Labelled compounds and methods for mass spectrometry-based quantification
Inventors
Reiter, Lukas • MÜLLER, Susanne Leslie
Assignees
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Publication Number
US-11293928-B2
Publication Date
2022-04-05
Expiration Date
Abstract
Methods for peptide and/or protein quantification by mass spectrometry using labeled peptides, wherein multiple labels lead to distinct fragments for the labeled peptides and their unlabeled variant, thus facilitating data analysis and enhancing the potential for quantification. Methods for selecting the label and label position are further given, as well as sets of labeled peptides resulting from or for use in the above-mentioned methods. The methods and substances are especially useful for data-independent or multiplexed parallel reaction monitoring proteomics applications involving peptide quantification.
Core Innovation
The described invention relates to mass spectrometry for absolute or relative quantitative analysis of proteins and/or peptides with or without post translational modification(s). An endogenous mixture of unlabeled proteins is digested and digestion products are selected, then fragmented, and a combined fragment spectrum is acquired comprising b-ions and y-ions.
At least one reference peptide is added to the mixture before and/or after digestion so that it is fragmented, acquired, and stored in the combined fragment spectrum together with b-ions and y-ions of the digestion products. The reference peptide is added in a known concentration for absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis.
The reference peptide is selectively isotopically labeled by incorporating one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and one isotopically labeled amino acid forming its very N-terminus or being one of the four terminal amino acids at the N-terminus. The description further includes refining aspects of the reference peptide and acquisition, including selecting label amino acid positions using peptide spectral libraries and label-cost considerations, and optionally simulating fragment collisions.
The reference peptides can allow acceptable post-translational modifications, and the workflow is supported for MS/DIA/mPRM instrument types such as LC-MS with settings consistent with DIA and multiplexed parallel reaction monitoring, including b-ions and y-ions in combined fragment spectra. Plasma experiments are reported to demonstrate improved fragment distinguishability, quantification accuracy, and identification scoring relative to single-labeled references.
Claims Coverage
The provided partial content includes one independent claim. The independent claim contains four inventive features focused on adding a selectively double-labeled reference peptide to an endogenous digest so that both b-ions and y-ions from the reference and sample are acquired and stored in a combined fragment spectrum, enabling absolute or relative quantification.
Reference peptide added to combined b/y fragment spectrum
At least one reference peptide is added to the endogenous mixture before and/or after digestion, is fragmented, acquired, and stored in the combined fragment spectrum comprising also b-ions and y-ions of the digestion products.
Known or constant reference peptide concentration for quantification
The at least one reference peptide is added in a known concentration for absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis.
Selectively double-terminal isotopic labeling of the reference peptide
The at least one reference peptide is selectively isotopically labeled by having incorporated one isotopically labeled amino acid forming its very C-terminus or being one of the four terminal amino acids at the C-terminus and one isotopically labeled amino acid forming its very N-terminus or being one of the four terminal amino acids at the N-terminus.
Isotopically labeled atoms confined to the amino acid and not the chemically modifying moiety
The isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety, comprising one or more atoms that are isotopically labeled such that the one or more atoms are present in the amino acid and not in the chemically modifying moiety.
Claim coverage in the provided partial content is grounded in the independent claim, where quantitative mass spectrometry is achieved by adding at least one selectively double-terminal isotopically labeled reference peptide into a workflow that produces a combined fragment spectrum containing b-ions and y-ions from both sample digestion products and the reference peptide.
Stated Advantages
Improved fragment distinguishability relative to single-labeled references.
Improved quantification accuracy relative to single-labeled references.
Improved identification scoring relative to single-labeled references.
Documented Applications
Human plasma experiments for MS-based quantitative proteomics using DIA and multiplexed PRM approaches, reporting improved fragment distinguishability, quantification accuracy, and identification scoring.
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