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Figure S1. Theoretical fragmentation patterns of the linearized branched form and cross-linked form of a SUMO1-modified peptide. (a) The linearized branched form of the SUMO1-modified peptide. Theoretical fragmentation of the linearized branched peptide would produce fragmentation ions similarly to a linear peptide. The sequence between the N-terminus and the modified lysine residue of the target peptide would produce incorrect fragmentation ions. (b) The cross-linked form of the SUMO1-modified peptide. Both the target peptide and the remnant would produce fragmentation ions during CID.

Figure S2. Workflow of UblSearch. (1) Identifying all the candidate peptides for a given MS/MS spectrum. UblSearch finds all the candidate peptides within a given mass tolerance, including linear and cross-linked peptides, from the created combinatorial database for each MS/MS spectrum. (2) Generating a theoretical fragment pattern for each candidate peptide. For the linear peptides, their theoretical fragment patterns are the same as normal database searches. For the cross-linked peptide, UblSearch initially considers the remnant as the variable modification on the miscleaved lysine residue within the target peptide (e.g., K2 or K7). A new fragmentation model for the Ub/Ubl conjugation peptides is used then to generate correspondingly theoretical fragment ions from the target peptides and the remnants (Supplementary Fig. 1B). (3) Scoring the candidate peptides and calculating the expectation value for the peptide with the highest score. UblSearch uses the X!Tandem scoring scheme to find the peptide matching best with the given MS/MS spectra and calculates the expectation value of the peptide identification.

Figure S3. Mass Spectrum ID 583 was successfully matched with MQIFVK[Ub_LysC]TLTGK by the improved workflow. Four of the top five peaks and 33% of the total intensity of all peaks in the spectrum were successfully matched with the fragment ions generated from the cross-linked form.

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