally, the dilution is automatically controlled. Any of the methods described herein can be carried out on the systems described herein.

[0020] The disclosure also relates to a method in which the analyte is an anti-CD22 antibody or a peptide fragment thereof, e.g., IYPGDGDTNYSGK and LSCAASGYEFSR, or both. The disclosure also relates to a method in which the analyte is PD-L1 or a peptide fragment thereof, e.g., an IgV domain fragment, e.g., LQDAGVYR, and optionally, the sample is obtained from a subject previously treated with an immune checkpoint molecule such as atezolizumab, and further optionally, the sample is a serum sample.

[0021] The Disclosure also relates to a method for detecting a peptide analyte of human PD-L1 in a biological sample derived from a human subject, comprising: (a) obtaining a biological sample (e.g., a serum sample) from the subject and performing affinity-based capture to isolate PD-L1 in the sample, optionally including the capture of PD-L1 complexed with an anti-PD-L1 antibody (e.g., atezolizumab); (b) digesting the captured protein with a protease to obtain a PD-L1 peptide analyte for detection (e.g., a PD-L1 IgV domain fragment such as LQDAGVYR); (c) performing a high-pH reversed-phase liquid chromatography (RPLC) survey scan on the PD-L1 peptide analyte at a predetermined concentration in the presence of an internal standard peptide, along with detection by mass spectrometry (MS), to determine the peak apex (Tapex) of the analyte; and (d) performing a high-pH reversed-phase liquid chromatography (RPLC) survey scan on the digested sample from (b) containing the peptide analyte. The method also includes (e) performing RPLC; (f) collecting at least a portion of the eluate from the high pH RPLC in (d) corresponding to the Tapex+ / - analyte, internal standard, or mean peak width of the peptide in the survey scan in (c), wherein the at least portion is optionally collected in a trap column; (g) performing low pH RPLC on the collected portion of the eluate from the high pH RPLC in (e); and detecting the PD-L1 peptide analyte by MS (e.g., multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem).

[0022] Please understand that both the above and the following further explanations are illustrative and descriptive only and do not limit the scope of the claims.

[0023] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (or more) embodiments and, together with the description, serve to explain the particular principles described herein.

Brief Description of the Drawings

[0024] [Figure 1] Shows the overall workflow of the reagent-independent targeted 2D-LC-MS / MS method.

[0025] [Figure 2] Shows the settings for determining the retention time window for the migration of analytes by high pH RPLC-MS / MD survey scan analysis. The retention time window is defined as Tapex + / - 0.3 minutes, where Tapex represents the retention time at the peak apex of the analyte.

[0026] [Figure 3] Shows a schematic workflow of the ultra-targeted 2D-LC-MS / MS with a targeted 2D configuration involving dilution. In the figure, the simple dashed line indicates the flow path of the high pH mobile phase, the solid line indicates the flow path of the low pH mobile phase, the composite of the dotted line and the dashed line indicates the flow path of the mixture of the high pH mobile phase and the low pH mobile phase, and the arrow indicates the position of the analyte. This method consists of the following five steps: (Step 1) Load the sample and equilibrate the column; (Step 2) Inject the sample and separate the analyte from the matrix molecules using a linear high pH RPLC gradient; (Step 3) Transfer the fraction / portion of the analyte within the retention time window of 0.6 minutes (Tapex + / - 0.3 minutes) to the trap column and dilute that fraction / portion with the secondary low pH mobile phase A; (Step 4) Elute the analyte through the analytical column using a linear low pH RPLC gradient for MS analysis, while eluting and discarding the matrix molecules from the high pH RPLC column; (Step 5) Wash the trap valve, tubing, and column using a high organic mobile phase.

[0027] [Figure 4]Compare the sensitivity between the analytical flow and microflow LC-MS / MS. Figure 4A shows the measurement using anti-CD22 spiked in monkey serum. Figure 4B shows the measurement using Herceptin spiked in mouse serum. The LC flow rates of the analytical flow and microflow LC-MS / MS were 300 μL / min and 8 μL / min, respectively. IS represents the internal standard.

[0028] [Figure 5] By analyzing anti-CD22 spiked in monkey serum, compare the sensitivity between microflow 1D-LC-MS / MS and 2D-LC-MS / MS. The anti-CD22 monoclonal antibody was spiked in monkey serum at 2-fold serial dilutions from 400 to 0.78 ng / mL. Each 50 microliter serum sample was subjected to protein A affinity capture and on-bead trypsin digestion. The same samples were analyzed by both 1D-LC-MS / MS (Figure 5A) and 2D-LC-MS / MS (Figure 5B) at the same LC flow rate of 8 μL / min. The ion transition m / z693.8→555.7 was monitored for the peptide IYPGDGDTNYSGK. IS represents the internal standard.

[0029] [Figure 6] Detection of the peptide IYPGDGDTNYSGK from monkey serum spiked with different concentrations of anti-CD22 from 0.78 ng / mL to 100 ng / mL is shown. IS indicates the internal standard. The double blank indicates monkey serum without analyte and IS; the serum blank indicates monkey serum spiked with IS but without analyte.

[0030] [Figure 7] Detection of the peptide IYPGDGDTNYSGK from human serum spiked with different concentrations of anti-CD22 from 0.78 ng / mL to 100 ng / mL is shown. IS indicates the internal standard. The double blank indicates monkey serum without analyte or IS; the serum blank indicates monkey serum spiked with IS but without analyte. IS represents the internal standard.

[0031] [Figure 8] The calibration curves for peptide IYPGDGDTNYSGK are shown. Figure 8A shows the curve for peptide IYPGDGDTNYSGK from monkey serum samples spiked with anti-CD22. Figure 8B shows the curve for peptide IYPGDGDTNYSGK from human serum samples spiked with anti-CD22. The analyte-to-internal standard peak area ratio (i.e., light-to-heavy peak area ratio) was plotted against the analyte concentration. Quality control was also included.

[0032] [Figure 9] This report describes the evaluation of analyte retention time reproducibility during high-pH RPLC separation using peptide IYPGDGDTNYSGK. 50 microliters of monkey serum spiked with 400 ng / mL anti-CD22 were subjected to protein A affinity capture and on-bees-trypsin digestion. These samples were analyzed three times on three different days (i.e., infusion 1 (day 1), 180 (day 3), and 320 (day 5)) in a run containing 320 samples by high-pH RPLC-MS / MS. IS represents the internal standard.

[0033] [Figure 10] This study evaluates the effect of sample volume on 2D-LC-MS / MS assay sensitivity. Monkey serum was spiked with anti-CD22 monoclonal antibody at 400 ng / mL, followed by 2-fold serial dilutions to obtain concentrations ranging from 400 to 0.78 ng / mL. Each serum sample was analyzed twice using different starting volumes (i.e., 25 μL vs. 50 μL). IS represents the internal standard.

[0034] [Figure 11] The calibration curves for the peptide LSCAASGYEFSR are shown. Figure 11A shows the calibration curve for the peptide LSCAASGYEFSR from monkey serum samples spiked with anti-CD22. Figure 11B shows the calibration curve for the peptide LSCAASGYEFSR from human serum samples spiked with anti-CD22. The analyte-to-internal standard peak area ratio (i.e., light-to-heavy peak area ratio) was plotted against the analyte concentration. Quality control was also included.

[0035] [Figure 12]The calibration curve for the peptide FTFSLDTSK from 50 μL of anti-CD22 spiked monkey serum is shown. The analyte versus internal standard peak area ratio (i.e., light / heavy peak area ratio) is plotted against the analyte concentration.

[0036] [Figure 13] This report evaluates PD-L1 binding by atezolizumab. 0.145 μM PD-L1 was incubated with 2.9 μM atezolizumab (molar ratio 1:20) at room temperature for 2 hours, and the sample was then subjected to protein A affinity capture (AC1). Following AC1, atezolizumab was added to the supernatant at the same concentration (2.9 μM), incubated for another 2 hours at room temperature, and then subjected to a second protein A affinity capture (AC2). Molecules captured by both AC1 and AC2 were digested and analyzed by 2D-LC-MS / MS. Figure 13A shows the 2D-LC-MS / MS analysis of the PD-L1 sample subjected to AC1 capture. Figure 13B shows the 2D-LC-MS / MS analysis of the PD-L1 sample subjected to AC1 capture and subsequent AC2 capture. No PD-L1 was detected in AC2, indicating that PD-L1 was completely bound by atezolizumab in AC1.

[0037] [Figure 14] This study compares the sensitivity of microflow 1D-LC-MS / MS and 2D-LC-MS / MS for the quantification of soluble PD-L1. Atezolizumab-PD-L1 complexes were spiked into normal human serum at 2-fold serial dilutions with PD-L1 concentrations ranging from 500 to 2 ng / mL (or 18 to 0.07 nM). Figure 14A shows analysis of spike-in samples by microflow 1D-LC-MS / MS. Figure 14B shows analysis of spike-in samples by 2D-LC-MS / MS. Serum blanks and LLOQ samples are shown here.

[0038] [Figure 15]This document describes the application of a targeted 2D-LC-MS / MS method to measure the total concentration of soluble PD-L1 variants bound to atezolizumab in post-treatment serum samples. Figure 15A shows the calibration curve for the peptide LQDAGVYR derived from the PD-L1 IgV domain. Figure 15B shows the molar concentrations of total soluble PD-L1 in 12 post-treatment serum samples. Figure 15C shows the mass concentration of soluble PD-L1 calculated based on the molecular weight of wild-type PD-L1. Figure 15D shows the correlation between 2D-LC-MS / MS and ELISA data. [Modes for carrying out the invention]

[0039] I. Definition Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings generally understood by those skilled in the art.

[0040] In this application, the use of “or” means “and / or” unless otherwise specified. In the context of multiple dependent claims, the use of “or” refers to only one or more preceding independent or dependent claims. Also, terms such as “element” or “component” include both elements and components containing one unit and elements and components containing more than one subunit, unless otherwise specified.

[0041] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include, unless otherwise indicated, any integers within the listed ranges, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer).

[0042] Units, prefixes, and symbols are shown in the form accepted by the International System of Units (SI). Numerical ranges include the number defining the range. The headings provided herein are not limitations on the various aspects of this disclosure that can be had by referring to this specification as a whole. Thus, the terms defined immediately below are more fully defined by referring to this specification as a whole.

[0043] When used in accordance with this disclosure, the following terms should be understood to have the following meanings unless otherwise indicated.

[0044] As used herein, “sample” refers to any specimen that may contain an analyte to be detected or quantified. In some embodiments, the sample is a “biological sample,” which is a sample taken from a biological organism, such as a “biological fluid sample.” As used herein, “biological fluid sample” refers to any biological fluid from an organism or subject that may contain an analyte for detection. Examples include tears, saliva, lymph, urine, serum, cerebrospinal fluid, pleural fluid, ascites, and plasma. The sample may be taken directly from its source (e.g., from a human or mammalian subject) or may be pre-treated to remove large debris. “Experimental sample” refers to a sample containing an undetermined amount of analyte from which the amount or concentration of the analyte is determined.

[0045] "Analyte" refers to the substance to be detected or quantified in a sample in the systems and methods of this specification. In some examples, the "analyte" is broken down into smaller fragments for detection. Thus, depending on the context, "analyte" refers to a common substance to be detected, such as a protein biomarker, as well as the peptide or other fragments of that protein biomarker that are actually separated by chromatography and / or analyzed by mass spectrometry.

[0046] "Liquid chromatography" or "LC" refers to the process of separating the components of a sample through the interaction of a stationary phase (e.g., a column of granular material) and a mobile phase (i.e., a fluid). LC may be performed in one dimension (1D-LC), meaning that one separation process is performed, or in two dimensions (2D-LC), meaning that the eluate or a portion thereof from the first separation is further separated in a second separation step using different separation means, such as using a different mobile phase. LC encompasses, for example, HPLC and reversed-phase HPLC. "High-performance liquid chromatography" or "HPLC" refers to a type of LC system in which the mobile phase flows through a stationary phase, such as a column, under pressure. The HPLC system may be coupled with a detector, such as a mass spectrometer. The HPLC process can be performed in "normal phase" ("NP" or "NP-HPLC") or "reverse phase" ("RP" or "RP-HPLC" or "RPLC"). In the RPLC process, the stationary phase (e.g., the column) is nonpolar, and the mobile phase is polar, such as a water / polar organic solvent mixture or gradient. In normal-phase HPLC, the stationary phase (e.g., the column) is polar, and the mobile phase is nonpolar.

[0047] "Mass spectrometry" or "MS" refers to the technique of measuring the mass-to-charge ratio (m / z) of one or more molecules in a sample. As used herein, "tandem MS" or "MS / MS" refers to the coupling of two MS analyzers. For example, a first MS analyzer may separate peptide ions by their m / z ratio, and then the peptide ions having a target m / z ratio may be further fragmented and analyzed by a second MS analyzer. There are several types of MS / MS systems, some of which are described further below.

[0048] Multiple reaction monitoring, or MRM, is a method of detecting analytes in a composite sample by tandem MS, for example, using a triple quadrupole MS instrument.

[0049] An "internal standard" refers to a molecule that is added to a sample in a known amount or concentration for detection and serves as a standard for determining the presence of an analyte in the sample or for quantifying its amount or concentration. In some embodiments, the internal standard is a molecule identical or similar to the analyte, except for the presence of heavy isotope labeling.

[0050] As used herein, the terms “marker” or “biomarker” refer to indicators that can be detected in a sample, such as predictive indicators, diagnostic indicators, and / or prognostic indicators. Markers or biomarkers may be proteins or polypeptides or nucleic acid molecules, as well as lipids or glycolipids or drugs or drug metabolites. Depending on the purpose of the assay, biomarkers may serve as indicators of the state of a sample or the organism or subject from which the sample is obtained, such as indicators of disease or disorder, the presence of a specific protein or nucleic acid or lipid, the presence of a drug or drug metabolite, the potential prognosis of a disease or disorder, or the potential responsiveness to a drug.

[0051] The terms "quantification" or "to quantify" in this specification mean determining numerically the level, quantity, number, or concentration of an analyte in a sample.

[0052] As used herein, the term “affinity-based capture” refers to a process by which an analyte is isolated in a sample by exposing the analyte to an affinity molecule, such as a ligand or antigen, and separating the analyte bound to the ligand or antigen or other affinity molecule from the rest of the sample. By isolating the analyte in such a process, the analyte can be enriched, concentrated, and / or removed from contaminating molecules, for example, before liquid chromatography.

[0053] The term "antibody" as used herein is used in its broadest sense and encompasses a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), nanobodies, diabodies, and antigen-binding fragments such as Fv, scFv, Fab, and (Fab')2, as long as they exhibit antibody-antigen binding activity.

[0054] The terms “polypeptide” and “protein” are used interchangeably and refer to polymers of amino acid residues. Such polymers of amino acid residues may contain, but are not limited to, natural and / or non-natural amino acid residues, and include peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. The term also includes polymers of amino acids that have modifications such as glycosylation, sialylation, or are complexed with other molecules. Protein biomarkers as used herein include, for example, disease-enriched or mutated proteins in diseased cells, natural and heterologous proteins such as oncogenic proteins, bacterial proteins, and viral proteins, as well as protein drugs and protein drug metabolites.

[0055] In this specification, "peptides," particularly those that can function as analytes or internal standards, are relatively short polymers of amino acids, such as those consisting of approximately 4 to 50 amino acids.

[0056] The terms “nucleic acid molecule” or “polynucleotide” include any compound and / or substance containing polymers of nucleotides. Each nucleotide is composed of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are often described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. The base sequence is typically represented from 5' to 3'. Nucleic acid biomarkers as used herein include, for example, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), particularly messenger RNA (mRNA), and other cellular RNA molecules such as small interfering RNA (siRNA), microRNA (miRNA), and non-coding RNA, as well as heterogeneous nucleic acids such as viral DNA or RNA or bacterial DNA or RNA, or drugs and metabolites containing DNA or RNA.

[0057] Generally, the “subject” as used herein refers to the individual in which a biological sample is tested for the presence of an analyte. In some embodiments, the subject is a human. However, in some embodiments, the subject may be another mammal, e.g., a livestock or animal species, e.g., a dog, cat, rabbit, horse, pig, cattle, goat, sheep, etc., or an experimental animal, e.g., a mouse or rat. Examples of mammals include, but are not limited to, livestock animals (e.g., cattle, sheep, cat, dog, and horse), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0058] As used herein, “automated” or “automatically controlled” processes are processes that can be performed by a computer-controlled system with appropriate software, in contrast to systems that require active manual intervention during or between at least one step, such as moving an analyte-containing sample from one part of the system to another. In some embodiments, the process is automated by software that controls the movement or position of one or more pumps, valves, and / or T-tubes during the liquid chromatography process, the movement or position controlling the flow of the mobile phase and eluting through the chromatography system.

[0059] In some embodiments of this specification, two or more steps or conditions may be "substantially the same." This means that they may differ from one another within the normal range of experimental error and within appropriate significant figures.

[0060] As used herein, "eluate" refers to a substance that has passed through a chromatography column and therefore eluted from the chromatography column.

[0061] As used herein, “retention time window” refers to a predetermined time in a liquid chromatography process during which an eluent, for example, an eluent generated in a first-dimensional liquid chromatography process and intended for further separation in a second-dimensional liquid chromatography process, is collected. II. Methods and Systems

[0062] This disclosure relates, for example, to a method and system for performing a two-dimensional liquid chromatography-mass spectrometry (e.g., tandem mass spectrometry) assay to characterize an analyte in a sample. In some embodiments, the method includes (a) performing a high pH reversed-phase liquid chromatography (RPLC) survey scan on the analyte at a predetermined concentration in the presence of an internal standard, along with detection by mass spectrometry (MS), to determine the peak apex (Tapex) of the analyte; (b) obtaining an experimental sample containing the analyte; (c) performing high pH RPLC on the sample from (b); (d) collecting at least a portion of the eluate from the high pH RPLC from (d) corresponding to the mean peak width of the Tapex+ / - analyte or internal standard in the survey scan from (a), wherein the at least portion is optionally collected in a trap column; and (e) performing low pH RPLC on the collected portion of the eluate from the high pH RPLC from (d), and detecting the analyte by MS.

[0063] In some embodiments, the analyte is a peptide. In some embodiments, when the analyte is a peptide, the method includes (a) obtaining an experimental sample and isolating the protein in the sample by affinity-based capture; (b) digesting the captured protein with a protease to obtain a peptide analyte for detection; (c) determining the peak tapex of the analyte by performing a high pH reversed-phase liquid chromatography (RPLC) survey scan on the peptide analyte at a predetermined concentration in the presence of an internal standard peptide, along with detection by mass spectrometry (MS); (d) performing high pH RPLC on the digested sample from (b) containing the peptide analyte; (e) collecting at least a portion of the eluate from the high pH RPLC from (d) corresponding to the average peak width of the peptide in the survey scan of Tapex+ / -(c), wherein the at least portion is optionally collected in a trap column; and (f) performing low pH RPLC on the collected portion of the high pH RPLC eluate from (e) and detecting the analyte by MS. In other cases, the captured protein is not digested before being subjected to liquid chromatography; instead, the captured protein sample is subjected directly to liquid chromatography.

[0064] For example, the examples herein describe a common reagent, super-targeted two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS / MS) method combining commercially available protein A affinity capture, isolation of targeted analytes by 2D-LC, and targeted detection by multiple reaction monitoring (MRM). This method was evaluated using anti-CD22 monoclonal antibodies spiked in monkey and human serum, achieving limits of quantification (LLOQ) of 0.78 ng / mL and 1.56 ng / mL, respectively. This represents an improvement of more than 100-fold in assay sensitivity compared to conventional 1D-LC-MS / MS methods. The performance of this method was further confirmed by analyzing another monoclonal antibody, bevacizumab, and soluble antigen circulating PD-L1 protein using this method, as described in the examples herein.

[0065] The disclosure herein also relates to a system for performing 2D-LC-MS analysis, wherein optionally the MS is tandem MS (MS / MS), and optionally the operation of the system can be automatically controlled. In some embodiments, the system comprises (a) an injection valve for injecting a sample containing an analyte into the system, connected to a high-performance liquid chromatography (HPLC) column and a first-dimensional pump, and controlling the flow of the sample and mobile phase through the column; (b) a trap column for collecting at least a predetermined portion of the eluate from the first-dimensional HPLC column for analysis in the second-dimensional HPLC process; (c) a trap valve for controlling the flow path of the mobile phase from the first-dimensional HPLC process to the second-dimensional HPLC process; (d) a second-dimensional pump for controlling the flow of the mobile phase through the second-dimensional HPLC column; and (e) a mass spectrometer for analyzing the analyte after the second-dimensional HPLC process.

[0066] Unlike previous methods using two independent HPLC systems, this system enables integrated control of each dimension of the LC process, which can be fully automated, and optionally uses microflow rates in both dimensions of the LC process. In some embodiments, this method can be performed on a single, integrated 2D-LC system. In some embodiments, the system can be partially or fully automated. For example, the mobile phase can be automatically controlled, for example, at a microflow rate, for the entire 2D-LC process. This system can also be highly targeted to identify and optionally quantify analytes using a narrow targeting window predetermined in a survey scan before performing 2D-LC-MS (e.g., 2D-LC-MS / MS). Furthermore, in some embodiments, optimizing 2D-LC conditions using a survey scan is straightforward and can be done, for example, in about 1-2 hours, whereas in conventional processes using two independent LC systems, optimizing conditions can take considerably longer.

[0067] The following subsections illustrate exemplary options and details of the methods and systems described herein. III. Exemplary methods for capturing samples and analytes

[0068] The methods described herein are applicable to any type of sample containing or thought to contain an analyte for analysis. In some embodiments, the sample is a biological sample. Examples of biological samples include cells, tissues, organs, bones, and fluids. In some embodiments, the sample is a biological fluid sample such as tears, saliva, lymph, urine, serum, cerebrospinal fluid, pleural fluid, ascites, whole blood, or plasma. In some embodiments, a biological sample may be a complex mixture of multiple components and may include proteins, carbohydrates, lipids, DNA, RNA, metabolites, small molecules, antigens, and / or toxins. An example of a biological sample is, for example, a biopsy sample. Examples of tissue samples include, for example, fresh or frozen tissue samples, as well as fixed paraffin-embedded (FPET) samples taken from any biological tissue or organ.

[0069] In some embodiments, the sample is not treated to isolate (i.e., enrich or concentrate) the analyte before liquid chromatography. In other embodiments, the sample is treated to isolate the analyte before liquid chromatography. Thus, in some embodiments, the sample is used directly without any treatment steps. In other embodiments, the sample is initially treated before use in the method herein, for example, to remove large particles by centrifugation or filtration, or to dissolve cellular material. In the case of tissue samples, the sample may be treated to homogenize the sample, lyse cells, and / or extract components from the sample that may contain analytes such as proteins or nucleic acids. Extraction or filtration or other such methods can be used at least in part to enrich or concentrate the analyte from the sample before the method herein. In some embodiments, the method includes a step of treating the sample to isolate the analyte before performing liquid chromatography. In some cases, the analyte can be isolated using an affinity-based capture process. In other cases, other methods can be used, for example, size exclusion chromatography or ion exchange chromatography processes.

[0070] For example, various affinity-based capture methods can be implemented. In some embodiments, the analyte can be captured and thus isolated by attachment to particles coated with a binding partner such as a ligand or antigen. Examples of particles used include glass, plastic, polystyrene, and agarose. The particles can be manufactured, for example, in the form of plates, beads, columns, matrices, gels, and resins. In some embodiments, the analyte can be captured in a bead suspension using, for example, magnetic beads. In some embodiments, the analyte can be captured in an immobilized affinity matrix. In some embodiments, the analyte can be captured using a chromatography column or a spin column.

[0071] In some embodiments, particles are coated with ligands of the analyte, such as proteins, peptides, antibodies, antigens, or small molecule ligands or cofactors. Examples include streptavidin, glutathione, concanavalin A, biotin, protein A, protein G, or chimeras of protein A and protein G. In some embodiments, if the analyte is an antibody, particles can be coated with protein A, protein G, and / or chimeras of protein A and protein G. In some embodiments, particles coated with protein A, protein G, and / or chimeras of protein A and protein G are magnetic beads. In some embodiments, particles coated with protein A, protein G, and / or chimeras of protein A and protein G are immobilized on a matrix, chromatography column, or spin column. In some embodiments, if the analyte is a nucleic acid, for example, particles for its capture can be treated with primers containing complementary sequences.

[0072] The methods described herein can be applied to a variety of analytes. In some embodiments, the methods described herein can be used to determine the levels of two or more different analytes in a sample, or the ratio of different analytes in a sample. For example, a capture method that captures more than one analyte may be used, such as similar proteins or modified and unmodified analytes. Alternatively, the sample may be divided, different analytes may be captured using different capture particles or different capture methods, and then each may be analyzed according to the 2D-LC-MS method described herein. In some cases, the methods described herein may be used to determine the levels of similar analytes in a sample, such as modified and unmodified proteins or nucleic acids or drugs or other analytes, such as alkylated and unalkylated analytes, drugs and their associated metabolites, glycosylated and unglycosylated protein or peptide analytes, or methylated and unmethylated nucleic acid stretches or the sum of classes of analytes. In some embodiments, the methods described herein enable the determination of the ratio of modified and unmodified analytes or two similar analytes in a sample. For example, by obtaining multiple measurements in different samples obtained as a change in conditions, it is also possible to track changes in the degree of a particular modification over the course of a change in conditions. For example, in some embodiments where analyte capture is used to obtain analytes before 2D-MS analysis, a capture method can be used that captures both unmodified and modified analytes, so that the quantities of both and their relative levels can be determined as part of the analysis. IV. Enzyme Digestion

[0073] In some embodiments, if the analyte to be detected is a relatively large molecule such as a protein or nucleic acid, the molecule is fragmented before liquid chromatography is performed. Therefore, in some embodiments, the sample is treated with one or more enzymes to at least partially digest the analyte, thereby reducing it to a size sufficient for liquid chromatography and mass spectrometry detection. Enzymatic digestion can be performed on the sample after affinity-based capture or other treatments for analyte isolation, or on samples that have not been treated in this manner.

[0074] If the analyte is a protein, it may be beneficial to digest the protein into smaller peptide fragments by protease treatment. Exemplary classes of proteases include, for example, serine proteases, cysteine ​​proteases, threonine proteases, aspartate proteases, glutamate proteases, metalloproteases, and asparagine peptide lyases. Exemplary proteases that can be used herein include trypsin, endoproteinase LysC, endoproteinase ArgC, Staphylococcus aureus V8, endoproteinase GluC, chymotrypsin, TEV protease, endopeptidase K, subtilisin, subtilisin A, alcalase®, maxatase®, sabinase®, esperase®, proteinase K, thermoravir proteinase K, and tori It contains psin-ultra (trademark), α-soluble protease, endoproteinase AspN, endoproteinase LysC, endoproteinase Arg-C, elastase, thermolysin, pepsin, pepsinogen, factor Xa protease, clostripine, carboxypeptidase A, carboxypeptidase B, carboxypeptidase Y, acylamino acid-releasing enzyme, pyroglutamate aminopeptidase, enteropeptidase, and papain.

[0075] Non-peptide analytes can also be digested at least partially by enzymes, if necessary, to produce molecules of a suitable size for detection. For example, nucleic acids can be treated with nucleases such as restriction endonucleases to cleave them into fragments of a size suitable for separation and mass spectrometry detection.

[0076] In some embodiments, enzymatic digestion is performed at high temperatures, depending on the efficiency of the enzyme. In some embodiments, enzymatic digestion is performed after isolation of the analyte by affinity capture. In some embodiments, enzymatic digestion is performed before removal of affinity-captured particles from the sample. In some embodiments, it is performed after removal of affinity-captured particles from the sample. V. Survey Scan

[0077] In some embodiments, a survey scan is incorporated into the method herein, for example, to predetermine the retention time window of the analyte of interest and / or an internal standard during the first-dimensional liquid chromatography process. In some embodiments, the survey scan is performed using the same type of sample and first-dimensional LC conditions as the first-dimensional complete 2D-LC process, i.e., using the same column, mobile phase and flow rate, as well as the same mass spectrometry detector and method. Thus, the retention time window determined by the survey scan more accurately reflects the time window during which the analyte being analyzed is eluted from the first-dimensional LC column.

[0078] In some embodiments, a survey scan can be used to determine the retention time window, as well as the peak width of the analyte and / or internal standard. For example, a survey scan may include performing a planned first-dimensional liquid chromatography process, followed by performing an intended mass spectrometry on a sample containing the analyte spiked with the internal standard and / or the internal standard of the analyte, to determine the retention time window for the process. In some embodiments, the survey scan also includes determining the Tapex of the analyte and / or internal standard, where Tapex is the retention time of the peak apex (i.e., the highest point of the peak) of the analyte and / or internal standard peaks. In some embodiments, a survey scan can be used to determine the mean peak width (PW) of the analyte and / or internal standard peaks in the sample as a means of defining a portion (corresponding to a retention time window or range) of the eluate from the first-dimensional LC process that is moved to a second-dimensional LC process for further separation. In some embodiments, for example, if the analyte is a peptide, the mean peak of the peptide in the sample may also be used to define the retention time window for moving from the first to the second dimension.

[0079] In some embodiments, the survey scan is repeated several times, for example, to improve accuracy. In some embodiments, the survey scan is performed at least three times, for example, four or five times. In some such cases, Tapex and PW can be determined, for example, from the mean or median of different scans. Furthermore, in some embodiments, PW is the average peak width of more than one peak in the sample, such as the analyte and / or internal standard and / or other similar molecules. Thus, in some embodiments, PW is the average peak width of peptides in the sample detected by the survey scan.

[0080] In some embodiments, once a suitable retention time window is obtained for the analyte in the survey scan, this window can be directly transitioned to the second-dimensional LC separation, allowing the 2D-LC process to be executed so that no mass spectrometry detection occurs between the first and second dimensions. This allows for the automatic control of the 2D-LC process in a system that, for example in some embodiments, automatically transitions the eluate from the first-dimensional separation within a suitable retention time range to the second-dimensional LC process, while discarding the remaining first-dimensional eluate as waste.

[0081] In some embodiments, the retention time window determined during the survey scan is equal to a multiple of Tapex ± PW, where Tapex represents the retention time at the peak apex of the analyte or internal standard, and PW represents the peak width of the analyte or internal standard. In some embodiments, the survey scan is used to identify retention windows equal to Tapex + / - 0.5 PW to 2 PW, or Tapex + / - 1 PW to 2 PW, or Tapex + / - 0.5 PW, 1 PW, 1.5 PW, or 2 PW. In some embodiments, the survey scan is used to identify a retention window equal to Tapex + / - 1 PW. For example, if the peak width of the peptide is 0.3 minutes, the retention time window equal to Tapex + / - 1 PW is Tapex + / - 0.3 minutes. For example, if the window is equal to Tapex + / - 1 PW and the peak width of the peptide is 0.2 minutes, the retention time window is Tapex + / - 0.2 minutes.

[0082] In some embodiments, the peak width of the analyte is, for example, 0.05 to 1 minute at a flow rate of 7 to 10 μL / min, depending on the analyte, mobile phase and column used, for example, 0.05 to 0.1 minutes, 0.1 to 0.15 minutes, 0.15 to 0.2 minutes, 0.2 to 0.25 minutes, 0.25 to 0.3 minutes, 0.35 to 0.4 minutes, 0.4 to 0.45 minutes, 0.45 to 0.5 minutes, 0.5 to 0.55 minutes, 0.55 to 0.6 minutes, 0.65 to 0.7 minutes, 0.7 to 0.75 minutes, 0.75 to 0.8 minutes, 0.8 to 0.85 minutes, 0.85 to 0.9 minutes, 0.9 to 0.95 minutes, or 0.95 to 1 minute. In some embodiments, depending on the PW, the retention time window is a window corresponding to Tapex+ / -0.2 min to Tapex+ / -0.5 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.4 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.25 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.5 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.4 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.3 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 to Tapex+ / -0.5 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 to Tapex+ / -0.4 minutes at a flow rate of 8 μl / min in a C18 column.In some embodiments, the retention time window corresponds to Tapex+ / -0.25 to Tapex+ / -0.30 minutes at a flow rate of 8 μl / min in a C18 column.

[0083] In all of the above cases, the listed retention time windows "corresponding" to the retention time window at a flow rate of 8 μl / min on a C18 column are the retention time windows determined when the survey scan is performed on a C18 column at a flow rate of 8 μl / min. In other words, this method is adaptable to various flow rates and columns depending on the analyte being detected. However, for the sake of comparison only, the retention time window corresponding to a specific Tapex+ / -X minutes at a flow rate of 8 μl / min on a C18 column corresponds to a specific Tapex+ / -X minutes when the survey scan is performed on a C18 column at a flow rate of 8 μl / min.

[0084] In some embodiments, the survey scan is performed at a microflow rate that can generally correspond to a flow rate of 2 to 100 μL / min. In some embodiments, the survey scan is performed at a flow rate of 4 to 10 μL / min, 5 to 10 μL / min, 6 to 9 μL / min, 7 to 10 μL / min, 6 to 8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, the selected flow rate is the same as or substantially the same as the flow rate used for the first-dimensional separation described later.

[0085] In some embodiments, the survey scan is a high-pH LC process, such as a high-pH RPLC process. In some embodiments, the high-pH RPLC is carried out at pH between 8 and 10, between 8 and 9, between 9 and 10, or at pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10. In some embodiments, the high-pH RPLC is carried out at pH between 8 and 10, between 8 and 9, between 9 and 10, or at pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10, using mobile phase A containing ammonium formate and mobile phase B containing ammonium formate and acetonitrile. In some embodiments, the pH is the same as or substantially the same as the flow rate for the first-dimensional separation described later.

[0086] In some embodiments, the mobile phase and column for the survey scan are selected to be identical to those of the first-dimensional LC process, as described below.

[0087] In some embodiments, the mobile phase, pH, column, and flow rate of the survey scan are identical or substantially identical to those used in the first-dimensional LC process. Generally, the temperature and pressure of the survey scan and the first-dimensional LC process are also expected to be the same or substantially identical. VI.2 D-LC-MS Process Steps

[0088] A two-dimensional LC process can be performed following the survey scan. Generally, as described above, the first dimension of the process is performed under the same conditions as the survey scan so that the retention time window identified in the survey scan predicts as accurately as possible when the analyte will elute during the first dimension of the LC process. Therefore, for example, in many embodiments, the survey scan and the first-dimensional LC can use the same flow rate, mobile phase, and / or the same column, and are performed at the same temperature and pressure.

[0089] In some embodiments, LC separation is an RPLC process. In RPLC, more polar compounds tend to have shorter retention times than hydrophobic or nonpolar compounds. When ionizable analytes are present, changes in the mobile phase pH can lead to dramatic changes in retention. Therefore, adjusting the buffer pH can significantly affect liquid chromatography separation. In some embodiments, each dimension is performed at a specific, relatively constant pH. In some embodiments, a high-pH buffer is used for the first dimension and a low-pH buffer for the second dimension. However, this method is also compatible with other separation methods, depending on the analyte, such as separation based on salt concentration rather than pH, or separation by a pH gradient in one or both dimensions.

[0090] In some embodiments, the first-dimensional LC is a high-pH LC process, such as a high-pH RPLC process. In some embodiments, the high-pH RPLC is carried out at pH between 8 and 10, between 8 and 9, between 9 and 10, or at pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10. In some embodiments, the mobile phase comprises a mixture of a salt-free or low-salt basic solution (e.g., a base in water) and a basic solution in an organic solvent. In some embodiments, ammonium formate is used as the base. In some embodiments, the organic solvent is acetonitrile. For example, in some embodiments, mobile phase A comprises a basic solution and mobile phase B comprises a basic solution in an organic substance such as acetonitrile. In some embodiments, mobile phase B comprises, for example, a solution of 80% to 90% of the organic phase and 10% to 20% of mobile phase A. In some embodiments, high pH RPLC is carried out at a pH between 8 and 10, between 8 and 9, between 9 and 10, or at pH 8.0, pH 8.5, pH 9.0, pH 9.5, or pH 10, using mobile phase A containing ammonium formate and mobile phase B containing ammonium formate and acetonitrile. In some embodiments, the mobile phase is 10 mM ammonium formate (pH 9.5) and 0-100% acetonitrile.

[0091] In some embodiments, the retention of the analyte is affected by the amount of organic solvent present in the buffer. In various embodiments, the buffer has 0-100% organic solvent (such as acetonitrile). In some embodiments, an acetonitrile concentration gradient is applied, and the gradient may be linear, step, increasing, and / or decreasing.

[0092] In some embodiments, the high pH process uses a mobile phase gradient rather than uniform concentration mobile phase separation. In some embodiments, the gradient is continuously changing rather than stepwise changing. For example, the continuous gradient can be automatically controlled. Thus, for example, mobile phases A and B can be mixed in different proportions as the process progresses. This mixing may be automatically controlled, for example, in some embodiments. In some embodiments, the process uses a high pH RPLC buffer gradient corresponding to a range of 0.1% to 90% buffer B (an organic, e.g., a basic solution containing ammonium formate and acetonitrile) over time, or 3% to 90% buffer B over time. In some embodiments, mobile phase A contains 10 mM ammonium formate and mobile phase B contains 10 mM ammonium formate in acetonitrile. In some such embodiments, mobile phase A contains 10 mM ammonium formate (pH 9.5) and mobile phase B is 10 mM ammonium formate (pH 9.5) in 90% acetonitrile.

[0093] In certain embodiments, for example at microflow rates, the following gradients are used: 3%B (0-3 mins), 3-10%B (3-4 mins), 10-35%B (4-14 mins), 35-90%B (14-14.5 mins), 90%B (14.5-15.5 mins), 90-35%B (15.5-15.6 mins), 35%B (15.6-17.5 mins), 35-3%B (17.5-17.6 mins), 3%B (17.6-20 mins). In some embodiments, mobile phase A contains 10 mM ammonium formate and mobile phase B is 10 mM ammonium formate in 90% acetonitrile. In some such embodiments, mobile phase A contains 10 mM ammonium formate (pH 9.5) and mobile phase B is 10 mM ammonium formate (pH 9.5) in 90% acetonitrile.

[0094] In some embodiments, a first-dimensional LC is performed based on a survey scan, such that the process includes collecting at least a portion of the eluate corresponding to the analyte obtained in the survey scan or an internal standard of Tapex+ / -0.5PW~2PW, or Tapex+ / -1PW~2PW, or Tapex+ / -0.5PW, 1PW, 1.5PW, or 2PW. In some embodiments, the first-dimensional LC is performed such that the process includes collecting at least a portion of the eluate corresponding to Tapex+ / -1PW.

[0095] In some embodiments, the peak width of the analyte is, for example, 0.05 to 1 minute at a flow rate of 7 to 10 μL / min, depending on the analyte, mobile phase and column used, for example, 0.05 to 0.1 minutes, 0.1 to 0.15 minutes, 0.15 to 0.2 minutes, 0.2 to 0.25 minutes, 0.25 to 0.3 minutes, 0.35 to 0.4 minutes, 0.4 to 0.45 minutes, 0.45 to 0.5 minutes, 0.5 to 0.55 minutes, 0.55 to 0.6 minutes, 0.65 to 0.7 minutes, 0.7 to 0.75 minutes, 0.75 to 0.8 minutes, 0.8 to 0.85 minutes, 0.85 to 0.9 minutes, 0.9 to 0.95 minutes, or 0.95 to 1 minute. In some embodiments, depending on the PW, the retention time window is a window corresponding to Tapex+ / -0.2 min to Tapex+ / -0.5 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.4 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.25 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.5 min at a flow rate of 8 μL / min in the C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.4 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.2 to Tapex+ / -0.3 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 to Tapex+ / -0.5 minutes at a flow rate of 8 μl / min in a C18 column. In some embodiments, the retention time window corresponds to Tapex+ / -0.3 to Tapex+ / -0.4 minutes at a flow rate of 8 μl / min in a C18 column.In some embodiments, the retention time window corresponds to Tapex+ / -0.25 to Tapex+ / -0.30 minutes at a flow rate of 8 μl / min in a C18 column.

[0096] Therefore, since Tapex+ / -0.5PW~2PW can be a fairly short time, less than one minute, in some embodiments the method is automatically controlled so that the eluate flow is collected only during this short time window, otherwise it can go to the waste container.

[0097] In some embodiments, the first dimension is performed at a microflow rate of, for example, 2 to 100 μL / min, such as 4 to 10 μL / min, 5 to 10 μL / min, 6 to 9 μL / min, 7 to 10 μL / min, 6 to 8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, the same flow rate is used for the survey scan and the first-dimensional LC process.

[0098] Many types of reversed-phase columns with a hydrophobic stationary phase can be used in 2D-LC. Examples of columns include silica-based columns and PS-DVB resin columns. In some embodiments, first-dimensional separation is performed using a C18 column or a C4 or C8 column. In some embodiments, a C18 column is used. In some embodiments, the column used for first-dimensional separation is the same as that used for survey scanning.

[0099] In some embodiments, the portion of the first-dimensional eluate to be collected for the second dimension, previously identified by the survey scan, is collected in a trap column or another suitable device, which can help separate and retain the unwanted portion of the first-dimensional eluate. The collected portion of the eluate can then be mixed with the mobile phase for the second-dimensional LC separation process. Simultaneously, the remaining portion of the first-dimensional eluate may be collected, for example, in a waste container. In some embodiments, a T-tube and / or valve or similar device can be incorporated into the system to control the eluate collected from the column so that the desired portion containing the analyte identified from the survey scan is collected for the second-dimensional analysis, and the remainder of the eluate is transferred to the waste. In some embodiments, the survey scan can be used to enable the system performing the method to automatically detect the start and end of the retention time window identified in the survey scan as containing the analyte, thereby allowing the eluate flow to automatically move from the waste to the collection (e.g., in a trap column or filter or similar device).

[0100] In some embodiments, the collected portion of the eluate from the first dimension is re-equilibriumated for further separation in the second dimension. For example, the collected analyte-containing portion may be mixed with the mobile phase of the second dimension for further separation in the second dimension. This buffer exchange can be performed, for example, using a trap column, a filter, or other means capable of retaining the analyte while exchanging the buffer.

[0101] In some embodiments, the second-dimensional LC is a low-pH LC, such as a low-pH RPLC. In some embodiments, the low-pH LC may be carried out at pH 2-6, 2-4.5, 2-4, 2-3, 2-2.5, 3-4, or at pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, pH 4.5, pH 5, or pH 6. In some embodiments, the pH may be between 2 and 2.5, for example, pH 2.0, pH 2.1, pH 2.2, pH 2.3, pH 2.4, and pH 2.5. In some embodiments, the low-pH RPLC may be carried out using mobile phase A containing formic acid and mobile phase B containing formic acid and an organic solvent such as acetonitrile or TFA. In some embodiments, mobile phase A may contain 0.1% (v / v) formic acid in water, and mobile phase B may contain 10% 0.1% formic acid (v / v) in 90% organic solvent such as acetonitrile. In some such embodiments, the pH of mobile phase A may be 2.4.

[0102] In some embodiments, a buffer gradient, such as a continuous buffer gradient, is used in the second-dimensional LC process, as opposed to a uniform or stepwise change in the mobile phase concentration. In some embodiments, low pH RPLC buffer gradients for use in microflow rates are as follows: 0.1%B (0-11 min), 0.1-10%B (11-11.5 min), 10-35%B (11.5-15.5 min), 35-90%B (15.5-15.6 min), 90%B (15.6-17.5 min), 90-0.1%B (17.5-17.6 min), 0.1%B (17.6-20 min). In some such embodiments, mobile phase A is 0.1% (v / v) formic acid in water, and mobile phase B is 0.1% (v / v) formic acid in acetonitrile.

[0103] In some embodiments, the second dimension is carried out at a microflow rate, for example, 2 to 100 μL / min, such as 4 to 10 μL / min, 5 to 10 μL / min, 6 to 9 μL / min, 7 to 10 μL / min, 6 to 8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, both the first and second dimension LC separations are carried out at microflow rates. In some embodiments, the same flow rate as the first dimension LC process is used for the second dimension LC process. In other embodiments, the flow rate for the second dimension is different from that of the first dimension.

[0104] Many types of reversed-phase columns with a hydrophobic stationary phase can be used in 2D-LC. Examples of columns include silica-based columns and PS-DVB resin columns. In some embodiments, second-dimensional separation is performed using a C18 column or a C4 or C8 column. In some embodiments, a C18 column is used.

[0105] Following the second-dimensional separation, the analyte can be collected for mass spectrometry. In some embodiments, the MS is tandem MS ("MS / MS"). Various MS options are available. In some embodiments, the MS apparatus comprises at least two quadrupole analyzers. In some embodiments, the MS apparatus comprises at least three quadrupole analyzers. In some embodiments, the MS is multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem. In some embodiments, the MS is MRM mass spectrometry. In some embodiments, the MS is SIM mass spectrometry.

[0106] In some embodiments, the 2D-LC-MS process is fully automated or carried out using a fully automated controllable system that allows the process to be fully automated, for example, from the start of first-dimensional separation to MS analysis. Systems according to this disclosure are described, for example, in the following sections and are further shown, for example, in Figures 2 and 3.

[0107] In some embodiments, the methods disclosed herein enable at least a 30-fold improvement in sensitivity compared to a microflow 1D-LC-MS / MS process, e.g., 30-100, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100. In some embodiments, the methods disclosed herein enable at least a 100-fold improvement in sensitivity compared to a conventional analytical flow 1D-LC-MS / MS, e.g., 100-150, 100-120, 120-140, or 130-150. In some embodiments, the methods herein can quantify analytes present in tissue samples at concentrations of, for example, less than 10 fmol / μg of total protein, less than 1 fmol / μg of total protein, 1 to 10 fmol / μg of total protein, less than 0.1 fmol / μg of total protein, 0.1 to 1 fmol / μg of total protein, or 0.01 to 0.1 fmol / μg of total protein. In some embodiments, the greatest improvement in sensitivity in tissue samples occurred with analytes at concentrations of, for example, 0.01 to 1 fmol / μg of total protein (e.g., 0.05 fmol / μg of total protein). In fluid samples, specific analytes could be detected at concentrations of less than 10 ng / mL, less than 1 ng / mL, less than 100 pg / mL, or less than 10 pg / mL, for example, 1 to 10 ng / mL, 100 pg / mL to 1 ng / mL, or 10 to 100 pg / mL. For fluid samples, the greatest improvement in sensitivity occurred with analytes of, for example, 10 pg / mL to 1 ng / mL, or 50 to 200 pg / mL or 100 pg / mL. VII.2D-LC-MS System

[0108] This disclosure also relates to systems for carrying out the methods described herein. In some embodiments, the methods described herein can be carried out using, for example, an integrated 2D-LC system coupled to a mass spectrometer. In some embodiments, such a system may be fully automated once a survey-scan process is performed to determine an appropriate retention time window for analyte collection between the first- and second-dimensional LC processes. Generally, a 2D-LC system may comprise two pumps, a first-dimensional column and a second-dimensional column, and appropriate tubing and valves to allow the flow of analyte and mobile phase through the system. In some embodiments, two separate pumps may be used to control the sample flow and / or mobile phase flow. Pumps may be used to facilitate the first and second-dimensional mobile phase gradients of the 2D-LC system. In some embodiments, the system includes a first-dimensional pump to control the flow of analyte and mobile phase through the first-dimensional column and a second-dimensional pump to control the flow of analyte and mobile phase through the second-dimensional column.

[0109] In some embodiments, the system is connected to one or more valves, which may include injection valves, switching valves, ports, ferrules, nuts, and autosamplers. The valves typically have multiple ports and positions (e.g., six ports and two positions) to accommodate various syringes, sample loops, and sample pumps. In some embodiments, the system includes an injection valve that allows for sample loading into a first-dimensional column.

[0110] In some embodiments, the system includes a trap column or other means to help retain the analyte between the first and second dimension separations. In such embodiments, the trap column is located between the first-dimensional and second-dimensional columns. In some embodiments, the trap column can collect a portion of the eluate from the first-dimensional column for analysis in the second dimension. In some embodiments, a trap valve controls the flow of analyte from the first-dimensional process to the second-dimensional process. In some embodiments, the eluate supplied to the trap column includes a portion of the first-dimensional eluate defined by a retention time window determined during the survey scan. In some embodiments, the flow of the first-dimensional eluate to the trap column or other means for retaining the analyte between dimensions is controlled via a trap valve (e.g., having several ports and positions) and / or a T-device for opening and closing a suitable tube for the flow of analyte-containing eluate to the trap column.

[0111] The 2D-LC system may be connected to a mass spectrometer or tandem mass spectrometer for analyzing the analyte after the second-dimensional LC process. In some embodiments, the MS is tandem MS ("MS / MS"). Various MS options are available. In some embodiments, the MS system comprises at least two quadrupole analyzers. In some embodiments, the MS system comprises at least three quadrupole analyzers. In some embodiments, the MS is multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem. In some embodiments, the MS is MRM mass spectrometry. In some embodiments, the MS is SIM mass spectrometry. Examples of tandem mass spectrometers include triple quadrupole (QQQ or triple quad), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap hybrid, and time-of-flight / time-of-flight instruments.

[0112] In some embodiments, the system is configured to use a microflow rate of, for example, 2 to 100 μL / min, such as 4 to 10 μL / min, 5 to 10 μL / min, 6 to 9 μL / min, 7 to 10 μL / min, 6 to 8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min. In some embodiments, LC separation is performed using a C18 column or a C4 or C8 column. In some embodiments, a C18 column is used.

[0113] An example of a 2D-LC-MS / MS system is shown in Figure 3, where an integrated 2D-LC system is connected to a tandem mass spectrometer. This figure illustrates five steps of the automated 2D-LC process according to this disclosure that may be performed on the integrated 2D-LC system after a suitable survey scan has been performed. The first step involves equilibrating the column and loading the analyte-containing sample into the system using an injection valve. In step 1 of Figure 3, the analyte-containing sample is transferred to the injection valve from a syringe or other external device. This figure also shows a first-dimensional pump for controlling the mobile phase during first-dimensional separation and a high-pH RPLC column for first-dimensional LC separation. The line from the first-dimensional pump through the high-pH RPLC column and through a trap valve to waste indicates that the eluate from the first-dimensional LC process can be sent to waste collection until the retention time reaches a window for analyte collection, as predetermined in the survey scan. Step 2 in the figure shows that the injection valve port is reset so that the analyte enters the high pH RPLC column for first-dimensional separation (see the thick bar at the left end of the column in the figure), and the mobile phase and eluate are collected as waste (see the line to the right of the column, which passes through the trap valve and is discarded). When the retention time window for collecting the analyte is reached, in Step 3, the port of the trap valve is reset so that the column eluate passes through a T-device (shown below the high pH column), the T-device is also opened to control the flow, and enters the trap column (shown above the high pH column), where the second-dimensional pump is used to mix the eluate collected in the trap column with the low pH mobile phase for second-dimensional separation. The collected analyte is shown as the thick bar at the left end of the trap column in Step 3 of the figure, and when the retention time window defined by the survey scan ends, the valve can be reset to collect the remainder of the first-dimensional eluate as waste (see, for example, the valve positions in Steps 3 and 4). In Step 4 of the figure, the second-dimensional LC process is performed using the second-dimensional pump. This process moves the analytes collected in the trap column to a low-pH column (analytical column) for further separation.Next, the eluate from this second-dimensional column can be transferred to a mass spectrometer for analysis. In step 5 of the figure, the injection and trap valve positions may be set to equilibrate both columns for a new run.

[0114] The system of this disclosure may also be configured to perform a survey scan using only the first-dimensional LC and mass spectrometer to determine the retention time window in the first-dimensional LC when the analyte elutes. Thus, in some embodiments, during the survey scan, the first-dimensional column can be reconfigured to connect directly to the mass spectrometer to perform the survey scan, and then the second-dimensional column can be connected to the mass spectrometer to perform the complete 2D-LC process. In some cases, the survey scan may be performed by placing the first-dimensional column in the normal position of the second-dimensional column and connecting it directly to the mass spectrometer. In other cases, the system can be configured so that the first-dimensional column is connected directly to the mass spectrometer in its normal position (i.e., its eluate bypasses the trap column or second column and is sent directly to the mass spectrometer).

[0115] In some embodiments, the system herein can operate so that all steps of the 2D-LC-MC process are automatically controlled (see, for example, the steps shown in Figure 3). In some embodiments, the system herein can also operate so that survey scans are automatically controlled. Generally, automatic control herein means that, for example, pumps, valves, and T-tubes in the system can be configured by software programming. Automatic control of these pumps, valves, and T-tubes that control the flow of the mobile phase and eluate in the system then allows the mobile phase and eluate of the chromatography process to move through the system's tubes and columns as appropriate. In some embodiments, the retention time window for collecting the portion of the first-dimensional eluate that will pass into the second dimension is automatically controlled. In some embodiments, one or more steps of the method may be performed without automatic control. For example, it is possible to perform the method herein without automatically controlling the transfer of the eluate of the completed 2D-LC process to the mass spectrometer. Therefore, in some embodiments, it is not necessary to directly connect the mass spectrometer to the 2D-LC system, but in other embodiments, the mass spectrometer may be directly connected. In some embodiments, the process may be carried out in an unintegrated system. In such cases, the separation of the first dimension can be automatically controlled by collecting the portion of the first-dimensional eluate that will be separated in the second dimension, although this portion of the eluate can be manually injected into a separate second-dimensional system optionally connected to the mass spectrometer. This second-dimensional system can be controlled automatically separately.

[0116] In some embodiments, the systems disclosed herein enable at least a 30-fold improvement in sensitivity compared to a microflow 1D-LC-MS / MS process, for example, 30-100, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 times. In some embodiments, the systems disclosed herein enable at least a 100-fold improvement in sensitivity compared to a conventional analytical flow 1D-LC-MS / MS, for example, 100-150, 100-120, 120-140, or 130-150 times. For example, in tissue samples, specific analytes (proteins or peptides) can be detected at concentrations of less than 10 fmol / μg of total protein, less than 1 fmol / μg of total protein, 1 to 10 fmol / μg of total protein, less than 0.1 fmol / μg of total protein, 0.1 to 1 fmol / μg of total protein, or 0.01 to 0.1 fmol / μg of total protein. In some embodiments, the greatest improvement in sensitivity in tissue samples occurred with analytes at concentrations of, for example, 0.01 to 1 fmol / μg of total protein (e.g., 0.05 fmol / μg of total protein). In fluid samples, specific analytes could be detected at concentrations of less than 10 ng / mL, less than 1 ng / mL, less than 100 pg / mL, or less than 10 pg / mL, such as 1 to 10 ng / mL, 100 pg / mL to 1 ng / mL, or 10 to 100 pg / mL. For fluid samples, the greatest improvement in sensitivity occurred with analytes of, for example, 10 pg / mL to 1 ng / mL, or 50 to 200 pg / mL or 100 pg / mL.

[0117] The methods and systems described herein are further illustrated by the following examples. These examples are intended to illustrate potential embodiments only and are not intended to limit the invention. [Examples]

[0118] This example describes a fully automated method for quantifying biopharmaceuticals and soluble targets in serum in the pg / ml range. The method combines (1) a common commercially available capture reagent—in this case, protein A, (2) targeted two-dimensional liquid chromatography (2D-LC) for analyte isolation, and (3) targeted detection by multiple reaction monitoring (MRM) mass spectrometry. Materials and methods

[0119] Spike-in experiments: Monoclonal antibodies were spiked into human, monkey, or mouse serum at concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, and 0.78 ng / mL by 2-fold serial dilutions, respectively. For soluble PD-L1 experiments, atezolizumab-PD-L1 conjugates were prepared by incubating 145 nM PD-L1 with 2.9 μM atezolizumab (molar ratio 1:20) at room temperature for 2 hours. Under these conditions, PD-L1 was completely bound by atezolizumab. Next, the atezolizumab-PD-L1 complex was spiked into normal human serum at 2-fold serial dilutions with PD-L1 molar concentrations of 18, 9, 4.5, 2.25, 1.125, 0.56, 0.28, 0.14, and 0.07 nM, respectively.

[0120] Protein A affinity capture and on-beads protein digestion: 25 μL or 50 μL serum aliquots were transferred to a 96-well plate containing 300 μL of HBS-EP buffer (GE Healthcare). Monoclonal antibodies or antibody-antigen complexes were captured from each sample by incubation with 50 μL of Protein A magnetic beads at room temperature for 1.5 hours. The beads were washed with 400 μL of HBS-EP buffer and 400 μL of water and transferred to a Protein LoBind® plate. The analytes captured by Protein A were denatured and reduced by adding 75 μL of 1 mg / mL RapiGest® SF Surfactant and 10 μL of 100 mM dithiothreitol to each sample and incubating at 60°C for 1 hour. The analytes were further alkylated by adding 25 μL of 100 mM iodoacetamide, incubating at room temperature in the dark for 30 minutes, followed by on-beads trypsin digestion overnight at 37°C. Next, the digestion reaction was quenched by adding 25 μL of 2N HCl solution containing an internal standard peptide of 8 pmol / mL. Magnetic beads were removed from each sample, and the supernatant was used for LC-MS / MS analysis. The magnetic beads were mixed, washed, collected, and transferred in the above steps using a KingFisher® 96 magnetic particle processor (Thermo Fisher Scientific).

[0121] High pH RPLC-MS / MS Survey Scan: Before developing the 2D-LC-MS / MS method, a high pH RPLC-MS / MS survey scan was performed to determine the retention time of the analytes. Briefly, 10 μL aliquots of the spike-in samples with the highest analyte concentration were analyzed using an ACQUITY UPLC® M-class 2D-LC system. Using a BEH 1.7 μm C18 column (300 μm id × 50 mm), peptides were separated using a linear gradient of 10 to 35% of mobile phase B (mobile phase A: 10 mM ammonium formate, pH 9.5; mobile phase B: 10 mM ammonium formate in 90% acetonitrile, pH 9.5) over 10 minutes at a flow rate of 8 μL / min. A QTRAP® 6500+ mass spectrometer with an Optiflow® Turbo V ion source was used to monitor both the analyte's signature peptide and internal standard peptide. Survey scan analysis was repeated 4-5 times until the retention time of the analyte peptide remained stable. Analyst 1.7 software was used for data acquisition and retention time determination. The retention time at the peak apex of the analyte was defined as Tapex.

[0122] Targeted 2D-LC-MS / MS: An online 2D-LC method was developed for targeted separation of analytes from matrix molecules, with a high-pH RPLC used for first-dimensional separation and a low-pH RPLC used for second-dimensional separation. To achieve targeted separation of analytes, fractions or portions within a retention time window of Tapex + / - 0.3 minutes were selectively transferred from the first dimension to the second. The detailed configuration of this targeted 2D-LC-MS / MS method is shown in Figure 3 and described below. Using two sets of binary pumps on an ACQUITY UPLC® M-class 2D-LC system, LC gradients were created for the high-pH and low-pH RPLCs at the same flow rate of 8 μL / min each. For the first-dimensional high-pH RPLC, the same column, mobile phase, and gradient were used as in the survey-scan analysis. Detailed high pH RPLC gradient information was as follows: 3%B (0-3 min), 3-10%B (3-4 min), 10-35%B (4-14 min), 35-90%B (14-14.5 min), 90%B (14.5-15.5 min), 90-35%B (15.5-15.6 min), 35%B (15.6-17.5 min), 35-3%B (17.5-17.6 min), 3%B (17.6-20 min). An HSS T3 1.8 μm C18 column (300 μm id × 100 mm) was used for second-dimensional separation by low pH RPLC, with mobile phase A being 0.1% formic acid and mobile phase B being 0.1% formic acid in acetonitrile. The LC gradients were 0.1%B (0-11 mins), 0.1-10%B (11-11.5 mins), 10-35%B (11.5-15.5 mins), 35-90%B (15.5-15.6 mins), 90%B (15.6-17.5 mins), 90-0.1%B (17.5-17.6 mins), and 0.1%B (17.6-20 mins). The trap valve position was switched to "Position 2" at Tapex-0.3 mins to begin the transfer of the analyte to the Symmetry 5μm C18 trap column (180μm id×20mm), and then returned to "Position 1" at Tapex+0.3 mins to end the transfer of the analyte. After 16.8 minutes, the system was switched back to "Position 2" to thoroughly clean the trap valve, trap column, and tubing, and then after 19 minutes, it was returned to "Position 1" to equilibrate the column.Using the method described above, all peptides present in this example were analyzed and found to be relatively hydrophilic, eluting before 11 minutes during high pH RPLC separation. For the analysis of hydrophobic peptides, a slight modification of the LC gradient was used to ensure that the analytes moved to the second dimension. Masslynx® V4.1 software was used to construct the method.

[0123] The QTRAP® 6500+ mass spectrometer was operated in positive ionization mode using an Optiflow® Turbo V ion source. The main source parameters were set as follows: curtain gas flow, 35; collision gas, medium; nebulizer gas, 25; ion spray voltage, 5000V; temperature, 300°C. The analyte-dependent ion transitions and quadrupole parameters of all peptides analyzed in this study are listed in Table 1 below. [Table 1]

[0124] Microflow 1D-LC-MS / MS: Digested samples were analyzed using an ACQUITY UPLC® M-class system connected online to a QTRAP® 6500+ mass spectrometer (Sciex). The M-class system was equipped with an HSS T3 1.8 μm C18 column (300 μm id × 100 mm). 10 microliters of each sample were loaded into the column, and peptides were separated at a flow rate of 8 μL / min using a 4-minute linear gradient of 10–35% acetonitrile in 0.1% formic acid. The QTRAP® 6500+ mass spectrometer was operated in the same manner as the targeted 2D-LC-MS / MS method.

[0125] Analytical flow LC-MS / MS: 10 microliters of each sample were loaded onto an Acquity UPLC® BEH 1.7 μm C18 column (2.1 × 50 mm), and chromatographic separation was performed using a Nexera X2 UPLC® system (Shimadzu) at a flow rate of 300 μL / min with a gradient of mobile phase A (0.1% formic acid) and mobile phase B (acetonitrile, 0.1% formic acid). A 3.4 minute gradient from 10 to 35% B was used. A QTRAP® 6500+ mass spectrometer (Sciex) was operated in positive ionization mode using an IonDrive® Turbo V ion source. The main source parameters were set as follows: curtain gas flow, 35; impacting gas, medium; nebulizer gas, 50; turbo ion spray gas, 50; ion spray voltage, 5500 V; temperature, 500 °C.

[0126] Data Analysis: Raw data acquired with QTRAP® 6500+ was processed using Skyline software (29). All data were manually inspected to ensure accurate peak integration. Target analytes were quantified using the analyte-to-internal standard peak area ratio. All extracted ion chromatograms (XICs) and calibration curves included in this study were created using Skyline.

[0127] ELISA: Soluble PD-L1 in serum samples was quantified by enzyme-linked immunosorbent assay (ELISA). Briefly, Nunc MaxiSorp® immunoplates were coated overnight at 2–8°C with 1 μg / mL monoclonal anti-human PD-L1 mouse antibody that binds to both unconjugated PD-L1 and PD-L1 in conjugation with atezolizumab. After washing, the plates were treated with 200 μL / well blocking reagent containing 0.5% BSA for 1–3 hours. Then, 100 microliters / well of calibrator and pre-diluted serum samples were added to the blocked plates and incubated for 1 hour. The calibration curve consisted of recombinant human PD-L1 and ranged from 0.64 ng / mL to 0.005 ng / mL. The minimum required dilution of serum samples was 1:10. The plates were washed and then incubated with 10 μg / mL atezolizumab for 1 hour to saturate the bound PD-L1 with atezolizumab. Unbound material was removed by washing. 0.5 μg / mL biotinylated mouse monoclonal anti-atezolizumab antibody was added as a secondary detection antibody and incubated for 1 hour, followed by incubation with streptavidin horseradish peroxidase (HRP). After the final washing step, 100 μL / well of TMB (3,3',5,5'-tetramethylbenzidine) peroxidase substrate was added and incubated for 25-30 minutes. The reaction was stopped with 1 M phosphate, and the plates were read using a SpectraMax plate reader at 450 nm read / 630 nm reference wavelength. PD-L1 concentrations in serum samples were quantified using a fitted calibration curve. result Common reagents, workflow, and basic principles of ultra-targeted 2D-LC-MS / MS.

[0128] The primary objective of this study was to develop a fully automated mass spectrometry-based method for quantifying monoclonal antibodies and their target antigens without significantly improving sensitivity and without developing high-quality capture or detection antibody reagents typically required for ligand-binding assays. With this objective in mind, the inventors developed a targeted 2D-LC-based method. The overall workflow of this method is shown in Figure 1. Monoclonal antibodies or antibody-antigen complexes are extracted from the sample matrix using a general protein A affinity capture method. After on-beads-trypsin digestion, a fully automated targeted 2D-LC method is performed to effectively separate the target analyte from the matrix molecules with high resolution and reproducibility. The significant reduction in matrix inhibition dramatically improves assay sensitivity.

[0129] The main advantage of this method over ligand-binding assays is its independence from high-quality reagents. Protein A affinity capture is used as a common sample preparation procedure applicable to most antibodies or antibody conjugates. Given that protein A is widely available and reasonably priced, our method is highly time-efficient and cost-effective, as it does not require the production of specific capture or detection antibody reagents.

[0130] Compared to commonly used comprehensive 2D-LC approaches, the method described herein is targeted to the analyte. High pH RPLC is used for first-dimensional separation, selectively isolating fractions / parts of the analyte within a narrow retention time window, and transferring them to the second dimension for further separation by low pH RPLC. To ensure that the analyte can be selectively transferred from the first dimension, a high pH RPLC-MS / MS survey scan is performed to determine the precise retention time and peak width of the analyte (Figure 2). The retention time window for analyte transfer is defined as Tapex+ / -1PW, where Tapex represents the retention time at the peak apex of the analyte and PW represents the peak width of the analyte. Under the LC conditions used in this study, the peak width of peptides is typically 0.25–0.3 minutes. Therefore, fractions / parts within a retention time window of Tapex+ / -0.3 minutes are transferred to the second dimension for all peptides analyzed in this study.

[0131] Following high pH RPLC-MS / MS survey-scan analysis, a targeted 2D configuration with dilution (Figure 3) is designed to selectively isolate the analyte using an automated method. This method consists of the following five steps: (Step 1) Load the sample and equilibrate the column; (Step 2) Inject the sample and separate the analyte from the matrix molecules using a linear high pH RPLC gradient; (Step 3) Transfer the target fraction / part within a specified retention time window (Tapex + / - 0.3 min) to the trap column while diluting that fraction / part with a second low pH mobile phase A; (Step 4) Elute the analyte from the trap column and further separate it from the remaining matrix components by the analytical column using a linear low pH RPLC gradient for MS analysis; (Step 5) Wash the trap valve (both position 1 and position 2), tube and column using a high organic mobile phase. The most critical step in this process is step 3, in which analyte migration is initiated by switching the trap valve position to "position 2" at Tapex-0.3 min and terminated by returning it to "position 1" at Tapex+0.3 min. In fact, the main variable in the 2D-LC method is the trap valve switching time. During the analyte migration process, the high pH fraction / part is diluted via a T-tube mixed with second-dimensional mobile phase A (0.1% formic acid) to ensure that the analyte can be captured and concentrated in the trap column (Figure 3).

[0132] Unlike comprehensive 2D-LC, which has a long timescale (typically several hours), the targeted 2D-LC method used in this study has a relatively short LC gradient. The total run time of this method is 20 minutes under fully automated conditions, enabling an analytical throughput of 70 samples per day. Targeted 2D-LC-MS / MS enables the quantification of monoclonal antibodies in serum in the pg / mL to low ng / mL range.

[0133] The performance of targeted 2D-LC-MS / MS was systematically evaluated using anti-CD22 monoclonal antibodies spiked into monkey and human serum using 2-fold serial dilutions ranging from 400 to 0.78 ng / mL. Two anti-CD22 signature peptides, IYPGDGDTNYSGK and LSCAASGYEFSR, were selected for LC-MS / MS, with the former showing a better response. Unless otherwise specified, the following methodological evaluations are based on the transition of peptide IYPGDGDTNYSGK from 693.8 to 555.7.

[0134] The inventors first evaluated the effect of LC flow rate on assay sensitivity. When anti-CD22 spiked in monkey serum was analyzed using conventional analytical flow LC-MS / MS (flow rate: 300 μL / min), the LLOQ was 100 ng / mL. In comparison, microflow LC-MS / MS (flow rate: 8 μL / min) analysis showed a four-fold improvement in assay sensitivity with an LLOQ of 25 ng / mL (Figure 4a). A similar phenomenon was observed for Herceptin spiked in mouse serum (Figure 4b). Based on this evaluation, the flow rate of the 2D-LC method was set to 8 μL / min.

[0135] Next, a direct comparison of sensitivity was performed between 1D- and 2D-LC-MS / MS at the same flow rate of 8 μL / min. As shown in Figure 5, the targeted 2D-LC-MS / MS method allowed for approximately a 6-fold increase in analyte intensity and approximately a 10-fold decrease in background intensity. This resulted in an approximately 60-fold increase in the signal-to-noise ratio (S / N ratio). The LLOQ for anti-CD22 in the 1D- and 2D-LC-MS / MS assays were 25 ng / mL and 0.78 ng / mL, respectively (Figure 5). In fact, the S / N ratio at 0.78 ng / mL was still approximately 9 in the 2D-LC-MS / MS method, suggesting that higher sensitivity (e.g., 400 pg / mL) is potentially achievable.

[0136] Next, the linearity of the targeted 2D-LC-MS / MS method was evaluated using anti-CD22 spiked in both monkey and human serum. Figure 6 shows the extracted ion chromatograms (XICs) of the transitions monitored for the peptide IYPGDGDTNYSGK in 50 μL cynomolgus monkey serum samples using 2-fold serial dilutions, with the upper and lower panels showing the responses of the analyte and internal standard (IS), respectively. The internal standard intensity remained stable across all spike-in samples (except for the double blank where the IS was not spiked), but a twofold increase in analyte intensity was observed, which correlated well with the concentration of anti-CD22. A similar phenomenon was demonstrated in human serum samples (Figure 7). Calibration curves were constructed by plotting the analyte-to-internal standard peak area ratio (i.e., light:heavy peak area ratio) against the analyte concentration. Excellent linearity was observed for monkey (Figure 8a) and human (Figure 8b) samples over the concentration ranges of 0.78–400 ng / mL and 1.56–400 ng / mL, respectively.

[0137] The precision and accuracy of the 2D-LC-MS / MS assays were further evaluated by analyzing quality control (QC) samples with low, medium, and high levels of anti-CD22. As shown in Table 2 below, the precision (%CV) was less than 10% and the accuracy (% difference from theoretical value) was less than 15% for both monkey and human serum samples across all QC levels, which met the acceptable criteria recommended by the Trade White Paper for Validation of LC-MS / MS Methods for Protein Therapeutic Agents (20). [Table 2]

[0138] The reproducibility of analyte retention times during high-pH RPLC separation is crucial for the automation of targeted 2D-LC-MS / MS methods. We evaluated this by injecting the same sample three times on three different days (i.e., injections 1, 180, and 320) in a run containing 320 samples. As shown in Figure 9, the retention times were relatively stable, with only slight shifts of 0.1 minutes after injection 180 and 0.15 minutes after injection 320. Considering that the analyte migrates from the first dimension with a retention time window of 0.6 minutes (Tapex + / - 0.3 minutes), this small shift in retention time does not result in significant loss of analyte or internal standard during the migration process. Even if some loss occurs, the accuracy and precision of the analysis are not affected, as quantification is based on the ratio of the analyte to the internal standard, which remains unchanged regardless of analyte migration and recovery. Nevertheless, it is recommended to verify and adjust the retention time window every 300 injections to allow the analyte to migrate to the second dimension in order to maintain analytical sensitivity. For small runs (i.e., fewer than 300 samples), there is no need to adjust the retention time window.

[0139] Carryover in the 2D-LC-MS / MS method was also evaluated by comparing the peak areas between the sample containing high concentrations of the analyte and the subsequent matrix blank. As shown in Table 3 below, the carryover was approximately 0.3% for both the analyte and IS. Similarly low carryover was observed for all other molecules analyzed by the inventors using this method (data not shown). [Table 3] For Table 3, 50 microliters of monkey serum spiked with 400 ng / mL anti-CD22 were analyzed using protein A affinity capture 2D-LC-MS / MS. Carryover was calculated based on the peak area of ​​this sample and the subsequent carryover blank. IS represents the internal standard.

[0140] The robustness of this method can be improved by maintaining a stable spray at microflow rates. For example, the Optiflow® ion source, optimized for microflow LC-MS / MS, can be used. In our recent evaluation, no spray problems were observed during a period in which more than 1200 serum samples were analyzed using the Optiflow® ion source.

[0141] The inventors also evaluated the effect of sample volume on assay sensitivity. As shown in Figure 10, when the serum volume was increased from 25 μL to 50 μL, the background intensity did not significantly increase in analyte retention time due to the excellent separation efficiency by 2D-LC, but the analyte intensity increased twofold. This observation indicates that the sensitivity of the 2D-LC-MS / MS assay can be enhanced by increasing the sample volume.

[0142] The second peptide, LSCAASGYEFSR, was also monitored for its anti-CD22 properties. Using the same general protein A affinity capture and targeted 2D-LC-MS / MS approach, LLOQ values ​​of 6.25 and 12.5 ng / mL were achieved for monkey serum samples and human serum samples, respectively (Figure 11). This represents a more than 30-fold improvement in assay sensitivity compared to microflow 1D-LC-MS / MS (data not shown).

[0143] The above evaluation using an anti-CD22 monoclonal antibody demonstrates that our method enables fully automated, reagent-independent, highly sensitive, and robust quantification of biotherapeutic drugs. We further confirmed the performance of this method using another monoclonal antibody, bevacizumab, and observed excellent linearity in monkey serum over the concentration range of 3.13–400 ng / mL at an LLOQ of 3.13 ng / mL (Figure 12). Targeted 2D-LC-MS / MS enables highly sensitive quantification of soluble targets of antibody therapeutics.

[0144] The inventors also used the Protein A affinity capture 2D-LC-MS / MS method to quantify soluble targets of monoclonal antibody therapeutics in human serum, which are frequently evaluated as predictive or therapeutic biomarkers (22-26). In blood, therapeutic monoclonal antibodies can specifically bind to their target antigens and form antigen-antibody complexes, which can be captured from the sample by protein A beads.

[0145] In this study, we measured the circulating concentration of PD-L1, a key immune checkpoint molecule, after treatment with atezolizumab, a humanized anti-PD-L1 monoclonal antibody. In post-treatment patient serum samples, atezolizumab was in large molar excess due to the high dose level, and soluble PD-L1 formed a complex with atezolizumab. To mimic the in vivo situation, a similar complex was prepared in vitro by incubating PD-L1 with atezolizumab at a molar ratio of 1:20 to ensure that all PD-L1 was bound by atezolizumab (Figure 13). This atezolizumab-PD-L1 complex was then spiked into normal human serum at 2-fold serial dilutions with PD-L1 concentrations ranging from 500 to 2 ng / mL (or 18 to 0.07 nM). Spike-in samples were initially tested using microflow 1D-LC-MS / MS, but the LLOQ was only about 60 ng / mL, which was insufficient to measure soluble PD-L1 in post-treatment samples (Figure 14a). In contrast, targeted 2D-LC-MS / MS enabled a sensitivity improvement of more than 30 times (Figure 14b) and allowed for accurate quantification of soluble PD-L1 in these samples. Multiple splice variants of PD-L1 exist in circulation (24, 27, 28), and it has been reported that atezolizumab binds to the PD-L1 IgV domain (29). To measure the total concentration of soluble PD-L1 variants bound by atezolizumab, the signature peptide LQDAGVYR derived from the PD-L1 IgV domain was selected for LC-MS / MS. Considering the different molecular weights of PD-L1 variants, a calibration curve was constructed using molar concentration rather than mass concentration, and excellent linearity was observed over the concentration range of 0.07–18 nM (Figure 15a). A total of 12 post-treatment serum samples were analyzed, and the total soluble PD-L1 concentration was measured at approximately 0.6–1.0 nM (Figure 15b). Assuming that the dominant form of PD-L1 is wild-type, the mass concentration of soluble PD-L1 in these samples was approximately 15–25 ng / mL (Figure 15c), which correlated well with the concentration determined by ELISA (Figure 15d). The results indicate that the concentration of circulating PD-L1 variants is not high enough to block the effect of atezolizumab.In fact, the absolute concentrations measured by 2D-LC-MS / MS were approximately 20-30% higher than those measured by ELISA. While the Protein A affinity capture 2D-LC-MS / MS method allows for the measurement of the total concentration of PD-L1 variants bound to atezolizumab, it should be noted that ELISA may underestimate the total soluble PD-L1 concentration because some variants may not be captured by the secondary antibody. conclusion

[0146] In this study, the inventors developed a method based on affinity capture and targeted 2D-LC-MS / MS that is more than 100 times more sensitive than conventional LC-MS / MS. Due to the significantly improved sensitivity, monoclonal antibody therapeutics can be accurately quantified by this method in the range of pg / mL to low ng / mL in both non-clinical and clinical serum samples, even without high-quality capture or detection antibody reagents. This method also successfully measured the total concentration of soluble antigen PD-L1 in post-treatment patient serum samples, which could not be quantified by microflow 1D-LC-MS / MS. This method is extremely robust, fully automated, and easy to implement. It has great potential to support the evaluation of pharmacokinetics and biomarkers during the drug development process. It is particularly useful when high assay sensitivity is required, especially when key reagents for ligand binding assays are unavailable. Example 2.

[0147] This example describes an exemplary method for capturing analytes from tissue samples. Tissue samples were suspended in ice-cold tissue lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with cOmplete® EDTA-free protease inhibitor cocktail tablets (SigmaAldrich), and then homogenized using 1.4 mm ceramic beads in an OMNI international Bead Ruptor® and Cooling Unit. The tissue lysates were transferred to 1.5 mL Protein LoBind® tubes (Eppendorf®) and centrifuged at 15000 g for 6 minutes at 4 °C. Protein concentrations in the supernatant were determined by the Pierce® BCA protein assay kit. Drug target proteins were captured from each sample by incubating the tissue lysates with antibodies against drug targets immobilized on Protein A magnetic beads. After Onby's-trypsin digestion, the signature peptides of drug target proteins, which may be in non-alkylated or alkylated forms, were analyzed by targeted 2D-LC-MS / MS. When the target protein is alkylated by a small molecule drug, both the free and alkylated targets can be quantified using the method described above. This allows for evaluation of alkylated target binding. 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Claims

1. A method for detecting analytes in a sample, (a) In the presence of an internal standard, the analyte is subjected to detection by mass spectrometry (MS) and a high-pH reversed-phase liquid chromatography (RPLC) survey scan at a predetermined concentration to determine the peak tapex of the analyte. (b) Obtain an experimental sample containing the analyte, (c) Perform high pH RPLC on the sample in (b), (d) In the survey scan of (a), collect at least a portion of the eluate of the high pH RPLC in (c) corresponding to the Tapex+ / - average peak width of the analyte or the internal standard, wherein the at least portion is optionally collected in a trap column, and (e)(d) Perform low pH RPLC on the portion of the eluate collected from the high pH RPLC. (f) Detection of the analyte by MS, Methods that include...

2. The method according to claim 1, wherein the analyte is a peptide.

3. A method for detecting peptide analytes in a sample, (a) Obtain an experimental sample and isolate the protein in the sample by performing affinity-based capture. (b) Digesting the captured protein with a protease to obtain a peptide analyte for detection. (c) In the presence of an internal standard peptide, the peptide analyte is detected by mass spectrometry (MS) and subjected to a high-pH reversed-phase liquid chromatography (RPLC) survey scan at a predetermined concentration to determine the peak tapex of the analyte. (d) Perform high pH RPLC on the digested sample of (b) containing the peptide analyte. (e) Collecting at least a portion of the eluate of the high pH RPLC in (d) corresponding to the Tapex+ / - of the analyte, the internal standard, or the average peak width of the peptide in the survey scan in (c), wherein the at least portion is optionally collected in a trap column. (f) Perform low pH RPLC on the collected portion of the eluate from the high pH RPLC in (e), and (g) Detecting the analyte by MS, Methods that include...

4. The method according to any one of claims 1 to 3, wherein the high pH RPLC and the low pH RPLC are each carried out at a microflow rate.

5. The method according to claim 4, wherein the high pH RPLC and / or the low pH RPLC are performed at a flow rate of 4 to 10 μL / min, 5 to 10 μL / min, 6 to 9 μL / min, 7 to 10 μL / min, 6 to 8 μL / min, 5 μL / min, 6 μL / min, 7 μL / min, 8 μL / min, 9 μL / min, or 10 μL / min.

6. The method according to any one of claims 1 to 5, wherein the high pH RPLC and / or the low pH RPLC are performed using a C18 column.

7. The method according to claim 6, wherein both the high-pH RPLC and the low-pH RPLC are performed using a C18 column.

8. The method according to any one of claims 1 to 7, wherein the high pH RPLC and survey scan are performed at a pH between 8 and 10, a pH between 8 and 9, a pH between 9 and 10, or a pH of 8.0, 8.5, 9.0, 9.5, or 10, using mobile phase A containing ammonium formate and mobile phase B containing ammonium formate and acetonitrile (for example, a 90% acetonitrile solution of ammonium formate).

9. The method according to any one of claims 1 to 8, wherein the high pH RPLC survey scan and the high pH RPLC performed on the sample or digested sample are performed at substantially the same flow rate, pH, temperature, pressure, and / or column type (e.g., C8, C18, etc.).

10. The method according to any one of claims 1 to 9, wherein the low pH RPLC is carried out at a pH between 2 and 4.5, between 2 and 4, between 2 and 3, between 2 and 2.5, between 2.5 and 3, between 3 and 4, or pH 2.0, pH 2.5, pH 3.0, pH 3.5, pH 4.0, or pH 4.5, using mobile phase A containing formic acid and mobile phase B containing formic acid and acetonitrile (for example, an acetonitrile solution of formic acid).

11. The method according to any one of claims 1 to 10, wherein the sample is a biological sample.

12. The method according to claim 11, wherein the sample is a biological fluid sample such as tears, saliva, lymph, urine, serum, cerebrospinal fluid, pleural fluid, ascites, or plasma.

13. The method according to claim 12, wherein the sample is a serum sample, and optionally the sample is treated to concentrate or extract components containing the analyte before being used in the method.

14. The method according to claim 11, wherein the sample is a tissue sample, and optionally the sample is treated to concentrate or extract components containing the analyte before being used in the method.

15. The method according to any one of claims 1 to 14, wherein the portion of the eluate of the high pH RPLC in which the low pH RPLC is performed corresponds to the portion containing Tapex + / - 0.25 min of the analyte at a flow rate of 8 μl / min in the C18 column.

16. The method according to any one of claims 1 to 14, wherein the portion of the eluate of the high pH RPLC in which the low pH RPLC is performed corresponds to the portion containing Tapex + / - 0.3 min of the analyte at a flow rate of 8 μl / min in the C18 column.

17. The method according to claim 15 or 16, wherein the portion of the eluate of the high pH RPLC in which the low pH RPLC is performed corresponds to the portion of the analyte consisting of Tapex + / - 0.25 to 0.30 minutes at a flow rate of 8 μl / min in the C18 column.

18. The method according to any one of claims 3 to 17, wherein the protein is captured by attachment to a particle coated with a binding partner of the protein, such as a ligand or antigen.

19. The method according to claim 18, wherein the particles are coated with protein A, protein G, or a chimera of protein A and protein G.

20. The method according to any one of claims 3 to 19, wherein the protein is an antibody.

21. The method according to any one of claims 2 to 20, wherein the analyte is a part of an antibody (for example, an antigen-binding fragment such as Fab, Fv, scFv, or F(ab')2 fragment).

22. The method according to any one of claims 2 to 19, wherein the analyte is an antigen or a drug-binding target.

23. A method according to any one of claims 3 to 22, wherein the protease comprises one or more of trypsin, endoproteinase LysC, endoproteinase ArgC, Staphylococcus aureus V8, endoproteinase GluC, chymotrypsin, or papain.

24. The method according to any one of claims 1 to 23, wherein the method detects more than one analyte in the sample, optionally determines the levels of two or more analytes, and optionally includes modified and unmodified molecular forms of the more than one analyte.

25. The method according to claim 24, wherein the method detects a modified form and an unmodified form of a molecule, and the modified form and the unmodified form include modified and unmodified proteins, for example, alkylated and non-alkylated proteins or glycosylated and non-glycosylated proteins, or molecules and metabolites of such molecules.

26. The method according to any one of claims 1 to 25, wherein the high pH RPLC and low pH RPLC and MS detection are automated.

27. The method according to any one of claims 1 to 26, wherein detecting the analyte by MS following the low pH RPLC quantitatively determines the amount or concentration of the analyte in the sample.

28. The method according to any one of claims 1 to 27, wherein the MS is a tandem MS ("MS / MS") (for example, multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem).

29. The method according to any one of claims 1 to 28, wherein at least a portion of the eluate from high pH RPLC is diluted with a low pH mobile phase before low pH RPLC is performed, and optionally the dilution is further automatically controlled.

30. The method according to any one of claims 1 to 29, wherein the method enables analyte detection sensitivity at a microflow rate that is at least 30 times or 30 to 100 times higher than that of a one-dimensional HPLC-tandem MS (1D-LC-MS / MS) process, and / or the method enables analyte detection sensitivity at an analytical flow rate that is at least 100 times higher than that of a one-dimensional HPLC-tandem MS (1D-LC-MS / MS) process.

31. The method according to any one of claims 1 to 30, wherein the method enables the detection of an analyte in a tissue sample at concentrations of less than 10 fmol / μg total protein, less than 1 fmol / μg total protein, 1 to 10 fmol / μg total protein, less than 0.1 fmol / μg total protein, 0.1 to 1 fmol / μg total protein, or 0.01 to 0.1 fmol / μg total protein, and / or the method enables the detection of an analyte in a fluid sample at concentrations of less than 10 ng / mL, less than 1 ng / mL, less than 100 pg / mL, or less than 10 pg / mL, for example, 1 to 10 ng / mL, 100 pg / mL to 1 ng / mL, or 10 to 100 pg / mL.

32. A system for performing a two-dimensional liquid chromatography method to detect analytes in a sample, (a) An injection valve for injecting a sample containing the analyte into the system, the injection valve being connected to a high-performance liquid chromatography (HPLC) column and a first-dimensional pump, and controlling the flow of the sample and mobile phase through the column, (b) A trap column for collecting at least a predetermined portion of the eluate from the first-dimensional HPLC column for analysis in a two-dimensional HPLC process, (c) A trap valve for controlling the flow path of the mobile phase from a one-dimensional HPLC process to a two-dimensional HPLC process, (d) A second-dimensional pump for controlling the flow of the mobile phase through the second-dimensional HPLC column, (e) A mass spectrometer for analyzing the analyte after a two-dimensional HPLC process, A system equipped with these features.

33. The system according to claim 32, which can carry out the method described in any one of claims 1 to 31.

34. The system according to claim 32 or 33, wherein the mass spectrometer is a tandem mass spectrometer.

35. The system according to any one of claims 32 to 34, wherein the positions of the injection valve and the trap valve may be automatically controlled during a two-dimensional liquid chromatography process, and the automatic control allows only a predetermined portion of the eluate from the first dimension to be separated in the second dimension.

36. The system according to any one of claims 32 to 35, wherein the predetermined portion of the eluate from the first dimension corresponds to the portion containing Tapex + / - 0.25 minutes of the analyte at a flow rate of 8 μl / min in the C18 column.

37. The system according to any one of claims 32 to 36, wherein the predetermined portion of the eluate from the first dimension corresponds to the portion containing Tapex + / - 0.3 min of the analyte at a flow rate of 8 μl / min in the C18 column.

38. The system according to claim 36 or 37, wherein the predetermined portion of the eluate from the first dimension corresponds to the portion of the analyte consisting of Tapex + / - 0.25 to 0.30 minutes at a flow rate of 8 μl / min in the C18 column.

39. The system according to any one of claims 32 to 38, wherein the system is further capable of performing a survey scan to determine the Tapex of the analyte in the first-dimensional HPLC column.

40. The system according to any one of claims 32 to 39, wherein the mass spectrometer performs multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem.

41. The system according to any one of claims 32 to 40, wherein the analyte is a peptide.

42. The system according to any one of claims 32 to 41, wherein the system can dilute a predetermined portion of the eluate from the one-dimensional process in a mobile phase buffer for the two-dimensional process, and optionally, the dilution is automatically controlled.

43. The method according to any one of claims 2 to 31, wherein the analyte is an anti-CD22 antibody or a peptide fragment thereof, for example, one or both of IYPGDGDTNYSGK and LSCAASGYEFSR.

44. The method according to any one of claims 2 to 31, wherein the analyte is PD-L1 or a peptide fragment thereof, for example, an IgV domain fragment, for example, LQDAGVYR, and optionally, the sample is obtained from a subject previously treated with an immune checkpoint molecule such as atezolizumab, and further optionally, the sample is a serum sample.

45. A method for detecting the peptide analyte of human PD-L1 in a biological sample derived from a human subject, (a) Obtaining a biological sample (e.g., a serum sample) from the subject and performing affinity-based capture to isolate PD-L1 in the sample, wherein the affinity-based capture optionally includes capturing PD-L1 complexed with an anti-PD-L1 antibody (e.g., atezolizumab). (b) Digesting the captured protein with a protease to obtain a PD-L1 peptide analyte for detection (e.g., a PD-L1 IgV domain fragment such as LQDAGVYR), (c) In the presence of an internal standard peptide, the PD-L1 peptide analyte is detected by mass spectrometry (MS) and subjected to a high-pH reversed-phase liquid chromatography (RPLC) survey scan at a predetermined concentration to determine the peak tapex of the analyte. (d) Perform high pH RPLC on the digested sample of (b) containing the peptide analyte. (e) Collecting at least a portion of the eluate of the high pH RPLC in (d) corresponding to the Tapex+ / - of the analyte, the internal standard, or the average peak width of the peptide in the survey scan in (c), wherein the at least portion is optionally collected in a trap column. (f) Perform low pH RPLC on the collected portion of the eluate from the high pH RPLC in (e), and (g) The PD-L1 peptide analyte is detected by MS (for example, multiple reaction monitoring (MRM), single ion monitoring (SIM), triple quadrupole (TSQ), quadrupole / time-of-flight (QTOF), quadrupole linear ion trap (QTRAP), hybrid ion trap / FTMS, time-of-flight / time-of-flight (TOF / TOF), or tandem-in-time MS / MS tandem). Methods that include...

46. The method according to any one of claims 1 to 31 or 43 to 45, as implemented in the system according to any one of claims 32 to 42.

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