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Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

机译：下下方法：通过质谱法进行序列和表征蛋白质/蛋白质的选择

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Owing to rapid growth in the elucidation of genome sequences of various organisms, deducing proteome sequences has become imperative, in order to have an improved understanding of biological processes. Since the traditional Edman method was unsuitable for high-throughput sequencing and also for N-terminus modified proteins, mass spectrometry (MS) based methods, mainly based on soft ionization modes: electrospray ionization and matrix-assisted laser desorption/ionization, began to gain significance. MS based methods were adaptable for high-throughput studies and applicable for sequencing N-terminus blocked proteins/peptides too. Consequently, over the last decade a new discipline called ‘proteomics’ has emerged, which encompasses the attributes necessary for high-throughput identification of proteins. ‘Proteomics’ may also be regarded as an offshoot of the classic field, ‘biochemistry’. Many protein sequencing and proteomic investigations were successfully accomplished through MS dependent sequence elucidation of ‘short proteolytic peptides (typically: 7–20 amino acid residues), which is called the ‘shotgun’ or ‘bottom-up (BU)’ approach. While the BU approach continues as a workhorse for proteomics/protein sequencing, attempts to sequence intact proteins without proteolysis, called the ‘top-down (TD)’ approach started, due to ambiguities in the BU approach, e.g. , protein inference problem, identification of proteoforms and the discovery of posttranslational modifications (PTMs). The high-throughput TD approach (TD proteomics) is yet in its infancy. Nevertheless, TD characterization of purified intact proteins has been useful for detecting PTMs. With the hope to overcome the pitfalls of BU and TD strategies, another concept called the ‘middle-down (MD)’ approach was put forward. Similar to BU, the MD approach also involves proteolysis, but in a restricted manner, to produce ‘longer’ proteolytic peptides than the ones usually obtained in BU studies, thereby providing better sequence coverage. In this regard, special proteases (OmpT, Sap9, IdeS) have been used, which can cleave proteins to produce longer proteolytic peptides. By reviewing ample evidences currently existing in the literature that is predominantly on PTM characterization of histones and antibodies, herein we highlight salient features of the MD approach. Consequently, we are inclined to claim that the MD concept might have widespread applications in future for various research areas, such as clinical, biopharmaceuticals (including PTM analysis) and even for general/routine characterization of proteins including therapeutic proteins, but not just limited to analysis of histones or antibodies.
机译：由于阐明了各种生物的基因组序列的快速增长，推断蛋白质组序列已成为必要的，以便改善对生物过程的理解。由于传统的EDMAM方法不适合高通量测序和N-末端改性蛋白质，质谱法（MS）的方法，主要基于软离子化模式：电喷雾电离和基质辅助激光解吸/电离，开始获得意义。基于MS的方法适用于高通量研究，并适用于抑制N-末端蛋白/肽。因此，在过去十年中，出现了一种名为“蛋白质组学”的新学科，其包括蛋白质的高通量鉴定所需的属性。 “蛋白质组学”也可能被视为经典领域的分支，“生物化学”。通过MS依赖性序列阐明的“短蛋白水解肽（通常：7-20个氨基酸残基）成功地完成了许多蛋白质测序和蛋白质组学研究，该序列被称为”霰弹枪“或”自下而上“的方法。虽然BU方法继续作为蛋白质组学/蛋白质测序的求战，但由于BU方法中的含苦，因此，在没有蛋白水溶性的情况下序列无蛋白质的序列序列的序列序列的序列序列的序列序列的序列序列序列是由于BU方法的歧义。 ，蛋白质推理问题，鉴定蛋白质ortms和后期修饰的发现（PTMS）。高通量TD方法（TD蛋白质组学）尚未在其初期阶段。然而，纯化的完整蛋白质的Td表征对于检测PTM是有用的。希望克服勃美的陷阱的陷阱，提出了另一个名为“下下（MD）”方法的概念。与Bu类似，MD方法还涉及蛋白水解，但以限制性的方式涉及产生比BU研究中通常获得的蛋白水解肽的蛋白水解肽，从而提供更好的序列覆盖。在这方面，已经使用了特殊的蛋白酶（OMPT，SAP9，IDES），其可以切割蛋白质以产生更长的蛋白水解肽。通过审查目前在文献中存在的充分证据，这些证据主要在于组蛋白和抗体的PTM表征，在本文中，我们突出了MD方法的突出特征。因此，我们倾向于要求MD概念在未来对于各种研究领域可能具有广泛的应用，例如临床，生物制药（包括PTM分析）甚至用于包括治疗蛋白的蛋白质的一般/常规表征，但不仅限于分析组蛋白或抗体。

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