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: Yeong Hee Ahn, Yong-Sam Kim, Eun Sun Ji, Ji Yeon Lee, Ji-Ae Jung, Jeong Heon Ko, Jong Shin Yoo
• Journal: Analytical chemistry (Anal Chem) Vol. 82 Issue 11 Pg. 4441-7 (Jun 01 2010) ISSN: 1520-6882 [Electronic] United States
• PMID: 20462175 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Desmoglein 2
• Glycoproteins
• Lectins
• Peptides
• Phytohemagglutinins
• Tissue Inhibitor of Metalloproteinase-1
• phytohemagglutinin L4
• N-Acetylglucosaminyltransferases
• alpha-1,6-mannosylglycoprotein beta 1,6-N-acetylglucosaminyltransferase
• Protein Tyrosine Phosphatases
• Trypsin

Topics

• Amino Acid Sequence
• Calibration
• Cell Line, Tumor
• Desmoglein 2 (metabolism)
• Glycoproteins (chemistry, metabolism)
• Glycosylation
• Humans
• Lectins (metabolism)
• Mass Spectrometry (methods)
• Molecular Sequence Data
• N-Acetylglucosaminyltransferases (metabolism)
• Peptides (chemistry, metabolism)
• Phytohemagglutinins (metabolism)
• Protein Tyrosine Phosphatases (metabolism)
• Tissue Inhibitor of Metalloproteinase-1 (metabolism)
• Trypsin (metabolism)