Mass spectrometric analysis

Abstract

The present disclosure provides isotopic mass tags for labelling peptides and other biomolecules, as well as methods of detecting an analyte by identifying by mass spectrometry one or more of the mass labels, or combinations thereof, described herein.

Description

033945.00485MASS SPECTROMETRIC ANALYSISCross-Reference to Related Application

[0001] This present application claims the benefit of priority to U.S. Patent Application No. 63 / 692,410, filed on September 9, 2024, the entire content of which is hereby incorporated by reference in its entirety.Technical Field

[0002] This disclosure relates to a method of assaying a target analyte by mass spectrometry, particularly biomolecules such as nucleic acids and proteins. Specifically, the disclosure relates to a method of multiplexed mass spectrometry using sets of mass labels. Within a given set, the mass labels are isotopologues, meaning they have the same structural formula and differ only in their isotopic composition. As common in the literature, such isotopologue labels will be referred to as isotopic labels in this disclosure.Background

[0003] The study of biological systems and particularly the understanding of human disease is dependent on the ability to detect changes caused in biological systems by or in response to a disease. Such changes provide means of diagnosis and offer insights into the targets for therapeutic compounds such as vaccines and medicines. A wide range of biological molecules need to be measured quantitatively to understand disease processes including nucleic acids, proteins, steroids, sugars and lipids. In this context, the ability to quantitatively detect such biomolecules using mass spectrometers has provided considerable advances in their study and application to human and also to veterinary disease. The same advances have also occurred in environmental analysis and monitoring, and in food and beverage manufacturing. In particular the use of stable isotopes to provide synthetic quantitative references has been developed in isotope dilution mass spectrometry for monitoring of all classes of biomolecules.033945.00485However, these methods have traditionally required an available synthetic standard, which is not always possible.

[0004] Recently, a range of chemical mass tags bearing heavy isotope substitutions have been developed to further improve the quantitative analysis of biomolecules by mass spectrometry. Depending on the tag design, members of tag sets are either isotopic having the same chemical structure but different absolute masses, or isobaric (i.e., often even isotopomeric), having both identical structure and absolute mass, but differ in the exact placement of heavy isotopes. Isotopic tags are typically used for quantification in MS mode whilst isobaric tags must be fragmented in MS / MS mode to release reporter fragments with a unique mass.

[0005] An early example of isotopic mass tags was the Isotope-Coded Affinity Tags (“ICAT”) (Gygi, S. P. et al., (1999) Nature Biotechnology, 17, 994-999). The ICAT reagents are a pair of mass tags bearing a differential incorporation of heavy isotopes in one (heavy) tag with no substitutions in the other (light) tag. Two samples are labelled with either the heavy or light tag and then mixed prior to analysis by LC-MS. A peptide present in both samples will give a pair of precursor ions with masses differing in proportion to the number of heavy isotope atomic substitutions.

[0006] The ICAT method also illustrates ‘sampling’ methods, which are useful as a way of reconciling the need to deal with small populations of peptides to reduce the complexity of the mass spectra generated while retaining sufficient information about the original sample to identify its components. The ‘isotope encoded affinity tags’ used in the ICAT procedure comprise a pair of biotin linker isotopes, which are reactive to thiols, for the capture peptides comprising cysteine. Typically, 90 to 95% or proteins in a proteome will have at least one cysteine-containing peptide and typically cysteine-containing peptides represent about 1 in 10 peptides overall so analysis of cysteine-containing peptides greatly reduces sample complexity033945.00485 without losing significant information about the sample. Thus, in the ICAT method, a sample of protein from one source is reacted with a ‘light’ isotope biotin linker while a sample of protein from a second source is reacted with a ‘heavy’ isotope biotin linker, which is typically 4 to 8 daltons heavier than the light isotope. The two samples are then pooled and cleaved with an endopeptidase. The biotinylated cysteine-containing peptides can then be isolated on avidinated beads for subsequent analysis by mass spectrometry. The two samples can be compared quantitatively: corresponding peptide pairs act as reciprocal standards allowing their ratios to be quantified. The ICAT sampling procedure produces a mixture of peptides that still accurately represents the source sample while being less complex than MudPIT, but large numbers of peptides are still isolated and their analysis by LC-MS / MS generates complex spectra. With 2 ICAT tags, the number of peptide ions in the mass spectrum is doubled compared to a label-free analysis. Further isotopic tags, such as IPCL, mTRAQ and dimethyl labeling allow for sample multiplexing in MS 1. Such isotopic labels increase the number of precursor ions as a function of the multiplexing level, i.e., a four-plex set of tags will create four times as many precursors and thus the amount of time needed for MS 1 -multiplexed data- dependent acquisition (DDA) analysis (Mertins, P. et al. (2012) Molecular & Cellular Proteomics 11) with potential large impacts on the level of protein coverage. In practical terms this has limited isotopic tag sets to maximally 4 members.

[0007] However, more recently, techniques for multiplexed analysis of proteins using isotopic labels have been developed using data independent acquisition (DIA) methods (e.g., plexDIA or mDIA) that increase throughput multiplicatively with the number of labels without reducing proteome coverage or quantitative accuracy (Derks, Jason, et al. (2023) Nature Biotechnology 41.1: 50-59, Thielert, Marvin et al., bioRxiv preprint doi: https: / / doi.org / 10.1101 / 2022.12.02.518917).033945.00485Brief Description

[0008] In view of the shortcomings of existing mass labels, there still remains a need for sets of isotopic tags for labelling peptides and other biomolecules that enable higher levels of sample multiplexing whilst retaining identical chromatographic retention times and providing unique mass-to-charge ratios for each version of the same peptide sequence. This is achievable within DIA strategies where relative quantification is performed at the MS 1 level of tandem mass spectrometry whilst identification of sets of peptides with similar mass-to-charge are made using windows of defined widths ranging from 100 daltons to less than 10 daltons, using the isotopic mass labels described herein. To maximize the multiplexing levels whilst retaining sufficient resolution each isotopic tag should be resolved from all other tags in the set by at least 0.003 daltons, by 0.006 daltons, by at least 1 dalton, and in some cases by at least 4 daltons.

[0009] In a first general aspect, the present disclosure relates to a set of two or more isotopic mass labels, wherein each label comprises the formula:X- Re wherein X is an isochemic structure having a unique mass relative to all other members of the set coding moiety having an exact mass and Re is a reactive functionality for attaching the mass label to an analyte or the analyte, and X comprises the following general formula:

[0010] In some aspects, the present disclosure relates to a set of two or more isotopic mass labels, wherein each mass label comprises the formula:033945.00485X- A wherein X is a coding moiety having an exact mass (see above), and A is the analyte, and wherein each mass label in the set has a different integer mass.

[0011] In some aspects, the set of two or more isotopic mass labels comprises mass labels having the following structure:wherein * represents that oxygen is18O, carbon is13C, nitrogen is15N, and hydrogen is2H, and wherein one or more * may be present. For clarity, substitutable hydrogens on carbon are not represented by * but may be substituted with2H in any position.

[0012] In some aspects, the set of two or more isotopic mass labels comprises n=6 mass labels having the following structures:033945.00485

[0013] In some aspects, the difference in exact mass between a mass label and the next heaviest mass label in the set of mass labels is at least 4 daltons, or at least 2 daltons, or at least 1 dalton. In some aspects, the difference in exact mass between a mass label and the next heaviest mass label in the set of mass labels is 4 daltons throughout the set of mass labels. In other aspects the differences in exact mass between a mass label and the next heaviest mass label in the set of mass labels varies and can be 4 daltons, 2 daltons, 1 dalton or less than 1 dalton.

[0014] In some aspects, the difference in exact mass between at least two of the mass labels is less than 100 millidaltons (“mDa”), less than 50 mDa, or less than 20 mDa. In some aspects, the difference in exact mass between at least two of the mass labels is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa.

[0015] In another aspect, the present disclosure relates to a method of mass spectrometry analysis, which method comprises quantifying an analyte by identifying by mass spectrometry ions with a mass-to-charge ratio equivalent to the analyte mass modified by the addition of one033945.00485 member of the set of isotopic mass labels, wherein the mass label is a mass label from a set of mass labels according to the disclosure, and wherein the analyte identification is obtained from data-independent acquisition mass spectrometry.

[0016] In some aspects, a method of mass spectrometry analysis comprises: a) providing a plurality of samples, wherein each sample is differentially labelled with an isotopic mass label or a combination of isotopic mass labels, each selected from a set of two or more isotopic mass labels as described herein; b) mixing the plurality of labelled samples to form an analysis mixture comprising labelled analytes; c) detecting the labelled analytes in amass spectrometer; d) isolating one or more labelled analytes in the mass spectrometer based on one or more isolation windows of >1 Da and then dissociating them to form labelled analyte fragments; e) detecting the analyte fragments; and f) identifying the one or more labelled analytes on the basis of the analyte fragments.

[0017] In some aspects, the one or more isolation windows comprise an isolation window that is <10 Da, <20 Da, <50 Da, and / or <100 Da.

[0018] In some aspects, the dissociation in step d) is collision-induced dissociation in a mass spectrometer. In some aspects, the dissociation is collision induced dissociation in a mass spectrometer with a resolution of greater than 60,000 at a mass-to-charge ratio of 400, optionally a resolution of greater than 100,000 at a mass-to-charge ratio of 400, or a resolution greater than 250,000 at a mass-to-charge ratio of 400.

[0019] In some aspects, the labelled analytes may be identified on the basis of the MS2 spectrum of the labelled analyte fragments and quantified using the MSI spectrum of the labelled analytes.

[0020] In some aspects, each isotopic mass label is resolved from all other tags in the set by at least 0.003 daltons, by 0.006 daltons, by 1 dalton, by 2 daltons or at least by 4 daltons.033945.00485Brief Description of the Drawings

[0021] FIG. 1 is a reaction scheme showing the synthesis of Benzyl 3-aminopropanoate hydrotosylate (H-[3Ala-0Bn * pTosOH).

[0022] FIG. 2 is a reaction scheme showing the synthesis of 3-tert- butoxy carbonylaminopropionic acid (Boc-bAla-OH).

[0023] FIG. 3 is a reaction scheme showing the synthesis of tert-butyl 4-(2-benzyloxy-2- oxo-ethyl)piperazine-l -carboxylate (Boc-Pip-Ac-OBn) (Fig. 3a), and 2-(4-tert- butoxycarbonylpiperazin-l-yl) acetic acid (Boc-Pip-Ac-OH) (Fig 3b).

[0024] FIG. 4 is a reaction scheme showing the synthesis of Methyl 2-aminoacetate hydrochloride (H-Gly-OMe * HC1).

[0025] FIG. 5 is a reaction scheme showing the synthesis of 2-(tert-Butoxycarbonylamino) acetic acid (Boc-Gly-OH).

[0026] FIG. 6 is a reaction scheme showing the synthesis of Methyl 2-(benzylamino)acetate hydrochloride (H-(Bzl)Gly-OMe * HC1).

[0027] FIG. 7 is a reaction scheme showing the synthesis of Methyl 2-[benzyl-[2-(tert- butoxycarbonylamino)-acetyl] amino] acetate (Boc-Gly-(Bzl)Gly-OMe).

[0028] FIG. 8 is a reaction scheme showing the synthesis of l-Benzylpiperazin-2,5-dione.

[0029] FIG. 9 is a reaction scheme showing the synthesis of 1 -Benzylpiperazine Dihydrochloride .

[0030] FIG. 10 is a reaction scheme showing the synthesis of Benzyl 2-bromacetate.

[0031] FIG. 11 is a reaction scheme showing the synthesis of Benzyl 2-(4-benzyl piperazin- 1-yl) acetate.

[0032] FIG. 12 is a reaction scheme showing the synthesis of benzyl 2-Piperazin-l-yl acetic acid.033945.00485

[0033] FIG. 13 is a reaction scheme showing the synthesis of 2-(4-tert- Butoxycarbonylpiperazin-l-yl) acetic acid (Boc-Pip-Ac-OH).

[0034] FIG. 14 is a reaction scheme showing the synthesis of Benzyl 3-[3-(tert- butoxycarbonylamino)propanoylamino]propanoate (Boc-pAla-pAla-OBn).

[0035] FIG. 15 is a reaction scheme showing the synthesis of Benzyl 3-(3- aminopropanoylamino)propanoate hydrochloride (H-pAla-[3Ala-OBn*HCl).

[0036] FIG. 16 is a reaction scheme showing the synthesis of tert-Butyl 4-[2-[[3-[(3- benzyloxy-3-oxo-propyl)amino]-3-oxo- propyl]amino]-2-oxo-ethyl]piperazine-l-carboxylate (Boc-Pip-Ac-pAla-pAla-OBn) .

[0037] FIG. 17 is a reaction scheme showing the synthesis of Benzyl 3-[3-[(2-piperazin-l- ylacetyl)amino] -propanoylamino] -propanoate Hydrochloride (H-Pip-Ac-pAla-pAla-OBn*HCl).

[0038] FIG. 18 is a reaction scheme showing the synthesis of Benzyl 3-[3-[[2-(4- isobutylpiperazin- 1 -yl)acetyl] amino] - propanoylamino] propanoate (Isobutyl-Pip-Ac-pAla- PAla-OBn).

[0039] FIG. 19 is a reaction scheme showing the synthesis of 3-[3-[[2-(4-isobutylpiperazin- l-yl)acetyl]amino]-propanoylamino]propanoate (Isobutyl -Pip-Ac-pAla-pAla-OH).

[0040] FIG. 20 is a reaction scheme showing the synthesis of (2,5-Dioxopyrrolidin-l-yl) 3- [3-[[2-(4-isobutylpiperazin-l-yl)-acetyl]amino]propanoylamino]propanoate (Isobutyl-Pip-Ac- PAla-pAla-OSu).

[0041] FIG. 21 illustrates the full mass spectrum results for the set of 6 mass labels as defined in Embodiment 1, showing a clean spectrum with no additional signals of relevant intensity and a visible mass shift of 4 daltons between each successive mass label in the set.

[0042] FIG. 22 illustrates the MS / MS spectrum of the unlabelled tryptic peptide HLVDEPQNLIK (3+) from bovine serum albumin.033945.00485

[0043] FIG. 23 illustrates the MS / MS spectrum of the mTRAQ labelled tryptic peptide HLVDEPQNLIK (3+) from bovine serum albumin.

[0044] FIG. 24 illustrates the MS / MS spectrum of the second generation plexDIA isotopic tag PD2-D0 439 labelled tryptic peptide HLVDEPQNLIK (3+) from bovine serum albumin. Sufficient structural ions are produced to allow confident sequence assignment.

[0045] FIG. 25 illustrates total ion chromatograms (TIC’ s) for HeLa tryptic digests unlabelled, or labelled with mTRAQ and PD2-D0 439.

[0046] FIG. 26 illustrates the precursor ion charge state distributions for HeLa tryptic digests unlabelled, or labelled with mTRAQ and PD2-D0 439.

[0047] FIG. 27 illustrates number of differently charged precursors, number of peptide spectral matches (PSM’s) and rates of sequence identifications for HeLa tryptic digests labelled with mTRAQ and PD2-D0 439.

[0048] FIG. 28 illustrates collision energy (nCE%) dependence of peptide sequence identification rates for precursors of different charge states in HeLa tryptic digests labelled with mTRAQ and PD2-D0 439.

[0049] FIG. 29 illustrates relative intensities of first (pD-iB-bAla) and second (PD2-D0 439) generation plexDIA tag fragments for precursors at different charge states for HeLa tryptic digests labelled with pD-iB-bAla and PD2-D0 439.

[0050] FIG. 30 illustrates relationship between nCE% and peptide sequence identification rates in a TIC of 3 rat liver / yeast lysate 6plex samples labelled with mTRAQ, pD-iB-bAla and PD2-D0 439.

[0051] FIG. 31 illustrates TICs of 3 rat liver / yeast lysate 6plex samples labelled with second generation plexDIA tags of Embodiment 1.

[0052] FIG. 32 illustrates results of 135-minute plexDIA analysis of three rat liver / yeast tryptic digests labelled with second generation plexDIA tags of Embodiment 1.033945.00485

[0053] FIG. 33 illustrates results of 35 -minute plexDIA analysis of three rat liver / yeast tryptic digests labelled with second generation plexDIA tags of Embodiment 1.

[0054] FIG. 34 illustrates quantitative accuracy for total yeast protein content of 135-minute plexDIA analysis of rat liver / yeast tryptic digests labelled with second generation plexDIA tags of Embodiment 1 using MSI precursor intensities.

[0055] FIG. 35 illustrates normalized ratios of yeast protein content from MSI precursor values in Fig. 34 where each of the channels is normalized to the PD2-D0_439 channel.

[0056] FIG. 36 illustrates quantitative accuracy for total yeast protein content of 135-minute plexDIA analysis of rat liver / yeast tryptic digests labelled with second generation plexDIA tags of Embodiment 1 using MS2 fragment intensities normalized to PD2-D0_439 channel.

[0057] FIG. 37 illustrates quantitative precision for total rat protein content of 135-minute plexDIA analysis of rat liver / yeast tryptic digests labelled with second generation plexDIA tags of Embodiment 1 using non-normalized MS2 fragment intensities.

[0058] FIG. 38 illustrates rates of protein identifications in a 6plex rat liver / yeast digest injected at amounts equivalent to 1 - 200 individual cells.

[0059] FIG. 39 illustrates rates of protein identifications for a 25 -minute DIA analysis of unlabeled single cell equivalent (250 pg) of rat liver / yeast digest compared with 35-minute DIA analysis of 6plex rat liver / yeast digest equivalent to 5 single cells and 20 cell trigger (6 ng), 5 cells + 35 cell trigger (10 ng) or 5 cells and 75 cell trigger (20 ng).Detailed DescriptionSets of Mass Labels

[0060] The present disclosure provides sets of isotopic reactive tags having mass differences in the range of millidaltons to several daltons for the purposes of labelling peptides and other biomolecules with potential multiplexing rates greatly in excess of 10-plex.033945.00485

[0061] The present disclosure also provides for methods of use of isotopic reactive tags that enable novel forms of analysis of labelled peptides, proteins and other biological molecules, particularly for the discovery of biologically significant differences between sets of biological samples.

[0062] In a first aspect, the present disclosure relates to a set of two or more mass labels, wherein each mass label comprises the formula:X-Re wherein:X is a mass coding moiety , andRe is a) a reactive functionality for attaching the mass label to an analyte or b) the analyte, wherein each mass label in the set has a unique mass, wherein the set comprises two or more mass labels, wherein each mass label has a coding moiety X comprising the following general formula:

[0063] The term “exact mass” refers to the theoretical mass of the mass label or of the coding moiety and is the sum of the exact masses of the individual isotopes of the entire mass label or coding moiety, e.g.12C=12.000000,13C=13.0033551H=1.007825,16O=15.994915. The “exact mass” takes account of mass defects.

[0064] In the literature the term “isotopic” often refers to species that have different masses and are not co-selectable for MS / MS but in the context of this disclosure the term “isotopic”033945.00485 refers to species that have different masses but which can still be co-selected for MS / MS using DIA.

[0065] The term “isotopologue” refers to a set of reagents having the same chemical structure but a different number of different isotopes. In the present invention, members of a set of mass labels are isotopologues.

[0066] The term “difference in exact mass between at least two of the mass labels” means the difference in exact mass between one label and the next heavier, or lighter member in the set of mass labels. In general, this will be the same value for all members of the set. In some instances, the difference in exact mass between at least two of the mass labels may be combinations of multiple daltons, single Dalton, and millidalton mass differences within a single set of mass labels.

[0067] The difference in exact mass between at least two of the mass labels in a set may be less than 100 millidaltons (“mDa”), less than 50 mDa, or less than 20 mDa. In some aspects, the difference in exact mass between at least two of the mass labels in a set is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa due to common isotope substitutions. For example, if a first label comprises a13C isotope, and in a second label this13C isotope is replaced by12C but a14N isotope is replaced by a15N isotope, the difference in exact mass between the two labels will be 6.3 mDa.

[0068] In the present specification, the term label is synonymous with the term tag.

[0069] It will be understood by the person skilled in the art that in order to achieve the desired masses for each tag within a set, one or both of the moieties X and the reactive functionality Re or the analyte may be modified with heavy isotopes. The heavy isotopes may be selected from2H,13C,15N or18O.

[0070] In some aspects, the aggregate molecular weight of the mass labels is 600 daltons or less, e.g., 500 daltons or less, 400 daltons or less, or from 300 to 500 daltons.033945.00485

[0071] The mass labels according to the disclosure are designed to be reacted with a biomolecule, such as a protein to form a labelled biomolecule, e.g. a labelled protein.

[0072] In some aspects, the bond between X and Re or between X and A comprises, without limitation, an amide bond, a urea linkage, an ester linkage or an ether linkage. In some aspects, the bond comprises an amide bond. In some aspects, the bond comprises a urea linkage . In some aspects, the bond comprises an ester linkage. In others, the bond comprises an ether bond. In some aspects, e.g., as illustrated by the mass label of Example 1, the bond comprises a carbon-carbon bond. In yet further aspects the bond is not cleavable by common fragmentation methods used in tandem mass spectrometry.

[0073] In some aspects, the mass tags additionally comprise a reactive functionality to allow the mass label to be conjugated to an analyte. The reactive functionality for attaching the mass label to the analyte is not especially limited and may comprise any appropriate reactive group.

[0074] The reactive functionality may react with an amino group on the analyte, for example the s-amino group of a lysine residue and the a-amino group of peptide or protein. In the simplest embodiments this may be an N-hydroxy succinimide ester or a tetrafluorophenyl ester. Other reactive functionalities are contemplated herein such as those which react with thiol groups in biological molecules. In particular these reactive functionalities are designed to react with the thiol group of a cysteine residue. Examples of reactive groups of the present disclosure which are able to react with cysteine residues are the maleimido, haloacetyl and 2- dithiopyridine groups. The thiol group of cysteine undergoes nucleophilic addition across the double bond of the maleimido group and undergoes nucleophilic substitution with the haloacetyl or 2-dithiopyridine group.

[0075] Reactive functionalities which are capable of reacting with carbonyl or hydroxyl groups in biological molecules are also contemplated herein. In particular, these reactive033945.00485 functionalities are designed to react with the carbonyl or hydroxyl groups of steroid hormones. Reactive groups of the present disclosure which are able to react with carbonyl or hydroxyl groups in a biological molecule are hydrazide or — CONH — (CH2)n — ONH2, wherein n is from 1 to 6 (e.g., wherein n is 3, i.e. aminoxypropyl amide). These groups react with carbonyl groups to form hydrazones or O-alkyloximes respectively. Examples of reactive functionalities are shown in WO 2011 / 036059, which reference is incorporated herein.

[0076] In some aspects, the reactive functionality is an N-hydroxy succinimide ester, a 2,3,5,6-tetrafluorophenyl ester or a sulphodichlorophenyl ester.

[0077] When the label has the structure X-A, A may comprise, e.g., amino acids, peptides, polypeptides, nucleotides, oligonucleotides, polynucleotides, carbohydrates, lipids, phospholipids or combination thereof.

[0078] In some aspects, mass labels according to the disclosure, and sets of two or more mass labels comprising heavy isotope mass series modifying groups, are illustrated by exemplary Embodiment 1 below.

[0079] Embodiment 1

[0080] The mass label has the structure:Exact Mass: 439.24Molecular Formula: C20H33N5O6 wherein * represents that oxygen may be18O, carbon may be13C, nitrogen may be15N, and hydrogen may be2H, and wherein one or more * may be present. For clarity, substitutable hydrogens on carbon are not represented by * but may be substituted with2H in any position.033945.00485

[0081] In some aspects, the mass modifier moiety * is13C or15N and the set comprises n=6 mass labels having the following structures:033945.00485Label: PD2-A20 459 Exact Mass: 459.28Molecular Formula: [13C]ieC4H33[15N]4NiO6

[0082] Embodiment 2

[0083] In some aspects, the mass modifier moiety * is13C or15N or2H and the set comprises n=9 mass labels having the following structures:033945.00485033945.00485Label: PD2-A28 467Molecular Formula: [13C]i6C4[2H]sH25[15N]4NiO6

[0084] In some aspects, a set of mass labels may comprise any of the fifteen structures shown in Embodiments 1 and 2 above, wherein one or more oxygen, carbon, nitrogen, and / or hydrogen components is replaced by an isotopic substituent. These alternatives offer a range of multiplexing levels for between 2 and 15 samples.033945.00485

[0085] Embodiment 3

[0086] In some aspects, the mass modifier moiety * is13C or15N or2H and the set comprises millidalton variants of each 4 Da variant tag from Embodiment 1 having the following structures:

[0087] Millidalton variants of PD2-A4 443

[0089] Millidalton variants of PD2-A 12 451033945.00485

[0091] It should be understood by the skilled practitioner that further millidalton variants may be possible using deuterium substitutions in place of any of the13C and15N substitutions represented in the examples above.

[0092] In a further embodiment, these millidalton variant PD2 tags may be combined with any of the tags provided in Embodiments 1 and 2 to provide higher multiplexing levels up to 30 and beyond.

[0093] Methods of Mass Spectrometry Analysis

[0094] The present disclosure also provides for a method of mass spectrometry analysis, which comprise quantifying an analyte by identifying by mass spectrometry ions with a mass-033945.00485 to-charge ratio equivalent to the analyte mass modified by the addition of one member of the set of isotopic mass labels, wherein the mass label is a mass label from a set of mass labels according to the disclosure, and wherein the analyte identification is obtained from data- independent acquisition mass spectrometry.

[0095] In some aspects, a method of mass spectrometry analysis comprises: a) providing a plurality of samples, wherein each sample is differentially labelled with an isotopic mass label or a combination of isotopic mass labels, each selected from a set of two or more isotopic mass labels as described herein; b) mixing the plurality of labelled samples to form an analysis mixture comprising labelled analytes; c) detecting the labelled analytes in amass spectrometer; d) isolating one or more labelled analytes in the mass spectrometer based on one or more isolation windows of >1 Da and then dissociating them to form labelled analyte fragments; e) detecting the analyte fragments; and f) identifying the one or more labelled analytes on the basis of the analyte fragments.

[0096] In some aspects, the one or more isolation windows comprise an isolation window that is <10 Da, <20 Da, <50 Da, and / or <100 Da.

[0097] In some aspects, the labelled analytes may be identified on the basis of the MS2 spectrum of the labelled analyte fragments and quantified using the MSI spectrum of the labelled analytes. Alternatively, they may be identified and quantified on the basis of the MS2 spectrum alone. In another aspect, the labelled analytes may be identified and quantified in MS2 spectra without first performing a survey (MSI) scan.

[0098] In some aspects, the dissociation in step d) is collision-induced dissociation in a mass spectrometer. In some aspects, the dissociation is collision induced dissociation in a mass spectrometer with a resolution of greater than 60,000 at a mass-to-charge ratio of 400, optionally a resolution of greater than 100,000 at a mass-to-charge ratio of 400, or a resolution greater than 250,000 at a mass-to-charge ratio of 400.033945.00485

[0099] The analytes may be identified on the basis of: i) the mass spectrum of the labelled analytes; or ii) the mass spectrum of the mass labels and / or analyte fragments comprising an intact mass label. When identification according to ii) occurs, the analyte fragment optionally comprises a b-series ion comprising an intact mass label (e.g., a bl ion).

[0100] Thus, in some aspects, the analytes may be identified on the basis of the mass spectrum of the labelled analytes.

[0101] In some aspects, the analytes may be identified on the basis of the mass spectrum of the mass labels and / or analyte fragments comprising an intact mass label. In some aspects, the analyte fragment comprising an intact mass label is a b-series ion comprising an intact mass label (e.g., a bl ion).

[0102] For the purposes of resolving all of the possible tags described herein, mass spectrometers with high resolution are required but the nature of the instruments is not particularly important to the practice of the methods described herein. Mass spectrometers using liner ion traps, quadrupoles, orbitraps and / or time-of-flight detectors are suitable for use with the tags of the present invention. Ions may be generated by electrospray or matrix assisted laser desorption ionization (MALDI) or any other ionization method known in the art. In addition, many of the tags that have been described in this application can still be resolved on instruments with only single dalton resolution as long as subsets of the possible tags that are separated by single dalton mass differences are used.

[0103] The mass labels and methods described herein are further explained by the following examples, which are merely illustrative and by no means limiting the scope of the invention claimed herein now or during prosecution of the present application.EXAMPLES

[0104] Example 1: Synthesis of (2,5-dioxopyrrolidin-l-yl) 3-[3-[[2-(4-isobutylpiperazin-l- yl)acetyl] amino]propanoylamino]propanoate .033945.00485

[0105] Step 1: Synthesis of Benzyl 3-aminopropanoate hydrotosylate (H-pAla-OBn * pTosOH) (FIG. 1).

[0106] A IL round bottom flask equipped with a Dean-Stark apparatus and a condenser is charged with 28.51g 3 -aminopropanoic acid (P-alanine), 199.64mL benzyl alcohol, 65.25g para-toluenesulfonic acid monohydrate-toluenesulfonic acid and 400 mL toluene. The reaction mixture is heated 5 hours under reflux and about 11 mL of water are separated, then about 200 mL toluene is distilled off. The solution is allowed to cool down, and at 45°C to 50°C 400 m L of diisopropyl ether is added slowly and the product starts to crystallize. The mixture is stirred for 1.5 hours, the temperature of the solution drops to 20°C-25°C and crystallization of the product goes to completion. The solid is fdtrated, washed with diisopropyl ether, and dried in vacuo to yield 111.08g product (0.316mol; 98.8%), as para-toluenesulfonate salt.

[0107] Step 2: Synthesis of 3-tert-butoxycarbonylaminopropionic acid (Boc-bAla-OH) (FIG. 2)

[0108] A solution of 16.32g sodium hydroxide in 163mL water is placed in the 2-L-two- neck flask. To this 15.0g 3 -aminopropanoic acid (P-alanine) and 150mL dioxane are added. A turbid solution is formed. Then, 53.47g di-tert-butyl-dicarbonate ((Boc)2O) are added. An exothermic reaction (55-57 °C) is observed, the pH value settles at about 9-10 and a solid precipitate. Stirring is continued for 5 hours. Leave to stand overnight and add 250mL water the next day so that a solution is formed. The reaction solution is washed twice with 250mL diisopropyl ether. The diisopropyl ether phases are discarded. The aqueous phase is adjusted to pH 3 with 2M hydrochloric acid (approx. 150mL) and the product is extracted 3 times with 200mL dichloromethane. The organic phases are dried over magnesium sulphate, fdtered, and concentrated to yield 29.78g product (0.155mol; 95.1%)

[0109] Step 3: Synthesis of 2-(4-tert-butoxy carbonylpiperazin- 1-yl) acetic acid (Boc-Pip- Ac-OH).033945.00485

[0110] Option 1: no heavy isotopes in piperazine ring.

[0111] Step 3,1: Synthesis of tert-butyl 4-(2-benzyloxy-2-oxo-ethyl)piperazine-l- carboxylate (Boc-Pip-Ac-OBn) (FIG. 3a).

[0112] Dissolve 20.0g Boc-piperazine in 200mL acetonitrile, add 15.3mL diisopropylethylamine (DIPEA), then slowly add 25.8g bromoacetic acid benzyl ester, thereby the temperature rises to 35°C. Stir for 3h at Rt. Dilute with 700mL ethyl acetate, wash twice with saturated NaHCO3 solution, dry and concentrate to yield 35.7g product as colorless oil (0.106mol; 99%).

[0113] Step 3,2: Synthesis of 2-(4-tert-butoxycarbonylpiperazin-l-yl) acetic acid (Boc- Pip-Ac-OH) (Fig 3b).

[0114] 1.75g 5%-Pd / C catalyst and 150mL methanol are placed under argon in a hydrogenation apparatus. Then, 35.7g Boc-Pip-Ac-OBn, dissolved in 50mL methanol are added, and hydrogenated at 25 °C until hydrogen uptake is complete. The reaction mixture is fdtered through Dicalite fdter aid to remove the Pd / C catalyst and the fdter residue is washed with methanol. The fdtrate is concentrated to dryness in vacuo to yield 25.3g of the product as white powder (0.104mol, 97%).

[0115] Option 2: heavy isotopes in piperazine ring.

[0116] The synthetic pathway is shown with heavy-isotope labeled versions of glycine and2-bromoacetic acid.

[0117] Step 3, 11: Synthesis of Methyl 2-aminoacetate hydrochloride (H-Gly-OMe * HC1)(FIG. 4)

[0118] Place 14.8g 2-aminoacetic acid (glycine) in lOOmL methanol in a 500mL three- neck flask. Add 33.8g thionyl chloride to the suspension within 20 minutes. Exothermic reaction, the temperature rises to 55°C. Then stir at reflux for approx. 2 hours, after approx. 1.5 hours a clear solution is obtained. The reaction solution is cooled to room temperature, thereby033945.00485 the reaction product precipitates. Add 300 mL diisopropyl ether and stir for approx. 30 minutes. The solid is filtrated, washed with diisopropyl ether, and dried in vacuo to yield 24.35g (0.189mol, 100% of theory) white crystals, as hydrochloride salt.

[0119] Step 3, 12: Synthesis of 2-(tert-Butoxy carbonylamino) acetic acid (Boc-Gly-OH) (FIG. 5)

[0120] A solution of 9.92g sodium hydroxide in HOmL water is placed in the 1-L-two- neck flask. To this, 9.21g 2-aminoacetic acid (glycine) and 92mL dioxane are added. A turbid solution is formed. Then, 36.01g di-tert-butyl-dicarbonate ((Boc)2O) are added. An exothermic reaction (55-57 °C) is observed, the pH value settles at about 9-10 and a solid precipitate. Stirring is continued for 5 hours. Leave to stand overnight and add 300mL water the next day so that a solution is formed. The reaction solution is washed twice with 250mL diisopropyl ether. The diisopropyl ether phases are discarded. The aqueous phase is adjusted to pH 3 with 2M hydrochloric acid and the product is extracted 3 times with 200mL ethyl acetate. The organic phases are dried over magnesium sulphate, filtered, and concentrated to yield 20.53g product as white solid (0.115mol; 97.7%)

[0121] Step 3, 13: Synthesis of Methyl 2-(benzylamino)acetate hydrochloride (H-(Bzl)Gly- OMe * HC1) (FIG. 6).

[0122] 24.29g (H-Gly-OMe * HC1) are placed in 120mL methanol in a IL three-neck flask.Add 26.88g diisopropylethylamine (DIPEA) and 21.12g benzaldehyde and stir for 1.5 hours at room temperature. A clear solution is formed, and the reaction solution is cooled to 0°C to 10°C. Then, 7.87g sodium borohydrate are added, the temperature should be kept between 0 and 15 °C. Stir for 3 hours and allow to come to room temperature. Leave to stand overnight. The next day, the reaction solution is poured onto 600 mL saturated sodium hydrogen carbonate solution and extracted with 300 mL ethyl acetate. The aqueous phase is saturated with sodium chloride and extracted twice with 200mL ethyl acetate. The organic layers are combined and033945.00485 washed with 500mL saturated sodium chloride solution, dried over magnesium sulphate, filtrated and concentrated. The residue is dissolved in 30mL diisopropyl ether and 57mL of 4M HC1 in dioxane are added. The hydrochloride salt of the product precipitates. 150mL diisopropyl ether are added and the mixture stirred for 1 hour. Then, the product is filtrated, washed with diisopropyl ether and dried. The product is then suspended with acetone and stirred for 30min, filtrated and dried in vacuo. 28.57g (0.131mol; 69.1%) white crystals are yielded.

[0123] Step 3, 14: Synthesis of Methyl 2-[benzyl-[2-(tert-butoxycarbonylamino)-acetyl] amino] acetate (Boc-Gly-(Bzl)Gly-OMe) (FIG. 7).

[0124] 20.53g Boc-Gly-OH are added to lOOmL of A. A-dimcthylfonnamidc in a 500mL round bottom flask. 44.59g diisopropylethylamine (DIPEA), 27.11g (H-(Bzl)Gly-OMe * HC1 and 24.65g 1 -hydroxybenzotriazole monohydrate (HOBt) are added. A clear solution is formed and 28.76g l-ethyl-3 -(3 -dimethyl amino-propyl)-carbodiimide hydrochloride (EDC) are added. Stir for 1.5 hours after addition. Then, the reaction solution is diluted with 400mL ethyl acetate and washed twice with 700mL semi-saturated sodium hydrogen carbonate solution, once with 400mL water and once with 400mL saturated sodium chloride solution. The organic phase is dried over magnesium sulphate, filtered and concentrated, to yield 37.87g product as white solid (0.111 mol, 96.2%).

[0125] Step 3, 15: Synthesis of l-Benzylpiperazin-2, 5-dione (FIG. 8).

[0126] 37.87g Boc-Gly-(Bzl)Gly-OMe are dissolved in 146mL dichloromethane in a500mL round bottom flask. Add 73mL 4M HC1 in dioxane are added. The reaction is slightly exothermic reaction and gas formation starts. The reaction is stirred for 2.5 hours, and reaction progress is monitored by HPLC analysis. Once completed, the reaction solution is concentrated. The residue is dissolved in 100mL tetrahydrofuran and 37.9mL diisopropylethylamine (DIPEA) are added. While stirring as room temperature, the reaction033945.00485 product precipitates. The reaction mixture is stirred for 30 minutes and left to stand overnight. The next day, the precipitate is filtrated, washed with dichloromethane and dried in vacuo. 20.39g of the product are yielded as white crystals (97.0mmol; 87.39% of theory).

[0127] Step 3, 16: Synthesis of 1 -Benzylpiperazine Dihydrochloride (FIG. 9).

[0128] Suspend 14.73g lithium aluminium hydride in 175mL tetrahydrofuran in a IL four- neck flask. 20.39g l-Benzylpiperazin-2,5-dione are added in several portions at under cooling at 0°C to 10°C. The reaction is exothermic and gas formation is observed. After the addition is complete, the cooling is removed. The temperature rises to approx. 45°C. The reaction is then stirred at reflux for 1.5 hours, the progress of the reaction is controlled by thin-layer chromatography. Then, the reaction solution is hydrolyzed at -10°C to 10°C with 17mL water, 51mL 15% sodium hydroxide solution and 17mL water. Stir for 1.5 hours and allow to warm to room temperature. The forming solid is removed by filter aid and washed with tetrahydrofuran. The filtrate is concentrated in a rotary evaporator. The residue is dissolved in 50ml diisopropyl ether and 61mL 4M HC1 in dioxane are added under cooling with an ice bath to generate the dihydrochloride. The solid is filtrated, washed with acetone and diisopropyl ether and dried in vacuo, to yield the product as yellowish powder, 22.85g (89.6mol; 92.3%).

[0129] Step 3, 17: Synthesis of Benzyl 2-bromacetate (FIG. 10).

[0130] Place 10g 2-bromoacetic acid in 150mL diisopropyl ether in a IL three-neck flask. Add 8.6g benzyl alcohol and 0.21g 4-dimethylaminopyridine (DMAP). Then cool to approx. - 10°C and add a solution of 16.25g N, A ’-dicyclohexyl carbodiimide in 75mL diisopropyl ether within 30 minutes. Stir for approx. 3 hours and allow the mixture to come to room temperature. The precipitated DCC-urea is filtered off and washed with diisopropyl ether. Add 75 mL of 5% citric acid to the filtrate and stir for 30 minutes. The layer is separated and the organic phase is washed twice with 200mL saturated sodium hydrogen carbonate solution, once with 200mL water, dried over magnesium sulphate, filtered and concentrated in vacuo. The residue is033945.00485 purified by chromatography on silica gel with n-hexane and n-hexane / diisopropyl ether 9: 1 to yield 15.1g product as colorless oil (65.56mmol, 92.3%).

[0131] Step 3, 18: Synthesis of Benzyl 2-(4-benzyl piperazin- 1-yl) acetate (FIG. 11).

[0132] 15.93g 1 -benzylpiperazine dihydrochloride are placed in a 500mL three-neck flask and 125mL acetonitrile are added to give a suspension. 42.64mL diisopropylethylamine (DIPEA) are added. A clear solution is formed and 15.1g benzyl 2-bromacetate are added. The temperature should be kept at 25-30°C. Then stir for 2 hours at room temperature. Then, approx. 75% of the acetonitrile is removed with a rotary evaporator. Add 250mL ethyl acetate and wash twice with 250mL saturated sodium hydrogen carbonate solution and once with 250mL saturated sodium chloride solution, dry over magnesium sulphate, filter and concentrate. (20.39g; 98.27%) The residue is purified by filtration on silica gel with approx. 500mL ethyl acetate. The product is yielded as yellow oil, 19.08g (57.41mmol; 91.9%) yellow oil.

[0133] Step 3, 19: Synthesis of 2-Piperazin-l-yl acetic acid (FIG. 12).

[0134] 0.38g 5%-Pd / C catalyst and 30.0mL methanol are placed in a hydrogenation apparatus under argon. Then 19.0g benzyl 2-(4-benzyl piperazin- 1-yl) acetate dissolved in 80mL methanol is added and hydrogenated at 25°C until hydrogen uptake is stopped, about half of the theoretical hydrogen amount of ~5 ,4L has been consumed. Then, the temperature is raised to 45°C to 55°C to initiate the removal of the second benzyl group. The second half of the hydrogen is consumed over a period of about 6 hours. After the hydrogen uptake is complete, the reaction is stirred for another 1 hour at room temperature. The reaction mixture is filtered through a filter aid to remove the Pd / C catalyst and the filter residue is washed with 1.5 litres of hot methanol. The filtrate is concentrated in vacuo to yield 7.7g of the product as yellowish solid (50.61mmol, 88.5%)

[0135] Step 3,20: Synthesis of 2-(4-tert-Butoxy carbonylpiperazin- 1-yl) acetic acid (Boc- Pip-Ac-OH) (FIG. 13).033945.00485

[0136] Place 7.7g 2-piperazin-l-yl acetic acid in a 500mL round bottom flask and add 77mL methanol to give a suspension. 7.2g 42.64mL diisopropylethylamine (DIPEA) are added, a clear solution is formed. Then add 11.6g di-tert-butyl-dicarbonate ((Boc)2O). The reaction is slightly exothermic and gas formation is observed. When the gas formation is complete, stir for approx. 1 hour. Then, the reaction solution is concentrated on a rotary evaporator. The residue is stirred with 50mL methyl tert-butyl ether, filtrated, washed with methyl tert-butyl ether and dried in vacuo. 11 ,26g of the product is yielded as yellowish powder (44.66 mmol; 88.17%).

[0137] Step 4: Synthesis of Benzyl 3-[3-(tert-butoxycarbonylamino) propanoylamino] propanoate (Boc-pAla-pAla-OBn) (FIG. 14).

[0138] In a 500mL flask, 12.71g Boc-bAla-OH, 20.78mL diisopropylethylamine (DIPEA), 21.70g H-pAla-OBn * pTosOH and 13.09g 1 -hydroxybenzotriazole monohydrate (HOBt) are added to about 125mL tetrahydrofuran, whereby a suspension is formed. Add 15.22g of 1- ethyl-3 -(3 -dimethyl amino-propyl)-carbodiimide hydrochloride (EDC) and the initial suspension goes into solution within 15-20 minutes and the temperature rises to about 30°C. Then stir for 7 hours at 25 °C and leave the mixture overnight at room temperature. Then, the reaction solution is concentrated on the rotary evaporator and the residue is dissolved in 200mL ethyl acetate. It is washed two times with 300mL saturated sodium hydrogen carbonate solution and once with 200mL semi-saturated sodium chloride solution. The ethyl acetate phase is dried over MgSO4, filtered over silica gel, and concentrated. A solid white residue remains as product, to yield 20.88g (58.3mmol; 95.4%).

[0139] Step 5: Synthesis of Benzyl 3-(3-aminopropanoylamino)propanoate hydrochloride (H-pAla-pAla-OBn*HCl) (FIG. 15).

[0140] Dissolve 7.18g Boc-bAla-bAla-OBn in 26mL dichloromethane and add 13mL 4M HC1 in dioxane. Gas evolution starts and stirring is carried out for about 2 hours at 25 °C. The033945.00485 reaction is monitored by HPLC-assisted reversed phase chromatography. After completion, the solution is concentrated on a rotary evaporator and dried, to yield the product quantitatively (5.89 g, 100%).

[0141] Step 6: Synthesis of tert-Butyl 4-[2-[[3-[(3-benzyloxy-3-oxo-propyl) amino]-3- oxo-propyl] amino] -2 -oxo-ethyl] piperazine- 1 -carboxylate (Boc-Pip-Ac-pAla-(3Ala-OBn) (FIG. 16)

[0142] 5.37g Boc-Pip-Ac-OH are dissolved in 60mL tetrahydrofuran. Add 5.89g H-[3Ala-PAla-OBn*HCl, 7.32mL diisopropylethylamine (DIPEA) and 4.29g 1 -hydroxybenzotriazole monohydrate (HOBt). Stir for 30 minutes and then add 4.98g l-ethyl-3-(3-dimethyl amino- propyl)-carbodiimide hydrochloride (EDC), thereby the temperature rises to approx. 40°C. Then stir for 5 hours at 25 °C and leave the mixture overnight at room temperature.

[0143] Remove approx, of the tetrahydrofuran on the rotary evaporator and add 150 mb of ethyl acetate to the residue. It is washed three times with lOOmL saturated sodium hydrogen carbonate solution and once with 150mL semi-saturated sodium chloride solution. The ethyl acetate phase is dried over MgSO4, filtered over silica gel, and concentrated. The residue is purified by chromatography on silica gel with ethyl acetate and ethyl acetate / methal 10: 1 as eluant. The product is yielded as yellowish oi, 9.23g (19.05mmol; 95.3%).

[0144] Step 7: Synthesis of Benzyl 3-[3-[(2-piperazin-l-ylacetyl)amino]- propanoylamino]- propanoate Hydrochloride (H-Pip-Ac-pAla-pAla-OBn*HCl) (FIG. 17).

[0145] Dissolve 5.38g Boc-Pip-Ac-bAla-bAla-OBn in 15mL mb dichloromethane and add 7.5 mb 4M HC1 in dioxane. Gas evolution starts and stirring is carried out for about 2h ours at 25 °C. The reaction is monitored by HPEC -assisted reversed phase chromatography. After completion, the solution is concentrated on a rotary evaporator and dried, to yield the product quantitatively (4.67g, 100%).033945.00485

[0146] Step 8: Synthesis of Benzyl 3-[3-[[2-(4-isobutylpiperazin-l-yl)acetyl]amino]- propanoylamino]propanoate (Isobutyl -Pip-Ac-pAla-pAla-OBn) (FIG. 18).

[0147] 4.67g H-pAla-pAla-pAla-OBn*HCl are dissolved in 50 mb of 1,2-dichloroethane under argon. Then, 3.01mL diisopropylethylamine (DIPEA) and 1.04mL isobutyraldehyde are added and stirred for 30 minutes to give a clear solution. Then, 4.71g of sodium triacetoxyborohydride are added, the temperature rises to about 40°C. Stirring is continued overnight at 25 °C. Then, remove about half of the 1,2-dichloroethane at the rotary evaporator and add lOOmL ethyl acetate to the residue. The organic layer is washed twice with lOOmL saturated sodium hydrogen carbonate solution and once with lOOmL semi-saturated sodium chloride solution, dried with MgSO4, fdtered over silica gel with approx. 500mL ethyl acetate and approx. 500mL ethyl acetate methanol 10: 1 and concentrated. 3.86g of the product is yielded as solid (8.68mmol, 78.2%).

[0148] Step 9: Synthesis of 3-[3-[[2-(4-isobutylpiperazin-l-yl)acetyl]amino]- propanoylamino]propanoate (Isobutyl-Pip-Ac-pAla-pAla-OH) (FIG. 19).

[0149] 0.19g 5%-Pd / C catalyst and 5mL methanol are placed under argon in a hydrogenation apparatus. Then, 3.86g Isobutyl-Pip-Ac-pAla-pAla-OBn, dissolved in 5mL methanol are added, and hydrogenated at 25 °C until hydrogen uptake is complete. The reaction mixture is filtered through Dicalite filter aid to remove the Pd / C catalyst and the filter residue is washed with methanol. The filtrate is concentrated to dryness in vacuo. The solid residue is suspended in methyl tert-butyl ether and stirred for 20min, filtrated, washed with methyl tertbutyl ether and dried in vacuo to yield 3.00g of the product as yellowish powder (8.56 mmol, 98.6%).

[0150] Step 10: Synthesis of (2,5-Dioxopyrrolidin-l-yl) 3-[3-[[2-(4-isobutylpiperazin-l- yl)-acetyl] amino] propanoylamino] propanoate (Isobutyl-Pip-Ac-pAla-pAla-OSu) (FIG. 20).033945.00485

[0151] In a 100 mL round botom flask, dissolve 0.70g Isobutyl-Pip-Ac-pAla-[3Ala-OH in 5 mL tetrahydrofuran at approx. 40-50°C. Add 0.27g A-hydroxysuccinimidc (NHS) and 0.48g N,N^ -dicyclohexyl carbodiimide (DCC) (3). The resulting DCC-based urea precipitates and the reaction is stirred for 1 hour at room temperature. The reaction solution is filtrated via filter aid, the residue is washed with tetrahydrofuran and the filtrate is concentrated on the rotary evaporator. The residue is dissolved in 2.7 mL ethyl acetate and 2.7 mL diisopropyl ether is added. The solution is stored overnight in a refrigerator at 4°C, whereby the product precipitates. The solid is isolated via a reverse frit, washed with diisopropyl ether and dried in vacuo. 0.79g of a yellowish powder is yielded (1.77 mmol; 88.3 % of theory).

[0152] Example 2: Synthesis of additional isotopic variants of (2,5-dioxopyrrolidin-l-yl) 3- [3- [ [2-(4-isobutylpiperazin-l-yl)acetyl] amino] propanoylaminojpropanoate PD2-D0 439).

[0153] After synthesis of undoped PD2-D0_439, we repeated synthesis with different precursors doped at different C, N & H positions as shown in Embodiments 1 - 3. The synthesis conditions were identical in all other respects. The final products were infused into an Orbitrap Fusion™ Tribrid™ mass spectrometer [Thermo Scientific] and the precursor mass and peak purity assessed (FIG. 21). All tags showed the expected mass and exhibited very good purity.

[0154] Example 3: Comparison of PD2-D0_439-labelled, mTRAQ-labelled and unlabelled BSA digests using data-dependent acquisition (DDA) mass spectrometry.

[0155] To demonstrate protein labeling and the effect of the new plexDIA tags on peptide fragmentation behaviour, we took 150 pmol of bovine serum albumin tryptic digest [New England Biolabs P8108S]. Peptides were labelled with PD2-D0 439 tag by Ih incubation in 100 mM TEAB buffer. Labelling was quenched with addition of 0.25% hydroxylamine solution and labelled peptides were purified by StageTip and eluates dried to completion using a SpeedVac centrifuge. In parallel, we prepared mTRAQ® [Applied Biosystems Inc.] labelled033945.00485BSA digests according to manufacturer’s instructions, and an unlabelled aliquot for comparison.

[0156] All three BSA digests were analyzed on an Orbitrap Fusion™ Tribrid™ mass spectrometer with on-line EASY-nLC™ 1200 UHPLC chromatography system [all Thermo Scientific] with spectra collected in data-dependent acquisition mode. The MS / MS spectra of a typical BSA unlabeled peptide (FIG. 22) was compared with the mTRAQ (FIG. 23) and PD2-D0_439-labled equivalent (FIG. 24) showing increased generation of informative structural ions to allow confident sequence assignment by PD2-D0 439 labelling.

[0157] Example 4: Analysis of HeLa tryptic digests using the PD2-D0 439 and mTRAQ labelling with data-dependent acquisition.

[0158]

[0159] Three replicate aliquots of commercially available HeLa tryptic digests (Thermo Scientific) were labelled with either PD2-D0_439 or mTRAQ tags (as described above) or left unlabeled for comparison. Samples were individually analysed by LC-MS / MS on an Orbitrap Exploris™ 480 mass spectrometer (Thermo Scientific) with on-line EASY-nLC™ 1200 UHPLC system. Peptides were eluted with an active acetonitrile gradient over 45 minutes with a range of different collision energies and using data-dependent acquisition. MS 1 survey scans were collected at 60K resolution and MS / MS spectra acquired in DIA mode with 10 Da isolation windows. The raw MS data file was searched in Proteome Discoverer software (v3.0) using the SEQUEST-HT node and UniprotKB H. sapiens library.

[0160] The total ion chromatograms for all three samples were similar (FIG. 25) suggesting the expected slight increase in retention times for labelled peptides. Labelled samples also produced a higher percentage of ions with charge states >+3 and surprisingly the tags showed even lower percentage of +2 ions compared to mTRAQ. (FIG. 26). The current tags also outperformed mTRAQ in terms of identification rates for 3+ to 5+ charge states, though was less successful in identifying the sequence of +2 ions (FIG. 27).033945.00485

[0161] We also defined the optimal collision energy (nCE %) for identification rates of the current tags compared to mTRAQ (FIG. 28). The rates for 3+ ions were similar between the tags at all collision energies tested, whereas the PD2-D0_439 labelled peptides with 4+ and 5+ charge states were superior to mTRAQ at all nCE% tested. Conversely, mTRAQ identification rates for 2+ ions were similar between nCE% values of 25 - 35 whilst the new tags required a higher nCE5 of 40 for maximal identifications, although this still remained below mTRAQ.

[0162] As the new tags require higher nCE% than previous tags, we also investigated the relative intensity of the main tag fragment (reporter ion) as a function of nCE%. For plexDIA it is preferable to keep the level of tag fragments as low as possible. As expected, this increased in line with nCE%, but remained below 20% of the total ion current at nCE% 35 which was defined as the preferred value for PD2-D0 439 labelled peptides. This was vastly superior to our previous plexDIA tags pD-iB-bAla described in US provisional 63 / 458629 (FIG. 29). Unfortunately, we were unable to compare this for mTRAQ as the reporter tag mass is below our scan range.

[0163] Example 5: Analysis of HeLa tryptic digests using the PD2-D0 439, pD-iB- bAla and mTRAQ labelling with data-independent acquisition.

[0164] To evaluate the performance of our new tags we analysed the same PD2-D0_439, pD-iB-bAla and mTRAQ labelled peptides using a 90-minute DIA method on an Exploris™ 480 mass spectrometer with the same LC system as described above. Replicate runs were performed using different isolation windows and nCE% values. For all tags, the best performance was seen at nCE% 25 with similar numbers of peptide identification from all tags. The fall in rate if identifications with increasing nCE% was similar between PD2-D0_439 and mTRAQ labelled samples, which were both much less affected than pD-iB-bAla-labelled samples (FIG. 30).033945.00485

[0165] Example 6: Preparation of rat and yeast spectral libraries for data- independent acquisition mass spectrometry.

[0166] Prior to performing multiplexed analysis of biological samples by data-independent acquisition (plexDIA) we first constructed spectral libraries to support peptide identification. In one aspect we created in silico predicted libraries using DIA-NN 1.9 software based on both rat (Uniprot KB R. norvegicus) and yeast (Uniprot KB .S', cerevisiae) protein sequence libraries. Base libraries were constructed for unlabelled peptides with fixed modification of N-termini and lysine by PD2-D0-439 (+324.21 Da). Variants were created for each of the other 5 tags from Embodiment 1 i.e. PD2-D4-443 (+4.01 Da), PD2-D8-447 (+8.01 Da), PD2-D12-451 (+12.03 Da), PD2-D16-455 (+16.03 Da) and PD2-D20-459 (+20.04 Da).

[0167] Separately, we have prepared an empirical focused database using narrow-window DIA. A mixed sample of rat liver (100) and yeast (6) lysates were labelled with PD2-D0-439 and analyzed on an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Scientific) with DIA windows of 2.5 - 5 Da and scan ranges from 300 - 1,000 Da. Raw files were processed in DIA-NN against the full rat and yeast in silico database and the resulting PD2-D0-439- labelled libraries saved in DIA format.

[0168] Example 7: Preparation of rat liver and yeast lysate plexDIA samples.

[0169] To demonstrate protein labeling, we prepared a lysate of frozen rat liver in UEPPS buffer (8M urea, 200 mM EPPS pH8.5, cOmplete™ Mini protease inhibitor cocktail (Roche)), which was then reduced (5 mM dithiothreitol), alkylated (14 mM iodoacetamide), diluted to 1 ,6M urea and then digested with trypsin (Promega).

[0170] Yeast lysate was purchased from Promega and diluted to 1.6M urea, then reduced (5 mM dithiothreitol), alkylated (14 mM iodoacetamide), and digested with trypsin (Promega). All generated peptides were desalted (SepPak tC18 cartridges, Waters) and dried under vacuum.

[0171] Volumes corresponding to 200 pg of rat liver lysate digests were spiked at five different concentrations with yeast digest (0%, 4%, 6%, 10%, 14%) to generate 18 samples as033945.00485 shown in the study design (Table 1). Each rat liver / yeast digest sample was mixed individually with one of the six PD2 reagents of Embodiment 1 (110 mM stock) at a final concentration of 16.5 mM and incubated for 1 hour at room temperature with shaking. The reactions were stopped by adding 0.25% hydroxylamine for 15 min at room temperature.

[0172] 75% of the individually labelled peptides were pooled at a ratio of 1: 1: 1: 1: 1: 1 to generate three 6plexes (PlexOl, Plex02, Plex03), half of which was then purified by tandem solid phase extraction (Oasis HLB (Waters) and SCX (SP Sepharose Fast Flow, Sigma)). To generate Plex04, a surrogate of a multiplexed single cell proteomics sample with 20 cell carrier, the individually labelled samples #1-6 were mixed at a ratio of 1: 1: 1: 1: 1:20 and purified completely by tandem SPE (HLB / SCX) as above. The bulk was then diluted to provide a final total protein amount of 0.5 pg / ml or 5 pg / ml respectively. The remainders of each individually labelled peptide sample were likewise purified by tandem SPE (HLB / SCX). All eluates were dried to completion under vacuum before transfer to LC / MS analysis.

[0173] Table 1. Study design

[0174] PlexOl

[0175] Plex02:033945.00485

[0176] Plex03

[0177] Example 8: DIA Mass Spectrometry of bulk rat liver / yeast plexDIA samples

[0178] Each of the PlexOl, Plex02 and Plex03, representing standard cell or tissue experiments were analysed by injecting 800 ng total labelled peptides onto a 25cm lonOpticks Aurora Ultimate column and eluted with increasing acetonitrile into an Orbitrap Exploris™ 480 mass spectrometer over 135 minutes with data collected in DIA mode with a scan range of 400 - 1,000 Da and variable window of 10 - 50 Da. Total Ion Chromatograms are shown in FIG. 31 and were consistent between the three different plexes. Raw fdes were processed in DIA-NN software vl.9.1 using the empirical focused database. On average a total of 3,054 proteins were identified from 100,361 precursors (FIG. 32). This equates to a daily throughput of 50 samples.

[0179] We also explored faster acquisition enabling a throughput of 180 samples per day with the 6plexDIA tags of Embodiment 1. Here 200 ng of protein were injected onto an 8 cm lonOpticks Rapid column and eluted as above using a shortened 35-minute gradient into the Exploris™ 480. Raw files were processed as above resulting in an average of 1,913 protein identifications from 51,912 precursors (FIG. 33).

[0180] Using data from the 135-minute run on the Exploris™ 480 we investigated quantitative accuracy by calculating the total intensity of unique yeast peptide precursors compared to the unique rat peptide precursors across the 3 x plexDIA samples from Example 7. Based on quantification at the MSI level the observed ratios were close to theoretical at -033945.004850.58 (4% yeast), 0 (6% yeast), 0.74 (10% yeast) and 1.22 (14% yeast). The raw data are shown in FIG. 34, and the same data normalized to the 6% yeast spike in PD2-D0_439 channel in each plex is shown in FIG. 35.

[0181] We also explored the quantitative accuracy of MS2-level quantification using the same DIA data. As seen with MSI -level quantification, the observed ratios of yeast precursors to rat precursors were close to theoretical. There was no obvious bias for any individual tag as shown by consistency of precursor intensities for each pair of yeast spike values in each of the 3 x plexDIA samples (FIG. 36)

[0182] Finally, we assessed the relative abundance of the rat proteome, where we expect all channels to have the same approximate total intensity. Across the three plexDIA samples the summed MS2 abundances were highly consistent and showed no inherent bias for higher or lower intensity for any of the plexDIA tags (FIG. 37).

[0183] Example 9: DIA Mass Spectrometry of pseudo single cell proteomics rat liver / yeast plexDIA samples

[0184] To investigate the utility of the plexDIA tags for analysis of single cell proteomes we prepared a sixplex sample using the previously prepared rat / yeast lysate mixtures from PlexOl of Example 7. In this case we mixed equal amounts of the PD2-D0 439 thru PD2- D16_455 labelled samples. We then added 20x the amount of the PD2-D20_459 sample to replicate a 20-cell trigger (Table 2).

[0185] Table 2. plexDIA sample for pseudo single cell proteomics analysis.

[0186] Plex04033945.00485

[0187] We initially tested different loading amounts of Plex04 on an 8-cm Ion Opticks Rapid column with a 35-minute DIA run, equivalent to analyzing 36 plexDIA samples per day. This represents 180 individual cells per day. For injection amounts of 0.5 - 6 ng we used a stock Plex04 at 0.5 ng / pl. We also prepared injection amounts of 6 - 50 ng using a Plex04 stock at 5 ng / pl. We estimated that HeLa cells have 250 pg total protein thus a plexDIA sample with 5 individual cells plus 20-cell carrier channel would contain approximately 6.25 ng. Raw mass spectrometer fdes were analyzed using DIA-NN v 1.9.1 searching against both in silico and empirical reference libraries.

[0188] As expected, the number of identified precursors and proteins increased with loading amount (FIG. 38), although the rate of increased declined rapidly above 6ng loaded on column, potentially reflecting the capacity of the smaller column used for high-throughput analysis. Interestingly, we saw that the 6 ng sample injected in 1.2 pl yielded approximately 16% higher protein identifications than for the equivalent amount loaded in 10 times the volume. This confirms that the tags are suitable for use for samples with very low total protein content.

[0189] We compared the protein identification rates using Plex04 at different loading amounts with 35-minute DIA to unlabeled Rat liver pus yeast spike (100:6.67) loaded at 250 pg with a 25-minute DIA run (equal to 48 cells per day). Using the same search parameters against the focused experimental reference library the unlabeled sample produced an average of 620 protein identifications compared to 1,373 for the plexDIA sample searched against the empirical reference library (FIG. 39).

[0190] Example 10: Time-of-flight DIA mass spectrometry analysis of human brain lysate spiked with yeast lysate

[0191] We further tested the performance of the 2ndgeneration plexDIA tags using a complex sample comprising human brain tissue lysate spiked with different concentrations of033945.00485 yeast digest on a fast asymmetric TOF mass spectrometer (Astral, Thermo Scientific). Six samples were prepared with constant human brain lysate spiked with different concentrations of a bulk lysate of .S' cerevisiae (Table 3).

[0192] Table 3. plexDIA samples for analysis on Orbitrap Astral mass spectrometer

[0193] Samples were mixed to form a single 6plex which was then analyzed by DIA on an Orbitrap Astral mass spectrometer with peptides loaded onto a Vanquish NEO LC system and eluted over 60 minutes. Two replicate runs were performed with different DIA window settings (Table 4).

[0194] Table 4. Run conditions for plexDIA analysis on Astral TOF.ScanOverlap, Range, Scan Max Inj Duty CycleWindow, Da Da m / z Events Time, ms approx, ms0.5 0.25 450 - 850 800 2.5 36001 0 400 - 1000 600 3.5 33001 0.25 400-1000 600 3.5 33002 0.5 360 - 1000 320 8 32004 0.5 360 - 1000 160 18 32008 1 360 - 1000 80 25 2160

[0195] Acquired raw data fries were processed with DIA-NN 1.9 software with searching performed against empirical spectral libraries prepared from separate human brain and yeast lysates labelled with the PD2-D0_439 tag. We used both MSI (Orbitrap, 240K resolution) and MS2 (Astral analyzer) quantification to assess quantitative accuracy of the yeast proteins and found no major differences compared with data collected on an an Exploris™ 480 Orbitrap system.033945.00485

[0196] The total number of precursors identified and quantified for yeast and human proteins were predictably higher using the Astral analyzer (Table 5) compared to Exploris 480 Orbitrap despite each Astral run being 50% shorter than the equivalent Exploris.

[0197] Table 5. Number of identified (yeast and human) and quantified (yeast) precursors and proteins in isotopically labeled sixplex sample analyzed on Orbitrap Astral Mass Spectrometer.

[0198] Example 11 - Synthesis of three additional isotopic tags with 2 Da spacing

[0199] To further exemplify the ability of modem mass spectrometers to resolve isotopically tagged peptides at 2 Da resolution we synthesized three additional tags using the same synthetic strategy as in Example 1 but using different precursors. The structures of the tags are:Label: PD2-A2 441Molecular Formula: [13C]2Ci8H33NsO6033945.00485Label: PD2-A10 449Molecular Formula: [13C]9CiiH33[15N]N4Oe

[0200] Synthesis of all three tags was achieved with comparable yields and high purity determined by HPLC. Masses were confirmed by mass spectrometry.

Claims

033945.00485CLAIMSWE CLAIM:

1. A set of two or more isotopic mass labels, wherein each label comprises the formula:X-Re wherein:X is an isochemic structure having a unique mass relative to all other members of the set coding moiety having an exact mass, andRe is a reactive functionality for attaching the mass label to an analyte or the analyte, and wherein X comprises the following general formula:

2. A set of two or more isotopic mass labels, wherein each mass label comprises the formula:X- A wherein:X is a coding moiety having an exact mass, andA is the analyte, and wherein each mass label in the set has a different integer mass.

3. The set of two or more isotopic mass labels according to claim 1 or claim 2, wherein each mass label has the following structure:033945.00485wherein * represents that oxygen is18O, carbon is13C, hydrogen is2H and nitrogen is15N, and wherein one or more * may be present.

4. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the set comprises n=6 mass labels having the following structures:033945.004855. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the difference in exact mass between a mass label and the next heaviest mass label in the set of mass labels is at least 4 daltons, at least 2 daltons, or at least 1 dalton.

6. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the difference in exact mass between a mass label and the next heaviest mass label in the set of mass labels is 4 daltons throughout the set of mass labels.

7. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the difference in exact mass between a mass label and the next heaviest mass label in the set of mass labels varies and can be 4 daltons, 2 daltons, 1 dalton, or less than 1 dalton.

8. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the difference in exact mass between at least two of the mass labels is less than 100 millidaltons (“mDa”), less than 50 mDa, or less than 20 mDa.

9. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the difference in exact mass between at least two of the mass labels is 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa.

10. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the set comprises four deuterated mass labels, each deuterated mass label comprising one or more hydrogen atoms substituted by2H.033945.0048511. The set of two or more isotopic mass labels according to any one of claims 1-3, wherein the set comprises twenty mass labels and the difference in exact mass between at least two of the mass labels is: a) less than 100 mDa, less than 50 mDa, or less than 20 mDa; or b) 2.5 mDa, 2.9 mDa, 6.3 mDa, 8.3 mDa, 9.3 mDa, or 10.2 mDa.

12. A method of mass spectrometry analysis, comprising: a) providing a plurality of samples, wherein each sample is differentially labelled with an isotopic mass label or a combination of isotopic mass labels, each selected from the set of two or more isotopic mass labels according to any one of claims 1-11; b) mixing the plurality of labelled samples to form an analysis mixture comprising labelled analytes; c) detecting the labelled analytes in a mass spectrometer; d) isolating one or more labelled analytes in the mass spectrometer based on one or more isolation windows of >1 Da and then dissociating them to form labelled analyte fragments; e) detecting the analyte fragments; and f) identifying the one or more labelled analytes on the basis of the analyte fragments.

13. The method of claim 12, wherein the one or more isolation windows comprise an isolation window that is <10 Da.

14. The method of claim 12, wherein the one or more isolation windows comprise an isolation window that is <20 Da.

15. The method of claim 12, wherein the one or more isolation windows comprise an isolation window that is <50 Da.

16. The method of claim 12, wherein the one or more isolation windows comprise an isolation window that is <100 Da.

17. The method of any one of claims 12-16, wherein the dissociation in step d) is collision- induced dissociation in a mass spectrometer.033945.0048518. The method of any one of claims 12-17, wherein the dissociation is collision induced dissociation in amass spectrometer with a resolution of greater than 60,000 at a mass-to-charge ratio of 400, optionally a resolution of greater than 100,000 at a mass-to-charge ratio of 400, or a resolution greater than 250,000 at a mass-to-charge ratio of 400.

19. The method of any one of claims 12-18, wherein the labelled analytes may be identified on the basis of the MS2 spectrum of the labelled analyte fragments and quantified using the MS 1 spectrum of the labelled analytes.

20. The method of any one of claims 12-18, wherein the labelled analytes may be identified and quantified on the basis of the MS2 spectrum of the labelled analyte fragments.

21. The method of any one of claims 12-19, wherein each isotopic mass label is resolved from all other tags in the set by at least 0.003 daltons, by 0.006 daltons, or at least 1 dalton.

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