Ahn Yeong Hee, Lee Ji Yeon, Lee Ju Yeon, Kim Yong-Sam, Ko Jeong Heon, Yoo Jong Shin
Division of Mass Spectrometry, Korea Basic Science Institute, Ochang-Myun, Cheongwon-Gun, Republic of Korea.
J Proteome Res. 2009 Sep;8(9):4216-24. doi: 10.1021/pr900269s.
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.
肿瘤相关血清中特定糖蛋白的糖基化水平或糖型变化已有广泛报道，可作为一种鉴别手段。然而，由于糖蛋白结构高度复杂且糖肽的质量灵敏度低，对其进行定量质谱分析较为困难。因此，需要更强大的技术来发现这些潜在的生物标志物。金属蛋白酶组织抑制剂1（TIMP1）是一种在血清中通常以低浓度存在的糖蛋白，已知在结肠癌细胞系中由于N-乙酰葡糖胺转移酶-V（GnT-V）添加β-1,6-N-乙酰葡糖胺（β-1,6-GlcNAc）而发生异常糖基化，据报道该酶在侵袭性/转移性癌细胞中上调。在本报告中，提出了一种高灵敏度方法，用于定量分析结直肠癌（CRC）患者血清中异常糖基化的TIMP1。具有以β-1,6-GlcNAc结尾的N-连接聚糖的糖蛋白通过植物血凝素-L(4)（L-PHA）进行富集，L-PHA是一种特异性识别N-连接聚糖的β-1,6-GlcNAc部分的凝集素。将L-PHA富集的糖蛋白在溶液中用胰蛋白酶消化。使用与磁珠共价连接的单克隆抗肽TIMP1抗体，从L-PHA富集的胰蛋白酶消化物中免疫富集TIMP1的独特靶肽（抗原），并通过多反应监测（MRM）质谱分析进行定量。L-PHA凝集素富集后接稳定同位素标准并通过抗肽抗体捕获（SISCAPA）与MRM质谱分析的系统联用，实现了阿托摩尔（10⁻¹⁸）浓度下TIMP1的定量。使用仅相当于一名CRC患者1.7微升血清的样品，定量了一种异常糖基化的亚化学计量TIMP1异构体，其血清浓度约为0.8纳克/毫升。这种方法为从含有多种蛋白质异构体的人血清中定量特定异常糖型提供了一种有用工具，并且可能有助于与蛋白质聚糖异质性相关的生物学功能研究。
