Future, it is foreseeable that derivatization and affinity purification may be
Future, it is foreseeable that derivatization and affinity purification may be increasingly combined to achieve purification and identification of novel PTMs. One example follows. Secretion of proteins from tissue, such as from the brain into CSF, is a major source of biomarkers in living subjects. Analysis by MS of proteins secreted from cells, tissue, or organisms under certain physiological or pathological conditions is referred to as secretomics. The AG-490 site secretome typically incorporates PTMs such as signal peptide cleavage, among others, and constitutes an important class of proteins that control and regulate multiple physiological processes such as endocrine, paracrine, and autocrine signaling. This makes it a clinically relevant source for biomarkers and therapeutic targets. Although promising, most secretome studies were carried out using in vitro tissue rather than in vivo [60]. In vitro, both contamination by nonstandard secreted proteins and release of intracellular proteins contribute to the proteome detected [61]. However, derivatization shows promise in overcoming these problems. As mentioned above, N-terminal peptide derivatization for identification has been applied to the secretome, and there is also a fascinating development recently in a method called secretome protein enrichment with click sugars (SPECS), which identified veritable in vivo beta secretase substrates in brain [62], using exactlyRen et al. Translational Neurodegeneration 2014, 3:23 http://www.translationalneurodegeneration.com/content/3/1/Page 5 ofthe combination of derivatization and affinity purification mentioned above.2.4 Identification by orthogonal parallel approaches, e.g. oxyblot/MSfollowing exposure to environmental toxicity which may precede loss of beneficial (or gain of toxic) function on the part of the modified protein [75].2.5 Peptide affinity purificationOxidative stress resulting in oxidative damage of proteins is a common event in neurodegeneration. A number of studies [63-73] utilize parallel detection by, for example, two-dimensional polyacrylamide (2D-PAGE) oxyblot analysis, in which carbonyl moieties (aldehydes and especially ketones, both of which normally are not present on proteins) are reacted with dinitrophenylhydrazine to form dinitrophenylhydrazone (DNP) adducts. Then, following two-dimensional electrophoresis and transfer to membrane, a DNP-recognizing antibody is capable of detecting oxidation event(s) on one or more protein spots, where each spot is thought to represent a single, unique protein species. Then, in parallel analysis, these studies proceed to identify the same PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/26437915 gel spots from a duplicate stained gel using MS [9]. Other versions of this parallel detection approach have been applied to the identification of, protein adducts occurring downstream of lipid peroxidation, where Michael adducts of alpha, beta-alkenal products of free radical mediated lipid peroxidation can attack proteins [69], and other modifications, such as advanced glycation end product protein conjugates. Since these modifications of proteins occur stochastically, and often on any of the 20 amino acid residues–as is the case for carbonylation–with limited site specificity as opposed to other PTMs which are imparted with enzymatic specificity, the abundance of any one chemically pure modification for detection by MS is limited, and thus a “guilt by association” approach may be the best available method for detection of these PTMs. A parallel immunobl.