Mamalian protein ubiquitination and SUMOylation are reversible post translationalmodifications, which are involved in a multitude of important complex regulatory processeswithin the cell. Current mass spectrometry approaches that involve bottom-up proteomics tocomprehensively analyse these modifications, have proved to be problematic. In this work,analytical approaches are carried out to improve and enhance the comprehensive analysis ofthese modifications.Tryptic proteolysis of ubiquitinated proteins results in the generation of isopeptides bearing adi-glycine (GG) remnant. Current mass spectrometry approaches used to identify theseisopeptides are predominantly reliant on detecting the signature mass shift of the GG remnant(114.043 Da). The lack of sequence information from the GG remnant post MS/MSacquisition results in database search algorithms falsely identifiying these isopeptides.Reductive methylation chemistry was employed to derivatize these isopeptides. Uponcollision induced dissociation of thes isopeptides two robust ions were released from the iso-N-terminus of the GG remnant ; i) an a1' ion at m/z 62.09, corresponding to the G of theremnant and ii) a b2' ion at m/z 147.11 , corresponding to the full GG remnant. Postacquisition data extraction of these unique diagnostic ions demonstrated enhanced selectivitytowards identifying these isopeptides.Tryptic proteolysis of SUMOylated proteins results in the generation of isopeptides bearing asubstantial iso-C-terminal SUMO remnant. The CRA(K) (Consecutive Residue Addition tolysines (K)) approach combined independant use of proteolytic enzymes and unbiasedconsecutive residue addition of amino acids pertaining to these iso-C-terminal SUMOremnants, on all lysine residues. This approach enabled the identification of analyticallyuseful novel wildtype isopeptides derived from the proteolysis of SUMO(1/2/3)ylatedproteins, bearing GG, TGG and QTGG remnants.The analytically useful isopeptides derived from proteolysis of SUMO(2/3)ylated proteinslacked robust diagnostic information from their iso-C-terminal bearing TGG and QTGGremnants. Reductive methylation chemistry was utilised to derivatize these isopeptides andenabled diagnostic a' and b' ions to be released from their iso-N-termini; i) a1' (m/z 133.13),b2' (m/z 262.17) and b4' (m/z 376.22) ions, corresponding to the QTGG remnant and ii) (m/z106.10), b2' (m/z 191.14) and b3' (m/z 248.14) ions, corresponding to the TGG remnant. Postacquisition data extraction of these unique diagnostic ions, enabled comprehensive structuralelucidation of these isopeptides and enhanced selectivity towards identification.