Harnessing chromatographic information in proteomics: comprehensive coverage of peptide post-translational and chemical modifications

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Rapid developments in bioanalytical chemistry have provided enormous benefits for many applied fields of modern science including human health, agriculture, energy, and environmental studies. Liquid chromatography - mass spectrometry (LC-MS) has become the method of choice for proteomic applications, as it is the most powerful technique for protein/peptide analysis. This technique relies on LC separation and MS analysis of smaller peptides: products of protein proteolytic digestion. Most of the developmental emphasis in recent years has been on the MS-component of the system. At the same time many researchers have recognized the ability of the LC component to provide very valuable information, given the ability to compute the retention properties of analyzed molecules. Overall this makes LC-MS a “double-edged sword”, where the outcomes from both methods can be used to improve the analysis output and design of the methods themselves. Several research teams have been developing peptide retention prediction models. My laboratory has been at the forefront of these developments: our Sequence Specific Retention Calculator (SSRCalc) model has become a benchmark tool in the field, attracting attention both from the proteomic/chromatographic community and from major vendors of proteomics equipment and software. So far, the vast majority of predictive approaches target peptides, which consist of 20 natural, non-modified amino acids. It is known, however, that proteins may carry numerous post-translational modifications, which often control protein functions in complex biological systems. These modifications cannot be predicted based on genomic sequence, which make proteomics a key methodology for their study. These modifications change both molecular weight and hydrophobicity of analysed peptides. While determining the effect of a modification on the MS properties (mass shift) of a peptide is straightforward, predicting the alteration in chromatographic properties is difficult. The focus of the next cycle of my Discovery Grant is to extend the SSRCalc prediction methodology to peptides with post-translational and chemical modifications. This will 1) provide proteomic researchers with new tools to effectively monitor these modifications; 2) help us to develop comprehensive solutions to this fundamental problem in separation chemistry while also gaining new insights into underlying chemical and biological processes; 3) provide extensive training for the next generation of bioanalytical chemists in this highly relevant field; 4) further establish Canada’s position as a world leader in proteomics and peptide chromatography research.