Comparative Quantitation of Aberrant Glycoforms by Lectin-Based Glycoprotein Enrichment Coupled with Multiple-Reaction Monitoring Mass Spectrometry
| Autor: | Yong-Sam Kim, Jong Shin Yoo, Eun Sun Ji, Ji Yeon Lee, Ji-Ae Jung, Jeong Heon Ko, Yeong Hee Ahn |
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| Rok vydání: | 2010 |
| Předmět: |
Glycosylation
Molecular Sequence Data Protein tyrosine phosphatase Matrix metalloproteinase N-Acetylglucosaminyltransferases Mass spectrometry Mass Spectrometry Analytical Chemistry Cell Line Tumor Lectins Humans Trypsin Amino Acid Sequence Phytohemagglutinins Glycoproteins TIMP1 chemistry.chemical_classification Desmoglein 2 Tissue Inhibitor of Metalloproteinase-1 biology Chemistry Selected reaction monitoring Lectin Tissue inhibitor of metalloproteinase Molecular biology Calibration biology.protein Protein Tyrosine Phosphatases Peptides Glycoprotein |
| Zdroj: | Analytical Chemistry. 82:4441-4447 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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