Plant et al. S1 Protein identifi cation was achieved by submitting the MGF fi les to a server running Mascot sequence database search software (version 2.2; Matrix Science Ltd.) and the mzXML fi les to a server running Sagen Sorcerer (version 1.0; Sagen Research Inc.), an implementation of the Sequest sequence database search software. Searches on both machines were run against a custom database created by adding the sequences of the synthesized rat Kv2. In each search, a peptide precursor mass tolerance of 5 ppm was used, allowing for modifi cations of peptide mass resulting from additions such as Gly-Gly (+114.04292 Th), methionine oxidation (15.99 Th), as-paragine deamidation (+0.984016 Th), and cysteine carbamido-methylation (57.021464), as well as up to three missed cleavages and strict adherence to tryptic digestion rules. The search results obtained from the two separate searches were then loaded into the Scaffold software package (version 6.09; Proteome Software), and peptides with scores of 95% confi dence or better were used to confi rm peptide assignments. Confi rmation that sumoylated peptides had been detected was achieved by programming the LTQ to specifi cally accumulate precursor masses of expected peptides and produce dedicated tandem MS of only those ions throughout a chromatographic run. A method was created using the LC conditions described to repetitively run a multi-event experiment consisting of a single MS scan event using the FT-ICR, and then up to three additional dedicated MS/MS scan experiments on the selected peptide masses. Data obtained from these experiments was then extracted as discussed previously and appended to the data from the earlier data-dependent runs and subjected to additional searches and manual evaluation. Such evaluation included assessment of molecular weight, spectral quality, and the consistency of the peptide retention time in relation to unmodifi ed versions of the peptide. Mass spectrometry (MS), detailed methods The cDNA sequence encoding rat Kv2.1 (available from Gen-Bank/EMBL/DDBJ under accession no. NP_037318.1) C-terminal residues 411–853 (Kv2.1 411-853) was cloned into a modifi ed pET28a vector (Invitrogen) carrying six N-terminal histidine residues and a tobacco itch virus (TEV) protease cleavage site in place of the thrombin site. Kv2.1 411-853 modifi ed by SUMO1 was produced using a dual vector system (Uchimura et al., 2004). Escherichia coli strain BL21 (DE3) was cotransformed with Kv2.1 411-853 in the modifi ed pET28 and pT-E1E2S1 (Uchimura et al., 2004) carrying mouse E1 (as a linear fusion product of Aos1 and Uba2) …