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Publication Number
US-10697976-B2
Publication Date
2020-06-30
Expiration Date
Abstract
The present invention provides a method for labelling one or more analytes in a sample, the method comprising: a) contacting the sample with one or more bifunctional linker reagents having the general formula Re1-L1-Re2, wherein Re1 is a first reactive group, L1 is a linker moiety and Re2 is a second reactive group, wherein Re1 reacts with an analyte to form a modified analyte; and b) contacting the sample with one or more mass labels, wherein Re2 of the bifunctional linker attached to the analyte reacts with a mass label to form a labelled analyte, wherein each mass label is relatable to an analyte by mass spectrometry.
Core Innovation
The invention provides a method for mass spectrometric analysis of one or more analytes in a sample by using bifunctional linker reagents having the general formula Re1-L1-Re2. Re1 reacts with an analyte to form a modified analyte, while Re2 is a protected reactive group that is deprotected to form a second reactive group, which then reacts with two or more mass labels to form a labelled analyte relatable to an analyte by mass spectrometry.
Each analyte is reacted with a bifunctional linker from a set of two or more bifunctional linkers, where each bifunctional linker in the set has a unique mass. The mass labels have the general formula V-L2-M, where V is a mass marker moiety, L2 is a linker cleavable by dissociation in a mass spectrometer, and M is a mass normalisation moiety that causes the mass label to have a selected aggregate mass. The mass label further comprises a reactive group Re3 that reacts with Re2 of the bifunctional linker attached to the analyte.
The disclosure further includes reversible solid phase capture and release concepts. In one arrangement, analytes are reversibly captured onto a solid phase support prior to contacting with the bifunctional linkers, and the labelled analyte is eluted from the solid phase support after labelling and prior to mass spectrometric analysis; in another arrangement, analytes are attached to a solid phase support by means of a functional group, cleaved by beta elimination, isolated, and reversibly captured onto a further solid phase support.
Where the analyte comprises an O-linked saccharide, the O-linked saccharide is reacted with an oxidising agent to form an aldehyde or a ketone, and Re1 of the bifunctional linker reacts with the aldehyde or ketone. The document also describes specific mass series modifier linker embodiments, including Re2 as a pyridyldithio group reduced to a free thiol, and mass tags coupling as MT–Re3.
Claims Coverage
The consolidated claim coverage includes four inventive features. Across the independent claims, the coverage centers on bifunctional linkers with protected reactive groups, deprotection-driven mass labeling with V-L2-M mass labels, unique-mass linker sets for distinguishable labeling, and optional solid-phase and O-linked saccharide workflows.
Bifunctional linker modification with protected reactive group
Contacting the sample with two or more bifunctional linker reagents having the general formula Re1-L1-Re2, where Re1 reacts with an analyte to form a modified analyte and Re2 is a protected reactive group, followed by deprotecting Re2 to form a second reactive group.
Mass label architecture with cleavable linker and selected aggregate mass
Contacting the sample with two or more mass labels, where the mass label has the general formula V-L2-M, with V as a mass marker moiety, L2 as a linker cleavable by dissociation in a mass spectrometer, M as a mass normalisation moiety causing a selected aggregate mass, and Re3 as a reactive group that reacts with Re2.
Unique-mass bifunctional linkers in a set
Reacting each analyte with a bifunctional linker from a set of two or more bifunctional linkers, where each bifunctional linker in the set has a unique mass.
Solid phase handling and O-linked saccharide coupling
Reversibly capturing analytes on a solid phase support before labeling and eluting the labelled analyte after labelling; alternatively attaching analytes to a solid phase support by means of a functional group, cleaving by beta elimination, isolating released analytes, and reversibly capturing them onto a further solid phase support; and, where the analyte comprises an O-linked saccharide, reacting it with an oxidising agent to form an aldehyde or a ketone for Re1 coupling.
The claims collectively define a mass spectrometric analysis workflow using bifunctional linkers with protected Re2 groups, deprotection to enable mass-label coupling, and mass labels defined by V-L2-M with a cleavable linker and mass normalisation to a selected aggregate mass. The claims also cover unique-mass linker sets, reversible solid-phase capture and elution, beta elimination release and recapture, and O-linked saccharide oxidation for Re1 coupling.
Stated Advantages
Supports multiplexing by labeling using sets of bifunctional linkers having unique masses and mass labels relatable to analytes by mass spectrometry.
Allows multiplexing by using sets of bifunctional linkers with unique masses and mass labels designed with marker/aggregate mass relationships.
Supports multiplexing by labeling using sets of bifunctional linkers having unique masses and mass labels relatable to analytes by mass spectrometry.
Allows multiplexing by using sets of bifunctional linkers with unique masses and mass labels designed with marker/aggregate mass relationships.
Documented Applications
Peptide/protein post-translational modification analysis, including phosphorylation and glycosylation, using the disclosed mass-labeling approach.
Mass spectrometric analysis workflows including affinity-based depletion of saccharide-group-containing analytes or phosphate-group-containing analytes prior to linker and mass label processing.
Multiplexing using the disclosed mass-labeling approach, including reduced reagent count and multiplexing sets.
Phosphopeptide analysis using phosphopeptide dephosphorylation workflows in which mass series modifier treatments are applied sequentially.
Protein redox/cysteine thiol oxidation-state analysis using sequential mass-series modifier treatments.
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