Simultaneous Analysis of Proteome, Phospho- and Glycoproteome of Rat Kidney Tissue with Electrostatic Repulsion Hydrophilic Interaction Chromatography

Piliang Hao,Tiannan Guo,Siu Kwan Sze,Y Adam Yuan

doi:10.1371/journal.pone.0016884

Abstract

Protein post-translational modifications (PTMs) are regulated separately from protein expression levels. Thus, simultaneous characterization of the proteome and its PTMs is pivotal to an understanding of protein regulation, function and activity. However, concurrent analysis of the proteome and its PTMs by mass spectrometry is a challenging task because the peptides bearing PTMs are present in sub-stoichiometric amounts and their ionization is often suppressed by unmodified peptides of high abundance. We describe here a method for concurrent analysis of phosphopeptides, glycopeptides and unmodified peptides in a tryptic digest of rat kidney tissue with a sequence of ERLIC and RP-LC-MS/MS in a single experimental run, thereby avoiding inter-experimental variation. Optimization of loading solvents and elution gradients permitted ERLIC to be performed with totally volatile solvents. Two SCX and four ERLIC gradients were compared in details, and one ERLIC gradient was found to perform the best, which identified 2929 proteins, 583 phosphorylation sites in 338 phosphoproteins and 722 N-glycosylation sites in 387 glycoproteins from rat kidney tissue. Two hundred low-abundance proteins with important functions were identified only from the glyco- or phospho-subproteomes, reflecting the importance of the enrichment and separation of modified peptides by ERLIC. In addition, this strategy enables identification of unmodified and corresponding modified peptides (partial phosphorylation and N-glycosylation) from the same protein. Interestingly, partially modified proteins tend to occur on proteins involved in transport. Moreover, some membrane or extracellular proteins, such as versican core protein and fibronectin, were found to have both phosphorylation and N-glycosylation, which may permit an assessment of the potential for cross talk between these two vital PTMs and their roles in regulation.

Highlights

As proteins and their modifications are directly involved in most biological processes, the identification and quantification of as many proteins and their post-translational modifications (PTMs) as possible from the same sample are the prerequisites for biological discovery
As peptides are more compatible with liquid chromatography (LC) separation and mass spectrometry (MS) detection, protein extracts are usually digested with a protease to yield a complex mixture of peptides in shotgun proteomics
Immunoprecipitation, immobilized metal affinity chromatography (IMAC), strongcation exchange (SCX) and titanium dioxide (TiO2) chromatography have become popular for phosphopeptide enrichment [10,11,12,13], and lectin-based affinity enrichment, hydrophilic interaction liquid chromatography (HILIC), SCX and hydrazide covalent chromatography have been extensively used in the enrichment of glycoproteins or glycopeptides [14,15,16,17,18]

Summary

Introduction

As proteins and their modifications are directly involved in most biological processes, the identification and quantification of as many proteins and their post-translational modifications (PTMs) as possible from the same sample are the prerequisites for biological discovery. Unmodified peptides are separated from phosphopeptides and sialylated glycopeptides to a significant extent, and concurrent analysis of proteome, phosphoproteome and glycoproteome is potentially achieved in one run (Figure 1C-1E).

Results

Conclusion