Phosphopeptide Enrichment Techniques: A Pivotal Step for Phosphoproteomic Studies

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.