Proteolytic Enzyme and Chemical Digestion with Trypsin and Acid
An elegant dual-digestion strategy involving trypsin and then microwave-assisted acid chemical digestion with 12.5% acetic acid to selectively target cleavages C- terminal to aspartic acid residues by acid hydrolysis has been developed . The selectivity involved in this approach facilitates a reduction in the length of these SUMO-(1)/(2)/(3) iso-chains of their respective SUMO-isopeptides in order to generate SUMO-isopeptides amenable to analytical analysis. Cleavage at the aspartic acid 86 position on the SUMO(1) iso-chain results in the generation of an iso-chain containing 11 amino acids, VIEVYQEQTGG. This is also the case when C-terminal cleavage of the aspartic acid residues at positions 85 and 82 on the SUMO(2) iso-chain and at positions 84 and 81 on the SUMO(3) iso-chains takes place, resulting in the generation of iso-chains containing either 8 (VFQQQTGG) or 11 amino acids (TIDVFQQQTGG). nLC-nESI-LTQ Orbitrap with low-energy linear ion-trap CID or ETD-based LC-MS/MS and MALDI- TOF/TOF with high-energy CID-based MS and MS/MS analysis have been employed to analyze these three isopeptides: di-SUMO(2)-isopeptides, di- SUMO(3)-isopeptides, and the SUMO(1)-isopeptide derived from the dual digestion of a SUMO(1)ylated E2-25K protein prepared in vitro. LC-MS/MS with ETD analysis of these three types of isopeptides facilitated ETD MS/MS spectra amenable to manual interpretation by matching theoretical ions generated from protein prospector. Since ETD fragmentation patterns occur in a predominantly sequence-independent manner, the ETD MS/MS displayed the generation of a characteristically comprehensive series of abundant c/c'- and z/z'-type product ions from both the backbone and iso-chains. This enabled the comprehensive structural elucidation of SUMO(1)/(2)/(3)-isopeptides (Figure 6.4).
Figure 6.4 An ETD MS/MS spectrum of a SUMO(1)-isopeptide derived from the aspartic acid cleavage/trypsin digestion of the SUMO(1)ylated E2-25K protein. Source: Omoruyi, 2012 . Reproduced with permission from Wiley.
By comparison, LC-MS/MS with low-energy CID and MALDI-TOF/TOF high-energy CID analysis of all three types of isopeptides facilitated CID MS/ MS with less complex spectra than observed when analyzing similar SUMO- isopeptides with full-length iso-chains with CID, enabling the comprehensive structural elucidation of these isopeptides. Both instrument platforms and subsequent fragmentation techniques were very effective and generated MS/ MS spectra with low complexity and predominantly facilitated a good balance of fragmentation of product ions between the backbone of the isopeptide and their respective iso-chains, critical for comprehensive structural elucidation. The results render this dual-digestion process using trypsin and 12.5% acetic acid in combination with LC-MS/MS with ETD and CID and MALDI-TOF/ TOF with high-energy CID as an effective approach to analyzing these SUMO(1)/(2)/(3)-isopeptides in simple digestion mixtures. Assessment of the diagnostic value of product-ion fragmentation from the iso-chains for postacquisition extraction could enable the demonstration of this strategy in more complex digestion mixtures where unknown SUMO-isopeptides are present. The selectivity of the acid hydrolysis can result in the generation of the multiple forms of a particular SUMO-isopeptide. This may have an adverse impact on the abundance of these isopeptides by diluting the ion current between multiple signals in data-dependent acquisitions (DDA) of more complex samples. In addition, the isopeptides generated using this approach contain multiple glutamine residues. The number of deamidation events available for input into a bioinformatic algorithm represents an inherent compromise. Therefore, if this approach was applied to the analysis of putative SUMO-isopeptides in more complex samples, it may benefit from data-independent acquisition (DIA) approaches where data acquisition is unbiased toward precursor ion signal abundance.