Gavin E. Reid Research Interests
The mixture complexity, dynamic range and diversity of transcriptional, translation and post translational modifications associated with protein expression, as well as the complex array of specific protein-protein interactions involved in the regulation of cellular function, presents a significant analytical challenge to mass spectrometry (MS) based proteomic methods employed for characterization of the hitherto unknown functional role of the thousands of genes identified from recent genome sequencing initiatives.
Research in our laboratory is broadly directed toward the development and application of novel mass spectrometry based chemistries and instrumentation to address these challenges. Aspects of this research include:
(i) systematic examination of the fundamental gas-phase ion chemistries of amino acids, peptides and proteins in order to determine the mechanisms and other factors influencing their fragmentation reactions;
The presence of post-translationally modified (PTM) amino acid residues within a peptide sequence often has a dramatic effect on the fragmentation behavior of its protonated ions under low energy collision induced dissociation (CID) tandem mass spectrometry (MS/MS) conditions. In many instances, the formation of 'non-sequence' ions, such as those corresponding to cleavage at the side chain of an amino acid residue containing a PTM, may dominate the product ion spectrum, while 'sequence' ions, formed by amide bond cleavages along the peptide backbone, are suppressed. Although non-sequence side chain cleavage product ions are diagnostic for the presence of the PTM residue within the peptide sequence, their formation in high relative abundance often results in the sequence of the peptide not being ammenable to identification by database interrogation or by manual interpretation. As current proteomics strategies are becoming increasingly reliant on the use of automated database search algorithms for identifying proteins from peptide MS/MS data, it is expected that determination of the factors that influence the formation of 'non-sequence' versus 'sequence' ions from the MS/MS of post-translationally modified peptide ions, and incorporation of the fragmentation "rules" arising from these studies into the database search algorithms, would aid in the development of more effective search and scoring algorithms for automated high throughput protein identification.
(ii) development of chemical derivatization reagents and associated methodologies to direct the dissociation of gas-phase peptide ions containing selected structural features (for example, those containing a particular amino acid or post translational modification) toward analytically useful fragmentation pathways.
We are developing a novel chemical derivatization strategy, termed 'Selected Extraction of Labelled Entities by Charge derivatization and Tandem mass spectrometry' (SELECT). This strategy is based on the formation of a 'fixed-charge' on the side-chain of a selected amino acid within a protein or peptide of interest, coupled with the use of tandem mass spectrometry to direct the dissociation of peptide ions containing the fixed charge toward formation of a single characteristic product ion, thereby allowing their selective identification from complex mixtures by neutral loss or precursor ion scan mode MS/MS. Further structural interrogation and characterization of the amino acid sequence of peptide ions identified by this fixed charge derivatization approach may be achieved by database analysis following multistage MS/MS (MS3) of the characteristic MS/MS product ion in a quadrupole ion trap mass spectrometer, or by energy resolved 'pseudo' MS3 in a triple quadrupole mass spectrometer. The incorporation of 'light' and 'heavy' isotopically encoded labels into the fixed-charge derivatives enables the application of this MS/MS based method to quantitative protein analysis, via measurement of the relative abundances of neutral loss product ions generated by dissociation of light and heavy labelled proteolytically derived peptide ions between two protein samples of interest;
(iii) development and application of 'top down' proteomics strategies for the identification, characterization and quantitation of proteins and protein complexes using sequence information derived from the gas-phase dissociation of intact protein ions.
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