title: Comparative quantitation of aberrant glycoforms by lectin-based glycoprotein enrichment coupled with multiple-reaction monitoring mass spectrometry.
abstract: Lectin enrichment-coupled multiple-reaction monitoring (MRM) mass spectrometry was employed to quantitatively monitor the variation of aberrant glycoforms produced under pathological states. For this, aberrant glycoforms of the tissue inhibitor of metalloproteinase 1 (TIMP1) and protein tyrosine phosphatase kappa (PTPkappa), previously known target proteins for N-acetylglucosaminyltransferase-V (GnT-V), were enriched by phytohemagglutinin-L(4) (L-PHA) lectin and comparatively analyzed in the conditioned medium of the WiDr colon cancer cell line and its GnT-V-overexpressing transfectant cells. Enriched glycoforms were digested, and the resultant peptides were comparatively quantified by MRM analysis. MRM quantitation data for the L-PHA-enriched samples revealed that the abundance of aberrant glycoforms of TIMP1 and PTPkappa was greatly increased (11.7- and 16.5-fold, respectively) in GnT-V-treated cells compared to the control cells, although the abundance of total TIMP1 and PTPkappa in GnT-V-treated cells was slightly different (1.1- and 0.5-fold, respectively) for unenriched samples compared to that in control cells. The dramatic variation in abundance of the aberrant glycoforms due to overexpressed GnT-V was confirmed quantitatively by comparative MRM analysis of lectin-enriched samples. This method is capable of comparatively quantitating the abundance of a protein of interest and its aberrant glycoform and will be useful for studying pathological mechanisms of cancer or verifying biomarker candidates.
authors.affiliation: Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun 363-883, Republic of Korea.
authors.forename: Yeong Hee
authors.initials: YH
authors.lastname: Ahn
authors.forename: Yong-Sam
authors.initials: YS
authors.lastname: Kim
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Ji
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Lee
authors.forename: Ji-Ae
authors.initials: JA
authors.lastname: Jung
authors.forename: Jeong Heon
authors.initials: JH
authors.lastname: Ko
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D000595:Amino Acid Sequence; D002138:Calibration; D045744:Cell Line, Tumor; D051301:Desmoglein 2 / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010835:Phytohemagglutinins / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D051301:Desmoglein 2; D006023:Glycoproteins; D037102:Lectins; D010455:Peptides; D010835:Phytohemagglutinins; D019715:Tissue Inhibitor of Metalloproteinase-1; C064103:phytohemagglutinin L4; D017351:N-Acetylglucosaminyltransferases; C052254:alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase; D017027:Protein Tyrosine Phosphatases; D014357:Trypsin