Tryptic peptide screening for primary immunodeficiency disease by LC/MS-MS.
Tryptic peptide screening for primary immunodeficiency disease by LC/MS-MS.
复制标题
通过 LC/MS-MS 进行原发性免疫缺陷病的胰蛋白酶肽筛查。
DOI：
10.1002/prca.201100096
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Hahn,SiHoun
中科院分区：
文献类型：
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作者：
Kerfoot,SandraA;Jung,Sunhee;Golob,Karin;Torgerson,TroyR;Hahn,SiHoun
目的早期诊断原发免疫缺陷疾病(PIDD)对于最大限度地提高患者存活率和临床预后至关重要。因此，人们对开发能够利用小血量识别PIDD患者的广泛、高通量、筛查方法非常感兴趣。实验设计我们开发了一种新的蛋白质组筛选方法，使用串联质谱仪同时识别来自跨膜分化蛋白簇3(CD3)ɛ的特定标志肽以及细胞内蛋白Wiskott-Aldrich综合征蛋白和Bruton酪氨酸激酶(BTK)，作为三种危及生命的PIDD的标志物；严重的联合免疫缺陷、Wiskott-Aldrich综合征和X连锁无球蛋白血症。用LC/MS-MS分析细胞系和白细胞(WBC)蛋白水解物中的标志性多肽。每种多肽的量由签名多肽峰面积与已知数量的标记标准多肽的面积之比确定。结果我们发现来自CD3ɛ、Wasp和Btk的标志性多肽很容易在蛋白分解的细胞裂解液中被检测到，它们的缺失可以正确地识别PIDD患者。结论和临床相关性这一概念验证研究证明了这种方法在PIDD筛查中的适用性，并提出了进一步多重分析以识别其他PIDD和潜在的其他疾病的可能性。
PurposeEarly diagnosis of primary immunodeficiency disorders (PIDDs) is critical for maximizing patient survival and clinical outcomes. Consequently, there is significant interest in developing broad‐based, high‐throughput, screening approaches capable of utilizing small blood volumes to identify patients with PIDD.Experimental designWe developed a novel proteomic screening approach using tandem mass spectrometry to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3)ɛ and the intracellular proteins Wiskott‐Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life‐threatening PIDDs; severe combined immunodeficiency, Wiskott‐Aldrich syndrome, and X‐linked Agammaglobulinemia. Signature peptides were analyzed by LC/MS‐MS in proteolytically digested lysates from cell lines and white blood cells (WBCs). The amount of each peptide was determined by the ratio of the signature peptide peak area to that of a known amount of labeled standard peptide. Peptide concentrations were normalized to actin.ResultsWe show that signature peptides from CD3ɛ, WASP, and BTK were readily detected in proteolytically digested cell lysate and their absence could correctly identify PIDD patients.Conclusions and clinical relevanceThis proof of concept study demonstrates the applicability of this approach to screen for PIDD and raises the possibility that it could be further multiplexed to identify additional PIDDs and potentially other disorders.