Quantitative mass spectrometric analysis of C-terminal 36 amino acid peptides of alpha-1 antitrypsin in plasma using survey spectra

IF 4.2 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods Pub Date : 2025-04-08 DOI:10.1016/j.ymeth.2025.04.004

Harald Tammen , Andreas Pich , Rüdiger Hess , Urszula Lechowicz , Sabina Janciauskiene , Joanna Chorostowska

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Abstract

C-terminal peptides of alpha-1 antitrypsin (AAT) may serve as biomarkers for diseases such as sepsis, chronic obstructive pulmonary disease, liver disease, and autoimmune disorders. In this study, we present a robust and straightforward MS (mass spectrometry)-based method for quantifying AAT peptides 388–418 (C36) and its polymorphic variant (E400D, C36D) in plasma samples. Absolute quantification was accomplished using MALDI-MS reflectron spectra and ESI-MS MS1 scans, implemented in two independent laboratories. Two plasma preparation methods, methanol precipitation and ultrafiltration, were evaluated, with methanol precipitation yielding significantly higher recovery rates. The impact of freeze–thaw cycles on C36 levels was also assessed, revealing a significant increase in C36 levels after each cycle. Comparisons between MALDI-MS and ESI-MS showed strong concordance in C36 and C36D measurements. Furthermore, C36 and C36D levels correlated strongly with post-precipitation protein content across both MS methods. Normalizing C36 levels to protein content effectively mitigated variability. This method should be straightforward to implement in other laboratories, facilitating clinical studies to evaluate the diagnostic and prognostic significance of C36 peptides across various diseases.

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血浆α -1抗胰蛋白酶c -末端36个氨基酸肽的定量质谱分析

α -1抗胰蛋白酶（AAT）的c端肽可作为脓毒症、慢性阻塞性肺疾病、肝脏疾病和自身免疫性疾病等疾病的生物标志物。在这项研究中，我们提出了一种基于质谱的可靠而直接的方法来定量血浆样品中的AAT肽388-418 （C36）及其多态性变体（E400D, C36D）。绝对定量使用MALDI-MS反射光谱和ESI-MS MS1扫描完成，在两个独立的实验室实施。对甲醇沉淀法和超滤法两种血浆制备方法进行了评价，甲醇沉淀法的回收率明显更高。冻融循环对C36水平的影响也被评估，揭示了每个循环后C36水平的显著增加。MALDI-MS和ESI-MS的比较显示C36和C36D测量值具有很强的一致性。此外，在两种MS方法中，C36和C36D水平与沉淀后蛋白质含量密切相关。将C36水平与蛋白质含量归一化有效地减轻了可变性。该方法应易于在其他实验室实施，便于临床研究评估C36肽对各种疾病的诊断和预后意义。

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来源期刊

Methods 生物-生化研究方法

CiteScore

9.80

自引率

2.10%

发文量

222

审稿时长

11.3 weeks

期刊介绍： Methods focuses on rapidly developing techniques in the experimental biological and medical sciences. Each topical issue, organized by a guest editor who is an expert in the area covered, consists solely of invited quality articles by specialist authors, many of them reviews. Issues are devoted to specific technical approaches with emphasis on clear detailed descriptions of protocols that allow them to be reproduced easily. The background information provided enables researchers to understand the principles underlying the methods; other helpful sections include comparisons of alternative methods giving the advantages and disadvantages of particular methods, guidance on avoiding potential pitfalls, and suggestions for troubleshooting.