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Home arrow Health arrow Analysis of Protein Post-Translational Modifications by Mass Spectrometry
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Mass Spectrometry Analysis of SUMO-Isopeptides Derived from Proteolytic Digestion

A key aspect in the proteomic workflow for the LC-nESI-MS/MS, direct ESI- MS/MS, or MALDI-MS/MS analysis of these two PTMs is the implementation of an effective sample preparation strategy. In the succeeding text, we will introduce the importance of digestion strategies and discuss the impact that different digestion strategies have on the type of Ub-isopeptide and SUMO- isopeptide that can be generated from the proteins that have been ubiquit- inated and SUMOylated and on their amenability to mass spectrometric analysis.

An important aspect that needs to be taken into consideration in amenability of SUMO-isopeptides and Ub-isopeptides to mass spectrometric analysis is the length and amino acid composition of their iso-chains. The length of the iso-chain is dependent upon the biological proteolytic or chemical digestion technique used in relation to which amino acids are preferentially (i) typically or (ii) atypically cleaved. (i) A typical amino acid cleavage site on a protein is one which the proteolytic enzyme or chemical being used preferentially cleaves at. (ii) An atypical amino acid cleavage site on a protein is one which the proteolytic enzyme or chemical being used preferentially cleaves at less frequently. It is important to remember that they both occur and can be of importance during the stages of mass spectrometric analysis and data interpretation. There are three types of digestion strategy that have been employed for the sample preparation of Ub-isopeptides and SUMO-isopeptides: (i) proteolytic enzyme digestion, (ii) dual proteolytic enzyme digestion, and (iii) proteolytic enzyme and/or chemical digestion.

 
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