Use of enrichment and depletion approaches to increase serum proteome coverage in quantitative proteomics experiments

摘要

The sera were independently processed using two different technologies, MARS-14 based depletion and ProteoMiner based enrichment. From SDS-PAGE analysis, we observed that both techniques are highly reproducible and each method showed its own unique protein profile. Tryptic peptides were desalted before iTRAQ labeling and iTRAQ labeled peptides were fractionated on a SCX column in a separate experiment. We observed more number of proteins can be identified by high pH RPLC fractionation. Desalted peptides were analyzed on a QTOF mass spectrometer coupled to Chip cube nanospray source. LC-MS/MS analysis led to identification of 257 proteins from the depletion method and 314 proteins from the enrichment method from SCX-based fractionated samples. In case of high pH RPLC fractionated peptides, 416 and 351 proteins were identified by MARS-14 and ProteoMiner, respectively. About 40percent of the proteins were common to the two approaches, most of which showed similar fold-changes. In total, we quantitated 546 serum proteins including many interesting novel proteins. For example, Proprotein convertase subtilisin/kexin type 9 belonging to the proteinase K subfamily of the secretory subtilase family was identified. The encoded protein is synthesized as a soluble zymogen that undergoes autocatalytic intramolecular processing in the endoplasmic reticulum. This protein plays a role in cholesterol homeostasis and may have a role in the differentiation of cortical neurons. To our knowledge this is the first quantitative proteomics study investigating differentially expressed proteins following bariatric surgery. Further investigation of the differentially expressed molecules post-bariatric surgery can lead to the identification of prognostic markers. Our data suggests that use of depletion and enrichment strategies increases the coverage of serum proteome which will specifically prove to be of use for serum biomarker discovery.