title: Deglycosylation and label-free quantitative LC-MALDI MS applied to efficient serum biomarker discovery of lung cancer.
abstract: BACKGROUND
  Serum 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.
  
  
  RESULTS
  Serum 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.
  
  
  CONCLUSIONS
  We demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.
authors.affiliation: Laboratory for Biomarker Development, Center for Genomic Medicine, RIKEN, Tsurumiku-Suehirocho1-7-22, Yokohama, Japan. k-ueda@riken.jp.
authors.forename: Atsuhiko
authors.initials: A
authors.lastname: Toyama
authors.forename: Hidewaki
authors.initials: H
authors.lastname: Nakagawa
authors.forename: Koichi
authors.initials: K
authors.lastname: Matsuda
authors.forename: Nobuhisa
authors.initials: N
authors.lastname: Ishikawa
authors.forename: Nobuoki
authors.initials: N
authors.lastname: Kohno
authors.forename: Yataro
authors.initials: Y
authors.lastname: Daigo
authors.forename: Taka-Aki
authors.initials: TA
authors.lastname: Sato
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Koji
authors.initials: K
authors.lastname: Ueda