Isobaric mass labels having n',n'-dimeihyl piperazine-2-carboxylic acid reporter moieties
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Publication Number
US-11453621-B2
Publication Date
2022-09-27
Expiration Date
Abstract
The present invention relates to a set of two or more mass labels, wherein each mass label comprises the formula: X-L-M-Re wherein X is a reporter moiety having an exact mass, L is a bond cleavable by collision in a mass spectrometer, M is a mass modifier, and Re 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 an integer mass, wherein each mass label in the set has the same integer mass, and wherein the set comprises two or more subsets of mass labels, each subset comprising one, two or more mass labels, and wherein, when the subset comprises two or more mass labels, the exact mass of the reporter moiety X of each mass label in the subset is different from the exact mass of the reporter moiety X of the mass labels in the same subset and in all other subsets, and wherein each mass label is distinguishable by mass spectrometry.
Core Innovation
The invention provides a set of mass labels in which each mass label comprises X-L-M-Re, where X is a reporter moiety having an exact mass, L is a bond cleavable by collision in a mass spectrometer, M is a mass modifier, and Re is a reactive functionality for attaching the mass label to an analyte or the analyte. Each mass label in the set has the same integer mass and is distinguishable by mass spectrometry.
The set comprises two or more subsets of mass labels, and when a subset comprises two or more mass labels, the exact mass of the reporter moiety X for each mass label in that subset is different from the exact mass of the reporter moiety X of the mass labels in the same subset and in all other subsets. The disclosure includes a set of n=5 mass labels, isotopic heavy-atom substitution options, and deuterated tag sets that are approximately isobaric with Embodiment Set 6, including co-selectable tags.
The invention also includes a second set of mass labels with formula X-L-M-Re, where X follows a generalized bicyclic/heterocycle scaffold defined by substituents R1–R5, and arrays of mass labels having sets with different integer masses across sets. A mass spectrometry analysis method is described in which analytes are detected by identifying mass labels or label combinations from either the first or second sets or from the array.
Claims Coverage
The independent claims cover a composition of mass labels and mass spectrometry analysis methods that use those labels. Across the claims, the central inventive features are the X-L-M-Re mass label design organized into subsets with distinguishable reporter-moiety exact masses but the same integer mass, and staged dissociation and detection of reporter moieties, intact mass labels, analyte fragments, and complement ions.
Subsets of isobaric mass labels with collision-cleavable linker and distinguishable reporter exact masses
A set of mass labels with formula X-L-M-Re, where X is a reporter moiety having an exact mass, L is a bond cleavable by collision in a mass spectrometer, M is a mass modifier, and Re is a reactive functionality for attaching the mass label to an analyte or the analyte; each mass label has the same integer mass; the set comprises two or more subsets with differing reporter-moiety X exact masses within each subset and across subsets; each mass label is distinguishable by mass spectrometry; and the set consists of n=5 mass labels.
Mass spectrometry analysis using differentially labeled samples and detection of intact mass labels and reporter moieties
A method of mass spectrometry analysis comprising providing a plurality of samples with one or more analytes differentially labelled with a mass label or a combination of mass labels, obtaining labelled analytes labelled with a set of n=5 mass labels of the X-L-M-Re structures; mixing to form an analysis mixture; dissociating labelled analytes to form mass labels and/or analyte fragments comprising intact mass labels; detecting the mass labels and/or analyte fragments comprising intact mass labels; optionally dissociating the mass labels to release reporter moieties and detecting the reporter moieties; optionally dissociating the reporter moieties to form fragments and detecting the fragments; and identifying analytes based on the mass spectrum of the labelled analytes and/or the mass spectrum of the mass labels and/or analyte fragments comprising an intact mass label and/or the mass spectrum of the reporter moieties or fragments.
Complement-ion remainder readout after releasing reporter moieties
A method of mass spectrometry analysis comprising providing a plurality of samples with one or more analytes differentially labelled with a mass label or a combination of mass labels, obtaining labelled analytes labelled with a set of n=5 mass labels of the X-L-M-Re structures; mixing to form an analysis mixture; detecting the labelled analytes in a mass spectrometer; dissociating the labelled analytes to release reporter moieties and detecting complement ions comprising the remainder of the mass label attached to the analyte or a fragment of the analyte; optionally further dissociating the complement ions to form fragments and detecting the fragments; and identifying analytes based on the mass spectrum of the labelled analytes and/or the mass spectrum of the complement ions and/or fragments thereof.
The claim set centers on an n=5 mass-label set defined by X-L-M-Re with a collision-cleavable linker L, a shared integer mass across labels, and distinguishable reporter moiety X exact masses arranged into subsets. The analysis methods then identify analytes by staged dissociation and mass spectral detection, using intact mass labels, reporter moieties, and complement-ion remainder after reporter release.
Stated Advantages
Increased multiplexing, including more than 10-plex and up to 28 with Sets 1+2, and up to 33 with Sets 1+2+3.
Reporter ions are discriminable by high-resolution FT/Orbitrap instrumentation due to specified minimum reporter ion mass separations.
Reduced spectral complexity.
Controlled fragmentation.
Each mass label is distinguishable by mass spectrometry.
The method can use complement ions comprising a remainder of the mass label attached to the analyte or a fragment for analyte identification.
Documented Applications
Mass spectrometry analysis of differentially labeled samples comprising one or more analytes, using mass label sets and identifying analytes based on mass spectra of labeled analytes and/or mass labels, analyte fragments, reporter moieties, and/or complement ions.
Quantification of peptides or proteins by mass spectrometry using reactive isobaric/isotopomeric mass labels.
Mass spectrometry analysis in which reporter moieties are released and complement ions comprising the remainder of the mass label attached to the analyte or analyte fragment are detected, optionally with further dissociation and fragment detection for identifying analytes.
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