Labelled compounds and methods for mass spectrometry-based quantification
Inventors
Reiter, Lukas • MÜLLER, Susanne Leslie
Assignees
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Publication Number
US-11754571-B2
Publication Date
2023-09-12
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 invention relates to mass spectrometry quantification of proteins or peptides using isotopically labeled reference peptides and a combined fragment spectrum acquired comprising b-ions as well as y-ions. The problem addressed is that single C-terminal heavy labeling can cause b-ion overlap between light and labeled variants, which prevents reliable N-terminal quantification and can skew fragment-intensity based identification or scoring.
To solve this, the invention proposes selectively isotopically labeled reference peptides designed so that both b-ions and y-ions differ in mass between light and labeled variants. In particular, the reference peptides incorporate an isotopically labeled amino acid forming the very C-terminus or one of four terminal amino acids at the C-terminus, and an isotopically labeled amino acid forming the very N-terminus or one of four terminal amino acids at the N-terminus.
The isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety in which isotopic atoms are present in the amino acid and not in the chemically modifying moiety. A procedure is used to select the label and label position so that they are optimized with respect to at least the availability of the labeled version of the corresponding amino acid at the respective position and the complexity of the incorporation of the labeled version at the respective position.
Claims Coverage
Independent claim clm-00001 covers four inventive features for absolute or relative quantitative analysis of proteins or peptides using mass spectrometry with a combined fragment spectrum containing b-ions and y-ions.
Selecting a selectively isotopically labeled reference peptide for b-ions and y-ions quantification
A method for selecting the label and label position of at least one suitable reference peptide for absolute or relative quantitative analysis of proteins or peptides using a mass spectrometry method acquiring a combined fragment spectrum comprising b-ions as well as y-ions, wherein at least one reference peptide is added and is fragmented, acquired, and stored in the combined fragment spectrum comprising also b-ions and y-ions of digestion products.
Optimizing terminal label position based on availability and incorporation complexity
The position of the label at the C-terminus or within the four terminal amino acids at the C-terminus, or the position of the label at the N-terminus or within the four terminal amino acids at the N-terminus, or both, is selected using a procedure which takes into account at least one of the availability of the labeled version of the corresponding amino acid at the respective position or the complexity of the incorporation of the labeled version at the respective position, wherein the label and label position is selected so as to be optimized with respect to these parameters.
Using reference peptides with isotopically labeled amino acids at both very C-terminus and very N-terminus
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, wherein the isotopically labeled amino acids are unmodified naturally occurring proteinogenic amino acids or amino acids carrying a chemically modifying moiety and comprise one or more atoms that are isotopically labeled present in the amino acid and not in the chemically modifying moiety.
Concentration definition for absolute or relative quantitative analysis
The at least one reference peptide is added in a known concentration in case of absolute quantification or in always the same concentration in a series of experiments for relative quantitative analysis.
Overall, the claim coverage centers on designing and selecting reference peptides with selectively isotopically labeled amino acids at terminal regions on both the C-terminus and N-terminus, acquiring a combined fragment spectrum containing b-ions and y-ions, and selecting the label position by optimization using labeled-amino-acid availability and incorporation complexity while supporting absolute or relative quantification via reference concentration.
Stated Advantages
Improved quantification/identification in combined fragment spectra by reducing fragment overlap between light and labeled variants.
Improved intensity correlation scores when using double-labeled reference peptides compared with single-labeled references.
Documented Applications
Absolute or relative quantitative analysis of proteins or peptides using mass spectrometry with a combined fragment spectrum comprising b-ions and y-ions.
Quantitative analysis in human plasma, as demonstrated for reduced overlap and improved intensity correlation scores.
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