Novel Strategies Enabling High-Throughput Proteomic Analyses
Bruno Domon
Institute of Molecular Systems Biology - ETH Zurich - Zurich, Switzerland
Shotgun proteomics strategies have successfully enabled identification andquantification of proteins in complex mixtures. Often only the most abundant proteinsare identified due to excessive sample complexity and under-sampling of currentinstrumentation. Novel strategies overcoming some of these issues will be presented,including a more effective analysis of post-translational modifications.
The existing information on identified peptides stored in a library was leveraged toestablish effective protocols for selected reaction monitoring (SRM), high-throughputexperiments on a triple quadrupole instrument. This technique enables rapid, routineanalysis of hundreds of peptides in multiples samples to determine their actualabundance. By measuring multiple transitions of each analyte of interest, the identity ofthe peptide can be established with confidence. Furthermore, by monitoring transitionscorresponding to modified and unmodified peptide fragment ions, possible modifications(such as phosphorylation or glycosylation) were readily detect and precisely localize.
In addition, modified peptides were analyzed by LC-MS using electron transferdissociation (ETD), implemented either on a linear ion trap or an orbitrap massspectrometer. The technique was applied to systematically sequence peptides anddetect phosphorylation or glycosylation sites of peptides present in specific isolates.More specifically, the oligosaccharide moieties of the N-glycosylated peptides werereleased enzymatically using endo-H (or endo-M), which leaves a GlcNAc residue on thepeptidic backbone, and were analyzed in ETD mode to unambiguously determine thepeptide sequence and localize the modification sites. In addition, the released glycanswere derivatized and analyzed by LC-MS using a porous graphite column to establishthe structure and the relative abundance of the various glycoforms.
These new mass spectrometry based strategies enable the structural analysis ofmodified peptides in complex mixtures with unprecedented sensitivity.
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