Deglycosylation and label-free quantitative LC-MALDI MS applied to efficient serum biomarker discovery of lung cancer

Atsuhiko Toyama,Koji Ueda,Nobuhisa Ishikawa,Yataro Daigo,Koichi Matsuda,Hidewaki Nakagawa,Nobuoki Kohno,Yusuke Nakamura,Taka-Aki Sato

doi:10.1186/1477-5956-9-18

Abstract

BackgroundSerum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results.ResultsSerum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (P < 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (P < 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9.ConclusionsWe demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.

Highlights

Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples
To elucidate the extent to which the ionization of peptides is interfered by glycopeptides, the first part of this study describes the changes caused by serum deglycosylation in the MS peak profiles obtained by label-free shotgun proteomic analysis
Quantitative MS analysis of deglycosylated serum In order to examine the effect of deglycosylation on data content and quality, tryptic digest of serum proteins was prepared with or without the removal of N-glycans and sialic acids (n = 6), and analyzed on the LC-MALDI

Summary

Introduction

Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. Since analyses of the serum proteome hold great promise for non-invasive detection of cancers and other diseases, various techniques for quantitative proteomic profiling have been developed to identify novel protein biomarkers [1,2] These include labeling methods using stable isotopes such as ICAT (Isotope-coded affinity tags) [3], 13CNBS (2-nitrobenzenesulfenyl) [4], SILAC (Stable isotope labeling with amino acids in cell culture) [5] and iTRAQ (Isobaric tags for relative and absolute quantification) [6]. Deglycosylation can be coupled with the incorporation of 18O stable isotope resulting in +3 Da mass shift of asparagine residues, which allows deterministic identification of glycosylation sites [13] As these studies indicated, most of serum proteins are heavily glycosylated, potential effect of glycopeptides on ionization suppression of coexisting peptides had been overlooked. Glycopeptides carry large, hydrophilic carbohydrate moieties, which can cause substantial ionization suppression [14], hampering precise quantification at low-concentration range

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