Development of a Sample Preparation Workflow for Sulfopeptide Enrichment: from Target Analysis to Challenges in Shotgun Sulfoproteomics.

Protein tyrosine O-sulfation is an important post-translational modification, as it has been correlated to inflammation, virus infection and signal pathways. Nevertheless, methods for the characterization of protein sulfation by sulfopeptide enrichment are currently limited. In this paper, two standard compounds, representative of mono- and di-sulfated peptides, were used to compare the enrichment capability of five sorbent materials: two commercial weak anion exchange mixed mode sorbents (Strata X-AW and Oasis WAX) and three phosphopeptide enrichment materials based on affinity chromatography to either immobilized metals (IMAC) or to metal oxides, i.e., Fe3+, TiO2 or Ti4+. The sulfopeptides were analyzed by UHPLC-multiple reaction monitoring analysis and were stable under all the tested experimental conditions. Recoveries of the enrichment step from spiked bovine serum albumin digests were above 80% for the commercial Fe-IMAC kit and the Strata X-AW sorbent. Shotgun proteomics was used on the same samples to evaluate the selectivity, calculated as the number of co-enriched peptides, and it was found better for the Fe-IMAC kit. Therefore, the Fe-IMAC protocol was embedded in a shotgun proteomics workflow and applied to serum spiked with the sulfopeptides before protein dephosphorylation and digestion. The recovery of the entire analytical workflow was 20%, which was compatible with previous data on TiO2 phosphopeptide enrichment. Despite the potential, no sulfopeptide was confidently identified in serum digests by conventional shotgun proteomics, probably due to very low abundance of native sulfoproteins, poor ionization efficiency of sulfopeptides in the positive mode, lack of unambiguous sulfopeptide identification by bioinformatics software. In this context, the use of negative ionization mode with high resolution mass spectrometry appeared promising to improve sensibility and allow sulfopeptide identification in complex samples.

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