Chemistry in Proteomics: An Interplay between Classical Methods in Chemical Modification of Proteins and Mass Spectrometry at the Cutting Edge

Abstract

This review deals with chemical approaches that facilitate proteomics research. The methods include chemical cleavage of peptide bonds, stepwise degradation of proteins, and site-specific modification enabling the discrimination between functional groups. These reactions could enhance the efficacy of mass spectrometry (MS) for identification, quantification, and sequencing of peptides and proteins. Particular stress is laid on the effect of each modification on mass spectra mainly with respect to the enhancement of peak intensities, the clarity of spectra, and the suppression of ambiguity in peak assignment. Chemical modifications targeting the terminal free amino and carboxyl groups are reviewed, because the charge or isotopic tagging on N-and C-termini has proved effective to identify a protein based on the terminal amino acid sequencing with tandem MS in combination with database searching. Such a tag that is designed to provide a peak with a characteristic sensitivity or pattern could corroborate the implication of mass spectra and facilitate the de novo protein sequencing of mature proteins, which are often subjected to post-translational modifications.

C‐terminomics: Targeted analysis of natural and posttranslationally modified protein and peptide C‐termini

“…The intrinsic difficulty associated with this strategy is the high similarity of the chemical properties of protein C‐terminal carboxyl groups and those present on the side‐chains of glutamates and aspartates. The oxazolone chemistry is thus far the only described way to selectively target C‐termini without affecting side‐chain carboxyl groups . In fact, automated C‐terminal sequencing relies on oxazolone chemistry that was further exploited to serve as a C‐terminal truncation strategy for C‐terminal ladder sequencing .…”

Terminal proteomics: N‐ and C‐terminal analyses for high‐fidelity identification of proteins using MS

“…To achieve the requisite tagging for characterization and identification of the protein C-terminus, it is indispensable to modify the C-terminal carboxyl group. As described in the section on stepwise C-terminal degradation, the chemistry of oxazolone could provide us with a variety of methods for the derivatization [57], However, the relatively low yield of oxazolone formation and the elaborate protocols for chemical manipulations, which are often unfriendly for proteomics researchers, need to be improved. Such hurdles inherent in oxazolone chemistry could be partially overcome by using MS for the detection of products with high sensitivity and throughput.…”

Site-specific protein labelling and immobilization mediated by microbial transglutaminase

“…For most, assigning functional roles will require methods for monitoring the activity and interactions of the gene products. Although a wide range of methods for the chemical modification of proteins have been developed, 2,3 these methods usually result in the selective labelling of a given functional group, rather than the site-specific labelling of one residue of a specific protein. However, by labelling proteins in living cells, critical information may be gained regarding their expression, localization and trafficking.…”