Post-translational modifications (PTMs) of proteins are fundamental processes that trigger, regulate and terminate most of the cellular mechanisms by covalent attachment of chemical moieties to substrate proteins. Conjugation to the Small Ubiquitin-like MOdifier (SUMO) is a highly conserved and regulated posttranslational modification that is often restricted to highly specific cellular events and a number of obligatory nuclear regulatory processes. Although much is known about the processes of SUMOylation and deSUMOylation, exactly how it mediates its various functions and 4 different human isoforms remains unclear. This is due in part to the extremely low abundance of modified proteins (about 2% of a given substrate will become modified), and the large expense of time and effort required to identify and characterize these SUMOylated proteins by current methods.
This thesis presents the development and application of an ion mobility mass spectrometry (IMMS)-based method of screening for isopeptides from SUMOylated substrates. The model conjugates poly-SUMO2 and poly-SUMO3 were digested by trypsin/chymotrypsin to produce small linear peptides and isopeptides with a QQQTGG tag on the substrate lysine. Using solution conditions to promote higher charge states and IMMS, mass spectra for the larger isopeptides with +3 charge state were extracted from the mass mobility plot. These isopeptides were confirmed using tandem mass spectrometry. Interestingly, a neutral loss of 17 Da was observed for all isopeptides, which resulted from the loss of ammonia due the acid-catalyzed rearrangement of the N-terminal glutamine residue of the isopeptide tag. The method was also applied to in vitro SUMOylated RanGAP1 and Sp100 fragments, two known SUMO substrates.
IMMS was also applied to the identification of tyrosine-sulfation in peptides using an approach that was developed for phosphorylated peptides. Analysis of a trypsin digest of phosphorylated a-casein showed the expected decrease in drift time. A similar reduction in drift time was observed for tyrosine sulfated synthetic peptide DY*MGWMDF-NH2 (Spp) relative to its unmodified analog (DSpp). This decrease in drift time was a result of the modified peptides becoming more compact due to electrostatic interaction between the negatively charged modification and the positively charged N-terminus. The tyrosine-sulfated peptide and its unmodified analog DSpp were spiked in equimolar amount into a digest of bovine serum albumin. The band corresponding to the modified peptide was clearly identified in the much more complex mass mobility plot with a significant shift in the drift time for the tyrosine-sulfated peptide.
IMMS is a highly promising method for analysis of PTMs due to its sensitivity and selectivity. In this work we demonstrated its utility in screening protein digests for isopeptides from SUMO-conjugated substrates and tyrosine-sulfated peptides using model analytes. Future work will involve testing the limits of these methods including peptide sequence, peptide size and detection limits in more complex cellular matrices.