Speaker
Description
Conventional mass spectrometry-based proteomics use positive polarity and provide a wealth of qualitative and quantitative information; however, these methods may not capture the true complexity of the proteome. Our work aims at alleviating this issue by resorting to ionization in negative polarity to account for the acidic portion of the intact proteome that preferentially ionizes as anions. Negative polarity bottom-up strategies have already shown utility in boosting coverage of acidic peptides and our objective was to extend this strategy to the top-down (i.e., intact protein) domain to discover acidic species at the proteoform level, as well. Standard polypeptides with molecular weights between 1-48 kDa were interrogated by HCD, CID and UVPD using both polarities, whereby cleavage propensities and the effect of charge density were determined. A unique feature of negative mode HCD and CID is their ability to cleave disulfide-linkages more readily than in positive polarity. While cleaving disulfides is appealing, using negative mode HCD and CID comes at the cost of producing a high ratio (>50% of the total ion current) of neutral losses, including series of consecutive ammonia, water and CO2 losses. These additional peaks make spectral interpretation challenging and negatively impact the p-score of proteoform anions. Measurements conducted on the LC timescale for a standard protein mixture (Pierce standard) using high-pH separation on a polymeric resin seem promising and we are working on introducing new bioinformatic tools for conducting database searches for negative mode data.
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