Quantitative analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-enrichment and SISCAPA with MRM mass spectrometry

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Quantitative analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-enrichment and SISCAPA with MRM mass spectrometry
Y H Ahn; J Y Lee; J Y Lee; Yong Sam KimJeong Heon Ko; J S Yoo
Bibliographic Citation
Journal of Proteome Research, vol. 8, no. 9, pp. 4216-4224
Publication Year
Variations in glycosylation levels or in the glycoprofile of a certain glycoprotein in tumor-related sera have been widely reported and can be used as a means of differentiation. However, quantitative mass analysis of glycoproteins is difficult because of their high structural complexity and low mass sensitivity of glycopeptides. Therefore, more powerful technologies are required for the discovery of these potential biomarkers. Tissue inhibitor of metalloproteinase 1 (TIMP1), a glycoprotein typically present at a low concentration in serum, is known to be aberrantly glycosylated in colorectal cancer cell lines as a result of the terminal addition of β-1,6-N- acetylglucosamine (β-1,6-GlcNAc) by N-acetylglucosaminyltransferase-V (GnT-V), which is reportedly up-regulated in invasive/metastatic cancer cells. In this report, a highly sensitive method is presented for the quantitative analysis of aberrant GlcNAcylated TIMP1 in the serum of colorectal cancer (CRC) patients. Glycoproteins having an N-linked glycan terminating with β-1,6-GlcNAc were enriched by phytohemagglutinin-L4 (L-PHA), a lectin that specifically recognizes the β-1,6-GlcNAc moiety of N-linked glycan. The L-PHA-enriched glycoproteins were digested in solution with trypsin. With the use of a monoclonal anti-peptide TIMP1 antibody linked covalently to magnetic beads, a unique target peptide (antigen) of TIMP1 was immuno-enriched from the L-PHA-enriched tryptic digests and analyzed quantitatively by multiple reaction monitoring (MRM) mass analysis. The systematic coupling of L-PHA lectin enrichment followed by stable isotope standards and capture by anti-peptide antibodies (SISCAPA) with MRM mass analysis afforded quantitation of TIMP1 at attomolar (10-18) concentrations. An aberrantly GlcNAcylated substoichiometric TIMP1 isoform was quantified at approximately 0.8 ng/mL serum, using sample equivalent to only 1.7 μL of serum from a CRC patient. This approach provides a useful tool for the quantitation of a specific aberrant glycoform from human serum containing a variety of protein isoforms and may be helpful in studies of biological function as it pertains to protein glycan heterogeneity.
Aberrant glycoformAnti-peptide antibodyL-PHAPeptide quantitationSISCAPATIMP1
Amer Chem Soc
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Synthetic Biology and Bioengineering Research Institute > Genome Editing Research Center > 1. Journal Articles
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