Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots
Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots
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DOI：
10.3389/fimmu.2018.02756
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发表时间：
2018-12-04
影响因子：
7.3
通讯作者：
Hahn, Si Houn
中科院分区：
文献类型：
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作者：
Collins, Christopher J.;Chang, Irene J.;Hahn, Si Houn
背景资料：原发性免疫缺陷疾病（PIDD）包括一组以免疫应答缺失或受损为特征的危及生命的先天性疾病。尽管事实上，有效的，治愈性的治疗方法是最佳的临床结果时，早期诊断，新生儿筛查不存在大多数这些疾病由于缺乏可检测的，具体的生物标志物或验证的方法，以人口为基础的筛选。肽免疫亲和富集结合选择反应监测质谱法（immuno-SRM）是一种灵敏的蛋白质组学测定法，涉及抗体介导的肽捕获，可同时定量多种分析物。该检测方法有望用于潜在的新生儿PIDDs筛查，在大多数情况下，PIDDs会导致靶蛋白减少或缺失。目的：确定和评价基于免疫SRM的多重检测是否能够可靠和精确地区分患有X连锁无丙种球蛋白血症（XLA）、Wiskott-Aldrich综合征（WAS）和CD 3 ε相关的严重联合免疫缺陷（SCID），以及来自未受影响的正常对照干血斑（DBS）样本。我们对来自WASp、BTK和CD 3 + T细胞的蛋白水解产生的肽进行了盲法、多重分析。（分别用于WAS、XLA和SCID）在来自42名PIDD患者、40名正常成人对照和62名正常新生儿的DBS样本中进行。肽ATP酶铜转运蛋白（ATP 7 B）1056同时监测质量保证purposes.Results：免疫SRM测定可靠地定量DBS中的靶肽，并准确区分受影响的患者从正常对照。特征肽的分析发现，在每种情况下，与对照组相比，受影响患者中的肽水平在统计学上显著降低或不存在（WASp和BTK：p = 0.0001，SCID：p = 0.05）。批内和批间精密度范围分别为11 - 22%和11 - 43%;线性（1.39-2000 fmol肽）和稳定性（对于WASp 274、CD 3 β 197、BTK 407和ATP 7 B 1056肽= 0.97）。DBS中来自WASp、BTK和CD 3 β的蛋白型肽的基于免疫SRM的定量将相关PIDD病例彼此区分并与对照区分，提高了采用这种方法进行选择性PIDDs的大规模多重新生儿筛查的可能性。
Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases.Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3 epsilon-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples.Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3 epsilon (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes.Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39-2000 fmol peptide), and stability (= 0.97 for the WASp 274, CD3 epsilon 197, BTK 407, and ATP7B 1056 peptides.Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3 epsilon in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.