title: MRM validation of targeted nonglycosylated peptides from N-glycoprotein biomarkers using direct trypsin digestion of undepleted human plasma.
abstract: A rapid, simple, and reproducible MRM-based validation method for serological glycoprotein biomarkers in clinical use was developed by targeting the nonglycosylated tryptic peptides adjacent to N-glycosylation sites. Since changes in protein glycosylation are known to be associated with a variety of diseases, glycoproteins have been major targets in biomarker discovery. We previously found that nonglycosylated tryptic peptides adjacent to N-glycosylation sites differed in concentration between normal and hepatocellular carcinoma (HCC) plasma due to differences in steric hindrance of the glycan moiety in N-glycoproteins to tryptic digestion (Lee et al., 2011). To increase the feasibility and applicability of clinical validation of biomarker candidates (nonglycosylated tryptic peptides), we developed a method to effectively monitor nonglycosylated tryptic peptides from a large number of plasma samples and to reduce the total analysis time with maximizing the effect of steric hindrance by the glycans during digestion of glycoproteins. The AUC values of targeted nonglycosylated tryptic peptides were excellent (0.955 for GQYCYELDEK, 0.880 for FEDGVLDPDYPR and 0.907 for TEDTIFLR), indicating that these could be effective biomarkers for hepatocellular carcinoma. This method provides the necessary throughput required to validate glycoprotein biomarkers, as well as quantitative accuracy for human plasma analysis, and should be amenable to clinical use.
  
  
  CONCLUSIONS
  Difficulties in verifying and validating putative protein biomarkers are often caused by complex sample preparation procedures required to determine their concentrations in a large number of plasma samples. To solve the difficulties, we developed MRM-based protein biomarker assays that greatly reduce complex, time-consuming, and less reproducible sample pretreatment steps in plasma for clinical implementation. First, we used undepleted human plasma samples without any enrichment procedures. Using nanoLC/MS/MS, we targeted nonglycosylated tryptic peptides adjacent to N-linked glycosylation sites in N-linked glycoprotein biomarkers, which could be detected in human plasma samples without depleting highly abundant proteins. Second, human plasma proteins were digested with trypsin without reduction and alkylation procedures to minimize sample preparation. Third, trypsin digestion times were shortened so as to obtain reproducible results with maximization of the steric hindrance effect of the glycans during enzyme digestion. Finally, this rapid and simple sample preparation method was applied to validate targeted nonglycosylated tryptic peptides as liver cancer biomarker candidates for diagnosis in 40 normal and 41 hepatocellular carcinoma (HCC) human plasma samples. This strategy provided the necessary throughput required to monitor protein biomarkers, as well as quantitative accuracy in human plasma analysis. From biomarker discovery to clinical implementation, our method will provide a biomarker study platform that is suitable for clinical deployment, and can be applied to high-throughput approaches.
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea; Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea; Graduate School of Science and Technology, Chungnam National University, Daejeon 305-333, Republic of Korea.
authors.forename: Mi Hee
authors.initials: MH
authors.lastname: Cheon
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea; Graduate School of Science and Technology, Chungnam National University, Daejeon 305-333, Republic of Korea.
authors.forename: Gun Wook
authors.initials: GW
authors.lastname: Park
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea.
authors.forename: Yeong Hee
authors.initials: YH
authors.lastname: Ahn
authors.affiliation: Department of Chemistry, Yonsei University, Seoul 120-749, Republic of Korea.
authors.forename: Myeong Hee
authors.initials: MH
authors.lastname: Moon
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, 804-1 Yangcheong-Ri, Ochang-eup, Cheongwon-Gun 363-883, Republic of Korea; Graduate School of Science and Technology, Chungnam National University, Daejeon 305-333, Republic of Korea. Electronic address: jongshin@kbsi.re.kr.
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood*; D006528:Carcinoma, Hepatocellular / Q000097:blood*; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood*; D009363:Neoplasm Proteins / Q000097:blood*; D010455:Peptides / Q000097:blood*; D010949:Plasma; D040901:Proteomics / Q000379:methods; D014357:Trypsin / Q000737:chemistry*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D010455:Peptides; D014357:Trypsin
keywords: Multiple reaction monitoring (MRM) validation; N-glycoprotein; Nonglycosylated peptide; Rapid digestion; Undepleted plasma