title: Analysis of N-glycoproteins using genomic N-glycosite prediction.
abstract: Protein glycosylation has long been recognized as one of the most common post-translational modifications. Most membrane proteins and extracellular proteins are N-linked glycosylated, and they account for the majority of current clinical diagnostic markers or therapeutic targets. Quantitative proteomic analysis of detectable N-linked glycoproteins from cells or tissues using mass spectrometry has the potential to provide biological basis for disease development and identify disease associated glycoproteins. However, the information of low abundance but important peptides is lost due to the lack of MS/MS fragmentation or low quality of MS/MS spectra for low abundance peptides. Here, we show the feasibility of formerly N-glycopeptide identification and quantification at MS1 level using genomic N-glycosite prediction (GenoGlyco) coupled with stable isotopic labeling and accurate mass matching. The GenoGlyco Analyzer software uses accurate precursor masses of detected N-deglycopeptide peaks to match them to N-linked deglycopeptides that are predicted from genes expressed in the cells. This method results in more robust glycopeptide identification compared to MS/MS-based identification. Our results showed that over three times the quantity of N-deglycopeptide assignments from the same mass spectrometry data could be produced in ovarian cancer cell lines compared to a MS/MS fragmentation method. Furthermore, the method was also applied to N-deglycopeptide analysis of ovarian tumors using the identified deglycopeptides from the two ovarian cell lines as heavy standards. We show that the described method has a great potential in the analysis of detectable N-glycoproteins from cells and tissues.
authors.affiliation: Department of Pathology, Johns Hopkins University , Baltimore, Maryland 21287, United States.
authors.forename: Shisheng
authors.initials: S
authors.lastname: Sun
authors.forename: Bai
authors.initials: B
authors.lastname: Zhang
authors.forename: Paul
authors.initials: P
authors.lastname: Aiyetan
authors.forename: Jian-Ying
authors.initials: JY
authors.lastname: Zhou
authors.forename: Punit
authors.initials: P
authors.lastname: Shah
authors.forename: Weiming
authors.initials: W
authors.lastname: Yang
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Levine
authors.forename: Zhen
authors.initials: Z
authors.lastname: Zhang
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Chan
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
mesh_terms: D002277:Carcinoma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D005260:Female; D015894:Genome, Human; D006023:Glycoproteins / Q000032:analysis* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007553:Isotope Labeling; D010051:Ovarian Neoplasms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D012984:Software
chemical_list: D006023:Glycoproteins