Comparative quantitation of aberrant glycoforms by lectin-based glycoprotein enrichment coupled with multiple-reaction monitoring mass spectrometry.
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.
Ahn
Yeong Hee
YH
Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun 363-883, Republic of Korea.
Kim
Yong-Sam
YS
Ji
Eun Sun
ES
Lee
Ji Yeon
JY
Jung
Ji-Ae
JA
Ko
Jeong Heon
JH
Yoo
Jong Shin
JS
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
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