Post-translational modifications (PTMs) play critical roles in biological processes and have significant effects on the structures and dynamics of proteins. Top-down proteomics methods were developed for and applied to the study of intact proteins and their PTMs in human samples. However, the large dynamic range and complexity of human samples makes the study of human proteins challenging. To address these challenges, we developed a 2D pH RP/RPLC-MS/MS technique that fuses high-resolution separation and intact protein characterization to study the human proteins in HeLa cell lysate. Our results provide a deep coverage of soluble proteins in human cancer cells. Compared to 225 proteoforms from 124 proteins identified when 1D separation was used, 2778 proteoforms from 628 proteins were detected and characterized using our 2D separation method. Many proteoforms with critically functional PTMs including phosphorylation were characterized. Additionally, we present the first detection of intact human GcvH proteoforms with rare modifications such as octanoylation and lipoylation. Overall, the increase in the number of proteoforms identified using 2DLC separation is largely due to the reduction in sample complexity through improved separation resolution, which enables the detection of low-abundance PTM-modified proteoforms. We demonstrate here that 2D pH RP/RPLC is an effective technique to analyze complex protein samples using top-down proteomics.

中文翻译：

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使用高 pH 值和低 pH 值反相液相色谱法对人细胞裂解液中的深度完整蛋白质组进行表征。

翻译后修饰 (PTM) 在生物过程中起着关键作用，并对蛋白质的结构和动力学产生重大影响。自上而下的蛋白质组学方法被开发用于研究人类样本中的完整蛋白质及其 PTM。然而，人类样本的大动态范围和复杂性使得人类蛋白质的研究具有挑战性。为了应对这些挑战，我们开发了一种 2D pH RP/RPLC-MS/MS 技术，该技术融合了高分辨率分离和完整蛋白质表征，以研究 HeLa 细胞裂解物中的人类蛋白质。我们的结果提供了人类癌细胞中可溶性蛋白质的深度覆盖。与使用 1D 分离时鉴定的 124 种蛋白质的 225 种蛋白质型相比，使用我们的 2D 分离方法检测和表征了 628 种蛋白质的 2778 种蛋白质型。许多具有包括磷酸化在内的关键功能 PTM 的蛋白型被表征。此外，我们首次检测到具有罕见修饰（例如辛酰化和脂酰化）的完整人类 GcvH 蛋白型。总体而言，使用 2DLC 分离鉴定的蛋白型数量增加主要是由于通过提高分离分辨率降低了样品复杂性，从而能够检测低丰度的 PTM 修饰型蛋白型。我们在此证明 2D pH RP/RPLC 是一种使用自上而下蛋白质组学分析复杂蛋白质样品的有效技术。使用 2DLC 分离鉴定的蛋白型数量增加主要是由于通过提高分离分辨率降低了样品复杂性，从而能够检测低丰度 PTM 修饰的蛋白型。我们在此证明 2D pH RP/RPLC 是一种使用自上而下的蛋白质组学分析复杂蛋白质样品的有效技术。使用 2DLC 分离鉴定的蛋白质型数量的增加主要是由于通过提高分离分辨率降低了样品复杂性，从而能够检测低丰度 PTM 修饰的蛋白质型。我们在此证明 2D pH RP/RPLC 是一种使用自上而下的蛋白质组学分析复杂蛋白质样品的有效技术。