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 κ (PTPκ), previously known target proteins for N-acetylglucosaminyltransferase-V (GnT-V), were enriched by phytohemagglutinin-L4 (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 PTPκ 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 PTPκ 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