Laboratory for Biomarker Development, Center for Genomic Medicine, RIKEN, Tsurumiku-Suehirocho1-7-22, Yokohama, Japan.
Proteome Sci. 2011 Apr 8;9:18. doi: 10.1186/1477-5956-9-18.
Serum is an ideal source of biomarker discovery and proteomic profiling studies are continuously pursued on serum samples. However, serum is featured by high level of protein glycosylations that often cause ionization suppression and confound accurate quantification analysis by mass spectrometry. Here we investigated the effect of N-glycan and sialic acid removal from serum proteins on the performance of label-free quantification results.
Serum tryptic digests with or without deglycosylation treatment were analyzed by LC-MALDI MS and quantitatively compared on the Expressionist Refiner MS module. As a result, 345 out of 2,984 peaks (11.6%) showed the specific detection or the significantly improved intensities in deglycosylated serum samples (P < 0.01). We then applied this deglycosylation-based sample preparation to the identification of lung cancer biomarkers. In comparison between 10 healthy controls and 20 lung cancer patients, 40 peptides were identified to be differentially presented (P < 0.01). Their quantitative accuracies were further verified by multiple reaction monitoring. The result showed that deglycosylation was needed for the identification of some unique candidates, including previously unreported O-linked glycopeptide of complement component C9.
We demonstrated here that sample deglycosylation improves the quantitative performance of shotgun proteomics, which can be effectively applied to any samples with high glycoprotein contents.
血清是生物标志物发现的理想来源，针对血清样本的蛋白质组学分析研究一直在进行。然而，血清的特点是蛋白质糖基化水平较高，这常常导致离子抑制，并通过质谱分析混淆准确的定量分析。在这里，我们研究了从血清蛋白中去除 N-糖基化和唾液酸对无标记定量结果性能的影响。
对经或未经去糖基化处理的血清胰蛋白酶消化物进行 LC-MALDI MS 分析，并在 Expressionist Refiner MS 模块上进行定量比较。结果，在去糖基化血清样本中（P < 0.01），2984 个峰中有 345 个（11.6%）具有特定的检测或显著增强的强度。然后，我们将这种基于去糖基化的样品制备方法应用于肺癌生物标志物的鉴定。在 10 名健康对照者和 20 名肺癌患者之间进行比较，鉴定出 40 个差异表达的肽（P < 0.01）。通过多重反应监测进一步验证了它们的定量准确性。结果表明，去糖基化对于一些独特候选物的鉴定是必要的，包括以前未报道的补体成分 C9 的 O-连接糖肽。
我们在这里证明，样品去糖基化提高了 shotgun 蛋白质组学的定量性能，这可以有效地应用于任何高糖蛋白含量的样品。
