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Oral Sessions
Day 2, June 23（Mon.） 16:10-16:25
Room C (Top of Yaima)
- 2C-O3-1610
Chromatography to the Rescue – Confident Differentiation Between Citrullination and Deamidation in Bottom-up Proteomic Experiments
(U. of Manitoba)
oOleg Krokhine
Confident differentiation between Arg citrullination and Asn deamidation, could be very complicated by using MS and MS/MS measurements as both resulting in identical +0.984 Da mass increase. In this work we aimed to investigate the possibility of confident delineation between these PTMs using chromatographic retention data in the framework of our Sequence-Specific Retention Calculator (SSRCalc) peptide retention prediction model.
Correspondent samples for retention dataset's LC-MS/MS acquisitions were obtained by spontaneous deamidation of complex tryptic digests (deamidation) and by enzymatic citrullination of model protein mixtures (citrullination). Tryptic digests were analysed by 1 and 2D-LC-MS/MS using a standard nano-flow RP HPLC-MS settings. First separation dimension featured several peptide separation modes, including high pH RP, HILIC, SCX
Average retention shifts for tryptic peptides in PR HPLC (formic acid) for citrullination and deamidation were found +1.7 and +0.5 %ACN on chromatographic elution scale, respectively. This difference was insufficient for confident differentiation between two modifications using SSRCalc retention time prediction. Subsequent utilization of the first-dimension retention data showed that strong cation exchange (SCX) possesses sufficient differentiation power due to charge elimination upon citrullination.