Search for KQ crosslinked peptides with Batch-Tag software in protein prospector version 6.2.1

The *.raw data files from the Orbitrap Fusion Lumos Tribrid were converted to *.mgf files using MSConvert (ProteoWizard Tools from SourceForge). The *.mgf files were analyzed using Batch-Tag Web [24] on the Protein Prospector website []

We created a specialized user database that included five abundant proteins from those identified in the Sus scrofa MAP-rich tubulin sample, plus neurofilament heavy polypeptide (see S1 Table). The six proteins were: tubulin alpha-1A chain (NP_001302639), tubulin beta-4B chain (XP_003122400), microtubule-associated protein 2 isoform X8 (XP_013839898), microtubule-associated protein 1B isoform X1 (XP_003134080), microtubule-associated protein Tau isoform X16 (XP_020922473), and neurofilament heavy polypeptide isoform (XP_005670835). Sequences were pasted into the User Protein Sequence window of Batch-Tag Web in FASTA format. MS/MS data were searched against this 6-protein database with Batch-Tag Web for isopeptide crosslinks between lysine (K) and glutamine (Q).

The search parameters were as follows. 1) Database: User protein. 2) User Protein Sequences: FASTA files from NCBI Protein Database for the user proteins were pasted into this window. 3) Precursor Charge Range: 2 3 4 5. 4) Masses: monoisotopic. 5) Parent Tol: 20 ppm. Frag Tol: 30 ppm. 6) Instrument: ESI-Q-high-res. 7) Digest: Trypsin. 8) Max missed cleavages: 3. 9) Constant Mods: Carbamidomethyl (C). 10) Variable Mods: Oxidation (M). 11) Expectation Calc Method: None. 12) Mass Modifications: range -18 to 3883 Da. (Formation of the isopeptide bond between K and Q is accompanied by loss of 17 Da due to loss of ammonia. The -18 mass modification allows for loss of water (-18) and loss of ammonia (-17). 13) Check mark in boxes K and Q. 14) Check mark in box Uncleaved. Checking the Uncleaved box avoids false candidates in which a C-terminal lysine is reported as the crosslinked lysine. Such reports are false because trypsin does not cleave modified lysines. 15) Crosslinking; Link Search Type: User Defined Link. 16) User Defined Link Parameters; Link AAs: K, Protein N-term>Q. 17) Bridge Elem Comp: N-1 H-3. S1 Fig is a screen shot of the Batch-Tag Web page showing the search parameters for KQ crosslinked peptides.

Protein Prospector identifies peptide crosslinks via a mass modification process wherein the mass of one peptide plus the crosslink mass is considered as a modification of the other peptide. This is an efficient process that minimizes the search space, but it does not recognize masses that contain fragments of both peptides, or fragment masses that involve unexpected amino acid modifications. Identifying these masses requires manual inspection of the fragmentation pattern.