PDF(Pubmed)
Abstract：
Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the \"bottom-up\" approach and the \"top-down\" approach. The \"shotgun\" method is a typical \"bottom-up\" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. \"Top-down\" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the \"bottom-up\" \"top-down\" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.
摘要：
蛋白质组学分析在生物医学研究中起着重要作用。蛋白质组学研究比基因组研究复杂得多,主要是因为蛋白质组样本的复杂性和多样性。高效液相色谱-质谱联用技术(HPLC-MS)是蛋白质组学研究的基础工具,决议,和敏感性。蛋白质组学研究目标从多肽和单个蛋白质到较大的蛋白质复合物,其分子量逐渐增加，导致结构和组成复杂性的持续增加以及分子性质的改变。因此,在处理蛋白质组学研究中的不同靶标以进行深入的蛋白质组学分析时，各种分离策略和固定相参数的选择至关重要。本文概述了实验室中常用的色谱分离策略，包括反相液相色谱（RPLC），亲水相互作用液相色谱（HILIC），疏水相互作用色谱（HIC），离子交换色谱（IEC），和尺寸排阻色谱法(SEC)，以及它们在各种生物大分子中的应用和选择性。目前,没有单一的色谱或电泳技术具有将这种复杂的混合物分解为单个组分所需的峰容量。多维液相色谱（MDLC），将不同的正交分离模式与MS相结合，在蛋白质组学研究中发挥着重要作用。在MDLC战略中，IEC,与RPLC一起，仍然是蛋白质组学分析中使用最广泛的分离模式;其他色谱方法也经常用于肽/蛋白质分馏。MDLC技术及其在各种蛋白质组学分析中的应用已经有了很大的发展。MDLC分离系统中的两种策略主要用于蛋白质组学分析：“自下而上”方法和“自上而下”方法。“shot弹枪”方法是一种典型的“自下而上”策略，该策略基于RPLC或MDLC分离全蛋白质样品消化物以及MS；它是鉴定大量蛋白质的出色技术。“自上而下”分析是基于完整蛋白质的分离，并提供其详细的分子信息;因此，该技术可能有利于分析蛋白质的翻译后修饰(PTM).在本文中,简要回顾了蛋白质组样本的“自下而上”和蛋白质-蛋白质相互作用（PPI）分析。描述了用于设置MDLC系统的不同色谱模式的不同组合，以及流动相和分析物之间的兼容性问题，在流动相和MS之间，分析了多维色谱中不同分离模式下的流动相之间。MDLC技术的新发展，如高丰度蛋白质消耗和色谱阵列，进一步讨论。在这次审查中,强调了研究人员在遇到兼容性问题时提出的解决方案。此外，研究了HPLC-MS结合各种样品预处理方法在外泌体和单细胞蛋白质组学研究中的应用。在外泌体隔离期间，超速离心和SEC的联合使用可以产生更高纯度的外泌体。使用具有超大孔径填充材料（200nm）的SEC可以分离外泌体亚组，和蛋白质组学研究揭示了这些亚组之间蛋白质组成和功能的显著差异。在单细胞蛋白质组学领域,研究人员已经解决了与减少样品处理量有关的挑战，防止样品损失，并避免样品制备过程中的污染。创新的方法和改进，例如利用毛细管进行样品处理和微芯片作为平台，以最小化液滴的接触面积，已被提议。这些技术与HPLC-MS的结合显示了一些进展。总之,本文重点介绍了HPLC-MS技术在蛋白质组学分析中的最新进展，为今后蛋白质组学领域的研究提供了全面的参考。
