Mining the acidic serum proteome utilizing off-gel isoelectric focusing and label free quantitative liquid chromatography mass spectrometry

Abstract

Serum remains an attractive source for the discovery of disease related biomarkers due to its intimate interaction with the majority of tissues within the body. The dynamic range of proteins present within serum has long complicated the ability to detect low level tissue leakage proteins that offer more promise as potential biomarkers due to their higher degree of specificity. Depletion strategies, using affinity based sorbents, to remove the most abundant serum proteins are routinely used for matrix simplification during discovery strategies focused on the serum proteome or glycoproteome. Glycoproteins bearing glycans with terminal sialic acid residues have been shown to be differentially expressed in a variety of cancers and are of interest due to the varied involvement of sialic acid in biological functions related to cancer development and metastasis. Herein, we describe the use of 14P serum depletion with subsequent off-gel isoelectric focusing using narrow pH range acidic strips, to facilitate simplification and enrichment of the acidic proteome and glycoproteome prior to label free liquid chromatography mass spectrometry (LC-MS) based proteomic analysis. The developed platform enabled the detection of proteins present within serum at sub-nanomolar concentrations while data analysis using a variety of bioinformatics resources suggested that many of the detected proteins were tissue leakage proteins or proteins associated with specific cellular compartments, rather than traditional secreted proteins. Comparison with theoretical isoelectric point (pI) values also revealed that some detected proteins had higher pI values than the separation range applied during the IEF step, suggesting that their presence may be due to glycans with high degrees of sialylation being attached to the protein backbone, as well as the presence of other post translational modifications. Such findings agree with offline glycomic profiling of the IEF fractions wherein increasing degrees of sialylation were detected across the pH strip from the basic towards the acidic end. Application of the platform using pooled serum from patients bearing gastric cancer, both before and after surgical intervention for tumour removal, revealed a number of differentially expressed proteins associated with cell recognition and cell signalling. The developed platform enabled excellent sensitivity and offers strong potential for application within biomarker discovery studies focused on the acidic proteome.

Keywords: Glycosylation; Isoelectric focusing; Liquid chromatography; Mass spectrometry; Proteomics; Sialic acid.