SPAP: Soluble Human Plasma Proteoform Analysis via Acetonitrile Precipitation and Top-Down Mass Spectrometry

Advances in liquid chromatography–mass spectrometry have significantly improved proteomic analyses of human plasma. However, information at the level of intact proteoforms remains limited due to the high dynamic range of protein abundance and the complexity of post-translational modifications. To address this challenge, we introduce soluble plasma proteoform analysis via acetonitrile precipitation (SPAP), a streamlined workflow for top-down mass spectrometry-based proteomics that isolates small, intact proteoforms from the acetonitrile-soluble plasma fraction, enabling direct measurement of proteoform diversity and post-translational modifications with high resolution. This simple and scalable method employs cold acetonitrile to precipitate abundant plasma proteins, thereby enriching the sample for lower-molecular-weight proteoforms. We first assessed the method’s performance using a reference plasma sample. To explore its clinical applicability, we applied SPAP to a cohort of 40 individuals, including 30 patients with liver cirrhosis and 10 healthy controls. In total, we report 3746 proteoforms from 255 proteins, including those with phosphorylation, truncation, and disulfide bond modifications. Reproducibility was confirmed with a coefficient of variation of <10% for the majority of enriched proteoforms, including those potentially associated with hemostasis, lipoprotein metabolism, cytoskeletal structure, and protease regulation. SPAP enabled effective stratification of the three cirrhosis stages, verifying previously published results and supporting the identification of candidate biomarkers. Although liver cirrhosis was used as a model system, the SPAP workflow is broadly applicable to human disease with proteoform-level resolution, offering a new path to stronger correlations in smaller cohorts and addressing key challenges in diagnostic and biomarker discovery.

液相色谱-质谱联用（liquid chromatography–mass spectrometry）技术的进步，显著推动了人类血浆蛋白质组学分析的发展。然而，由于蛋白质丰度的动态范围极广，且翻译后修饰（post-translational modifications）复杂多样，完整蛋白质变体（proteoforms）层面的信息仍较为有限。为应对这一挑战，本研究提出了基于乙腈沉淀的可溶性血浆蛋白质变体分析方法（soluble plasma proteoform analysis via acetonitrile precipitation, SPAP）：这是一种用于自上而下质谱（top-down mass spectrometry）蛋白质组学研究的简化流程，可从乙腈可溶性血浆组分中分离小型完整蛋白质变体，从而实现高分辨率下蛋白质变体多样性与翻译后修饰的直接检测。该方法简便且可规模化，通过冷乙腈沉淀丰度较高的血浆蛋白质，实现低分子量蛋白质变体的样品富集。本研究首先采用参考血浆样本对该方法的性能进行评估。为探究其临床应用潜力，我们将SPAP应用于由40名个体组成的队列，其中包括30名肝硬化患者与10名健康对照者。本研究共从255种蛋白质中鉴定得到3746个蛋白质变体，涵盖发生磷酸化、截短以及二硫键修饰的蛋白质变体。多数富集得到的蛋白质变体的变异系数均低于10%，证实了该方法具备良好重复性；这些蛋白质变体潜在与止血、脂蛋白代谢、细胞骨架结构以及蛋白酶调控等生物学过程相关。SPAP可有效区分肝硬化的三个病程阶段，验证了既往已发表的研究结果，并为候选生物标志物的鉴定提供了支撑。尽管本研究以肝硬化作为模型体系，但SPAP流程可广泛适用于需要蛋白质变体层面分辨率的人类疾病研究，为小样本队列中更强相关性的分析提供了新途径，同时解决了诊断与生物标志物发现领域的关键挑战。