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DUBs, ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) and the ubiquitin-specific peptidase 14 (USP14), were detected in the cytoplasm of a high proportion of DLBCL tumor cells [329]

DUBs, ubiquitin carboxyl-terminal hydrolase L5 (UCHL5) and the ubiquitin-specific peptidase 14 (USP14), were detected in the cytoplasm of a high proportion of DLBCL tumor cells [329]. to the proteome through the addition of biochemical moieties to specific residues of proteins, altering their structure, function and/or localization. Mass spectrometry (MS)-based techniques are at the forefront of PTM analysis due to their ability to detect large numbers of modified proteins with a high level of sensitivity and specificity. The low stoichiometry of altered peptides means fractionation and enrichment techniques are often performed prior to MS to improve detection yields. Immuno-based techniques remain popular, with improvements in the quality of commercially available modification-specific antibodies facilitating the detection of altered proteins with high affinity. PTM-focused studies on blood cancers have provided information on altered cellular processes, including cell signaling, apoptosis and transcriptional regulation, that contribute to the malignant phenotype. Furthermore, the mechanism of action of many blood malignancy therapies, such as kinase inhibitors, entails inhibiting or modulating protein modifications. Continued optimization of protocols and techniques for PTM analysis in blood malignancy will undoubtedly lead to novel insights into mechanisms of malignant transformation, proliferation, and survival, in addition to the identification of novel biomarkers and therapeutic targets. This review discusses techniques utilized for PTM analysis and their applications in blood cancer research. and wild-type -lytic protease (WaLP) [137,138,139]. A method derived to avoid the limitations associated with mutant SUMO peptides, protease-reliant identification of SUMO modification (PRISM), involves the use of His-tagged SUMO, acetylation and SUMO-specific proteases for sumoylation site identification [140]. Furthermore, Hendriks et al. recognized 14,869 SUMO2/3 sites in human cells by incorporating a serial digestion workflow using Lys-C and Asp-and peptide-level immunoprecipitation followed by LCCMS/MS [139]. SUMO-interacting motifs (SIMs) and recombinant SUMO-binding entities (SUBEs) have been adopted for the enrichment and identification of endogenous poly-SUMO proteins [141,142]. SILAC, iTRAQ and LFQ have been utilized for quantitation of sumoylation [138,143,144]. Numerous bioinformatic tools such as SUMmOn, SUMOhydro, SumSec, etc., have been launched for sumoylation site identification [129,145,146,147]. 2.5. Acetylation and Methylation Acetylation and CZC-25146 hydrochloride methylation are prominent PTMs that play functions in many cellular processes including cell signaling, metabolic pathways and most notably, DNA-protein interactions. The acetylation of histone proteins is usually a crucial process that influences the convenience of DNA to the transcriptional machinery. The transfer of an acetyl group to the -amino group at the N-terminus of the protein is an irreversible modification, whereas acetylation at a lysine residue is usually reversible. Acetylation is usually catalyzed by acetyltransferases and lysine acetylation may be reversed by lysine deacetylases [148]. Methylation mainly occurs on lysine and arginine amino acids. However, other residues such as histidine, CZC-25146 hydrochloride proline, and glutamine may also be subject to methylation. Methylation is usually catalyzed by lysine or arginine methyltransferases and reversed by demethylases [149] Research on acetylation and methylation is usually often focused on histone modifications which requires specific sample preparation methods, as explained above [22]. Radiolabeling of proteins with radiolabeled acetyl groups from 14C or 3H-acetyl CoA allows the detection of acetylated proteins by autoradiography after gel electrophoresis is performed [150,151]. However, the use of this technique has dropped in recent years due to the extra security precautions required when using radioactive molecules [148]. Immuno-based techniques are commonly used to detect acetylation with many highly specific and sensitive anti-acetyl lysine antibodies currently available [152]. Several recent studies have expanded on standard FCM for acetylation analysis using FCM-based techniques including single-cell imaging circulation cytometry and mass cytometry (CyTOF) [153,154,155]. Once again, acetylated proteins are enriched prior to LCCMS/MS. Immunoaffinity purification of acetylated peptides using specific antibodies is the most common and effective method of enrichment [22,156,157,158]. Combined fractional diagonal chromatography (COFRADIC) is also a popular technique CZC-25146 hydrochloride that specifically enriches N-terminal acetylated proteins through the derivatization of main amines [159,160]. The relatively new method, stable-isotope protein N-terminal acetylation quantification (SILProNAQ) allows Rabbit polyclonal to RAB4A the direct quantitation of N-terminal peptides, although few studies have reported using this technique [161,162]. The previously discussed fractionation technique, SCX is commonly utilized for acetylation analysis. Tandem MS identifies acetylation by the presence of a 42 Da mass shift on.