Department of Chemistry, Chemistry of Life Processes Institute and Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.
Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 34134, South Korea.
J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1659-1670. doi: 10.1021/jasms.1c00026. Epub 2021 May 27.
Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values ( = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C ( = 0.5) and glycated ApoA-I with higher fasting glucose ( = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single -acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference ( ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.
同一基因的不同蛋白形式产物与健康和疾病的关联可能不同，它们的修饰模式可能为个体之间表型差异提供更精确的标志物。然而，目前用于蛋白质生物标志物发现和定量的工具，如自上而下的蛋白质组学和 ELISA，大多对蛋白形式不敏感。此外，目前通过液相色谱-质谱（LCMS）进行定量自上而下蛋白质组学的蛋白形式水平分析的通量与需要强大、更快的蛋白形式分析的人群水平生物标志物调查不兼容。为此，我们开发了免疫沉淀与样品流质谱（IP-SampleStream-MS）相结合的高通量、自动化技术，用于蛋白形式的靶向定量。我们将 IP-SampleStream-MS 应用于 25 名个体的血清样本，以评估载脂蛋白 A-I（ApoA-I）和 C-III（ApoC-III）的蛋白形式丰度。将 ApoA-I 的结果与这些个体的 LCMS 结果进行比较，IP-SampleStream-MS 的通量高出>7 倍，分析变异度高出>50%。通过 IP-SampleStream-MS 测量的蛋白形式丰度与基于 LCMS 的值高度相关（=0.6-0.9），并产生收敛的蛋白形式与表型的关联，即典型 ApoA-I 的丰度与较低的高密度脂蛋白胆固醇（=0.5）和糖化 ApoA-I 与较高的空腹血糖（=0.6）相关。我们还观察到 ApoC-III 的蛋白形式与表型的关联，本研究中鉴定了 22 种糖基化 ApoC-III 蛋白形式。单个乙酰己糖胺（HexNAc）修饰的 ApoC-III 的丰度与 BMI、体重和腰围等肥胖指数相关（∼0.7）。这些数据表明，IP-SampleStream-MS 是一种强大的、可扩展的工作流程，用于高通量地将蛋白形式与表型关联起来。
