Home Health Analysis of Protein Post-Translational Modifications by Mass Spectrometry
Concluding Remarks and Recommendations
Wild-type mammalian protein ubiquitination and SUMOylation are highly regulated PTMs, involved in an extensive range of important biological functions both independently and codependently with each other, and a range of other PTMs. Dysregulation of ubiquitination and SUMOylation pathways has been shown to have a substantial impact on the progression of a number of disease states.
MS-based analysis is a key analytical technique in the identification and detection of the isopeptides derived from these proteins that have been targeted by these two PTMs, essentially enabling critical information to be obtained on a high-throughput scale in order to increase the depth of our understanding of their biological roles. However, MS-based analysis is challenging due to the inherent physicochemical nature of each modification. The MS analysis of each PTM presents different challenges, which effectively render the isopeptides they are present on difficult to robustly structurally elucidate and detect by MS-based proteomic analyses. There have, therefore, been significant efforts to develop improved MS-based proteomic strategies.
First, the complexities arising from MS-based and bioinformatic analyses resulting from SUMO-isopeptides containing highly charged full-length tryptic iso-chains and generating highly complex low-energy CID MS/MS spectra was negated by the development of the following strategies to improve the overall analytical amenability (i) by use of alternative low-energy-based fragmentation techniques such as IRMPD, ECD, and high-energy CID to facilitate an improvement in the complexity of their MS/MS spectra; (ii) the MS-based and bioinformatically CRA(K)-enabled analyses of more analytically amenable SUMO-isopeptides derived from atypical tryptic cleavage, under low-energy CID conditions; (iii) the MS-based analysis of analytically amenable SUMO-isopeptides derived from dual-digestion strategies, under low-energy
CID, ETD, and high-energy CID conditions; and (iv) the MS-based analysis of chemically derivatized SUMO-isopeptides derived from atypical tryptic cleavage, in order to facilitate the generation of modification-specific diagnostic product ions providing direct sequence coverage of the QTGG and TGG isochains under low-energy CID conditions. Strategies involving (ii)-(iv) demonstrated marked improvements in the generation of analytically amenable SUMO-isopeptides and their subsequent structural elucidation. In particular, one of the chemical modification-based strategies involved in (iv), termed MEDUSA, enabled the simultaneous detection and quantification of Ub- isopeptides in MS mode and also in MS/MS mode based on the generation of the robust QTGG and TGG iso-chain modification-specific diagnostic ions.
Second, the complexities arising from the MS-based and bioinformatic analyses of Ub-isopeptides containing GG iso-chains, predominantly resulting from CID conditions, were negated by the development of the following strategies to enhance the overall MS analysis of Ub-isopeptides: (i) alternative digestion strategies to produce a longer LRGG iso-chain in order to facilitate the generation of a modification-specific diagnostic ion from the iso-chain under low-energy CID conditions; (ii) chemical modification of Ub-isopeptides in order to facilitate the generation of a series of diagnostic ions indicating the presence of GG iso-chain modification under high-energy CID conditions; and (iii) chemical derivatization of Ub-isopeptides in order to facilitate the generation of modification-specific diagnostic a'/b'-type product ions in RUbI and MEDUSA approaches, providing specific sequence coverage of the GG iso-chain under low-energy CID conditions. The strategies involved in (ii) and (iii) were marked improvements in enhancing the analysis of Ub-isopeptide structure and subsequent robust and confident identification. In particular, one chemical modification-based strategy involved in (iii), termed MEDUSA, enabled the simultaneous detection and quantification of Ub-isopeptides in MS mode and also in MS/MS based on the generation of the robust GG isochain modification-specific diagnostic ions.
In addition to the improvements made by the development of these methods to the MS-based proteomic analysis of SUMO and Ub-isopeptides, additional progress has been made in the development of effective enrichment strategies for the biological- and chemical-based enrichment of Ub- and SUMO-isopeptides to assist with improving global MS-based proteomic analyses of protein ubiquitination and SUMOylation. The most effective enrichment strategies include the (i) utilization of monoclonal antibodies to specifically enrich for both Ub- and SUMO-isopeptides with internal lysine residues bearing a GG iso-chain, (ii) the development of a COFRADIC-based strategy to specifically enrich for approach-generated Ub-G-isopeptides, and (iii) a protease-reliant strategy termed PRISM for the analysis of protein SUMOylation.
Recommendations for the improvement of SUMO- and Ub-isopeptide analysis would involve (i) digestion strategies, which are capable of generating a distinguishable single version of a SUMO(1) iso-chain and a SUMO(2/3) iso-chain, which are both analytically amenable to MS-based proteomic analyses; (ii) development of additional monoclonal antibodies that recognize lysine residues bearing both of these iso-chains and/or the chemically derivatized iso-N-terminal forms of these iso-chains to reap the additional benefits of the chemically facilitated diagnostic ions under low-energy CID conditions for both enhanced detection and subsequent quantification in MS/MS mode (this would also apply to Ub-isopeptides); and (iii) development of bioinformatic software to analyze the potentially large list of putative SUMO and Ub- isopeptide candidates that would be generated from postacquisition ion chromatogram extraction of diagnostic ions generated in the RUbI and MEDUSA approaches, should these approaches be applied to MS-based global analyses.
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