Abstract 4569: Serum proteome analysis by label-free quantification system and LC-MALDI mass spectrometry for the discovery of novel biomarkers for lung cancer
Authors: Toyama Atsuhiko , Ueda Koji , Nakagawa Hidewaki , Daigo Yataro , Sato Taka-Aki , Nakamura Yusuke 
Published: February 2011

Abstract
Lung cancer is the leading cause of cancer death in Japan and throughout the world, and highly specific and sensitive serum biomarkers applicable for screening are urgently required. Here we report our label-free quantification study conducted on the serum samples from 10 healthy volunteers, 10 patients at early stages and 10 patients at advanced stages of lung adenocarcinoma, aiming to identify serum proteins that can be used as lung cancer biomarkers.
The workflow was divided into four sections: 1) serum sample preparations involving immunodepletion of 14 high-abundance proteins, enzymatic deglycosylation in H218O, trypsin digestion and nano-reversed phase liquid chromatography; 2) acquisition of matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) data from 30 cases; 3) label-free quantification and statistical analysis using Genedata Expressionist® platform; and 4) candidate biomarker protein identification by retrospective MS/MS acquisition.
Deglycosylation was the critical step in the sample preparation phase as it could reduce spectral complexity caused by the presence of glycopeptides with heterogeneous glycan structures. Here we utilized H218O to incorporate 18O into aspartic acid residues that occurred during the deglycosylation reaction, and thus the deglycosylation sites resulted in the specific +3 Da mass shift allowing unambiguous determination of deglycosylated peptides and their glycosylation sites.
We have shown here that 7.5 % of the identified peptides were N-glycosylated. This high level of glycopeptide occurrence, attributable to the fact that over 90 % of proteins in serum are glycoproteins, suggests that deglycosylation process would significantly simplify the peptide composition, which is critical for in-depth mass spectrometric quantification analyses of serum samples.
As the result of label-free quantification analysis, approximately 15,000 peptide species (as peak clusters) were detected from 30 serum samples. 25 peptides were screened by one-way analysis of variance to be differentially represented among the three sample groups (normal, early and advanced lung cancer cases, p &lt; 0.01). 16 peptides were specifically detected in normal serum and 9 showed increased levels in advanced stage cases, of which 2 were also detected at higher levels in early-stage cases than normal sera (p &lt; 0.05). These peptides were subjected to MS/MS acquisition, referring back to the original data to select maximally detectable spot on the archived MALDI target plate for increased chance of identification.
In conclusion, the combination of H218O deglycosylation, MALDI MS and label-free quantification can work effectively for precise quantification of glycoprotein-rich clinical specimens, allowing for the screening of serum biomarkers for cancer.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4569.