Quantitative Analysis of an Aberrant Glycoform of TIMP1 from Colon Cancer Serum by L-PHA-Enrichment and SISCAPA with MRM Mass Spectrometry

Abstract

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 beta-1,6-N-acetylglucosamine (beta-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 beta-1,6-GlcNAc were enriched by phytohemagglutinin-L(4) (L-PHA), a lectin that specifically recognizes the beta-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 microL 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.

Online High-Flow Peptide Immunoaffinity Enrichment and Nanoflow LC-MS/MS: Assay Development for Total Salivary Pepsin/Pepsinogen

“…Although the original SISCAPA work relied on online nanoliter immunoaffinity columns based on Poros supports linked to nanoflow LC and nanospray mass spectrometry (6 ), off-line magnetic bead-based enrichment of enzymatically released peptides followed by either nano-or capillary LC-MS/MS has become a popular approach (7)(8)(9)(10)(11)(12). Bead-based sample preparation has the advantage that the antibody capture step can be done in parallel for multiple samples, and this process can be automated on a magnetic particle processor (11 ) or liquid-handling robotics, as shown for immunoaffinity enrichment of proteins (13,14 ).…”

Applications of targeted proteomics in systems biology and translational medicine

“…Ahn and coworkers used a variation of SISCAPA with a combination of phytohemagglutinin‐L 4 (L‐PHA) for N‐linked glycan capture and a monoclonal anti‐peptide TIMP1 antibody conjugated to magnetic beads to quantitate the cancer candidate biomarker TIMP1, which is present at approximately 0.8 ng/ml in serum using only 1.7 μl of serum from a patient with colorectal cancer 108. Using online chromatography for affinity capture instead of offline magnetic beads, Neubert and coworkers quantified a marker for gastroesophageal reflux disease, pepsin/pepsinogen, in the saliva of healthy volunteers down to a range of 0.17 to 0.67 ng/ml, providing the most sensitive and specific test to date 109.…”

Glycoprotein Disease Markers and Single Protein-omics

“…Recent studies suggest that such methods are, in general, feasible; analysis of single proteins (e.g. immunoglobulins) by LC-MS-MRM methods was reported, and additional examples are likely to follow (51,(95)(96)(97). Quantifications of some of the glycoforms may be limited to the analysis of isolated proteins, and not all glycoforms will be within methodological limits of detection, but successful applications can be expected.…”