Quantitative Analysis of Single Amino Acid Variant Peptides Associated with Pancreatic Cancer in Serum by an Isobaric Labeling Quantitative Method

Single amino acid variations are highly associated with many human diseases. The direct detection of peptides containing single amino acid variants (SAAVs) derived from nonsynonymous single nucleotide polymorphisms (SNPs) in serum can provide unique opportunities for SAAV associated biomarker discovery. In the present study, an isobaric labeling quantitative strategy was applied to identify and quantify variant peptides in serum samples of pancreatic cancer patients and other benign controls. The largest number of SAAV peptides to date in serum including 96 unique variant peptides were quantified in this quantitative analysis, of which five variant peptides showed a statistically significant difference between pancreatic cancer and other controls (p-value VAVVK from serotransferrin were detected between pancreatic cancer and controls, which was further validated by selected reaction monitoring (SRM) analysis. The novel biomarker panel obtained by combining α-1-antichymotrypsin (AACT), Thrombospondin-1 (THBS1) and this variant peptide showed an excellent diagnostic performance in discriminating pancreatic cancer from healthy controls (AUC = 0.98) and chronic pancreatitis (AUC = 0.90). These results suggest that large-scale analysis of SAAV peptides in serum may provide a new direction for biomarker discovery research.

单氨基酸变异与诸多人类疾病存在紧密关联。直接检测血清中源自非同义单核苷酸多态性（SNPs）的携带单氨基酸变异体（single amino acid variants，SAAVs）的肽段，可为与SAAVs相关的生物标志物发现工作提供独特契机。本研究采用同量异位素标记定量策略，对胰腺癌患者及其他良性对照人群的血清样本中的变异肽段进行鉴定与定量分析。本次定量分析共定量到目前为止血清中已报道的数量最多的SAAV肽段，涵盖96种独特变异肽段，其中5种变异肽段在胰腺癌组与其他对照组间呈现统计学显著差异。一株来自转铁蛋白、序列为VAVVK的独特变异肽段在胰腺癌组与对照组间被检出，并通过选择反应监测（selected reaction monitoring，SRM）分析得到进一步验证。将α1-抗胰凝乳蛋白酶（α-1-antichymotrypsin，AACT）、血小板反应蛋白-1（Thrombospondin-1，THBS1）与该变异肽段相结合所获得的新型生物标志物组合，在区分胰腺癌与健康对照人群（AUC=0.98）以及慢性胰腺炎患者（AUC=0.90）方面展现出优异的诊断性能。上述结果表明，对血清中的SAAV肽段进行大规模分析，可为生物标志物发现研究提供全新方向。