Harvard College

Scott Anthony Gerber

Steven P Gygi

Carlos Augusto Gartner

The invention provides reagents, kits and methods for detecting and/or quantifying proteins in complex mixtures, such as a cell lysate. The methods can be used in high throughput assays to profile cellular proteomes. In one aspect, the invention provides a peptide internal standard labeled with a stable isotope and corresponding in amino acid sequence to the amino acid sequence of a subsequence of a target polypeptide. In another aspect, the peptide internal standard is labeled at a modified amino acid residue and is used to determine the presence of, and/or quantitate the amount of a particular modified form of a protein.

Absolute quantification of proteins and modified forms thereof by multistage mass spectrometry

The invention provides non-affinity based isotope tagged peptides, chemistries for making these peptides, and methods for using these peptides. In one aspect, tags comprise a reactive site (RS) for reacting with a molecule on a protein to form a stable association with the peptide (e.g., a covalent bond) and an anchoring site (AS) group for reversibly or removably anchoring the tag to a solid phase such as a resin support. Anchoring may be direct or indirect (e.g., through a linker molecule). Preferably, the anchoring site comprises a biotin compound. Preferably, the tag comprises a mass-altering label, such as a stable isotope, such that association of the tag with the peptide can be monitored by mass spectrometry. The reagents can be used for rapid and quantitative analysis of proteins or protein function in mixtures of proteins.

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Brookline, MA, United States of America

Scott Anthony Gerber