Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Campinas, Brazil.
Adv Exp Med Biol. 2022;1382:17-27. doi: 10.1007/978-3-031-05460-0_2.
Many pathological conditions are caused by dysregulation of cell signaling, which can generate a cascade of abnormal responses and completely change the functions of a cell or tissue. A large portion of the regulation of these signals is via protein phosphorylation, in which cell responses can be activated or inhibited. Proteins that are both downstream and upstream of a phosphorylated protein can be modified, altering metabolism and other biological processes. Recently, the number of phosphoproteomic studies based on mass spectrometry has increased, constantly aiming to obtain a higher coverage of proteins and increase the number and location of their phospho-sites, as well as better understand their respective phosphorylation states. In this way, it is possible to better understand biological processes as a whole and their roles in cellular dysfunctions and diseases. To study changes at the phosphoproteome level, the stochiometric imbalance between the non-phosphorylated and phosphorylated peptides must be overcome, since higher quantities and comparatively better ionization of non-phosphorylated peptides can suppress the ion signals of the phosphorylated peptides. It is for this reason that phosphophopeptides are rarely found in samples that did not pass through a phospho-enrichment step, highlighting the importance of performing enrichment steps in phosphoproteomic studies. The numbers of identified phosphopeptides and phosphorylation sites are extremely important to the quality of a phosphoproteomic analysis; therefore, the efficiency of the enrichment process is critical. Here, phospho-enrichment techniques are presented to offer insight into the applicability of these methods to different experiment types and consequently support the growth of phosphoproteomic studies overall.
许多病理状况是由细胞信号转导失调引起的，这会产生一连串的异常反应，彻底改变细胞或组织的功能。这些信号的大部分调节是通过蛋白质磷酸化实现的，细胞反应可以被激活或抑制。磷酸化蛋白质的下游和上游的蛋白质都可以被修饰，改变代谢和其他生物过程。最近，基于质谱的磷酸蛋白质组学研究数量增加，其目的始终是获得更高的蛋白质覆盖率，并增加其磷酸化位点的数量和位置，以及更好地了解它们各自的磷酸化状态。通过这种方式，可以更好地了解整个生物过程及其在细胞功能障碍和疾病中的作用。为了研究磷酸蛋白质组水平的变化，必须克服非磷酸化和磷酸化肽之间的化学计量不平衡，因为非磷酸化肽的数量较高且相对较好的离子化可以抑制磷酸化肽的离子信号。正是由于这个原因，未经磷酸化富集步骤的样品中很少发现磷酸肽，这突出了在磷酸蛋白质组学研究中进行富集步骤的重要性。鉴定的磷酸肽和磷酸化位点的数量对磷酸蛋白质组分析的质量极为重要；因此，富集过程的效率至关重要。本文介绍了磷酸化富集技术，以期深入了解这些方法在不同实验类型中的适用性，并最终支持整体磷酸蛋白质组学研究的发展。
