Formic acid

Urea, DL-dithiothreitol, iodacetamide, IPG buffer, and formic acid were purchased from GE (USA); SDS (sodium dodecyl sμlfonate), Tris (Tris(hydroxymethyl)aminomethane), TCA (Trichloroacetic acid), AP (Ammonium persμLfate) and tetramethylethylenediamine were purchased from Amresco (USA); TEAB (triethylammonium bicarbonate) and Coomassie Brilliant Blue G-250 were purchased from Sigma (USA); trypsin was purchased from Promega (USA); acetonitrile, HPLC grade and H₂O were purchased from Thermo (USA).

Cross-linking was performed with either succinimidyl 4,4′-azipentanoate (SDA, Thermo Scientific) or succinimidyl 6-(4,4′-azipentanamido)hexanoate (LC-SDA, Thermo Scientific) reagent at an equimolar ratio with total TRAPP lysines in a buffer consisting of 20 mM HEPES pH 7.5, 150 mM NaCl, 4.6% glycerol, 0.02% CHAPS, and 0.5 mM TCEP, following the method described in ref. ^{57 (link)}. Briefly, available lysines were coupled to the reagent in a 10 min reaction at room temperature followed by 5 s of pulsed laser photolysis at 355 nm. Reactions were then quenched with 50 mM ammonium bicarbonate. Cross-linked samples were denatured, alkylated with 40 mM chloroacetamide (Sigma), and then digested with trypsin (MS-grade; Thermo Scientific) at a 1:20 protein-to-enzyme ratio for 4 h at 37 °C. Digestion was quenched with 0.5% formic acid (Thermo Scientific) and size exclusion chromatography (SEC) used to enrich for cross-linked peptides (Superdex Peptide PC 3.2/30 SEC column, GE Healthcare), using a mobile phase of 30% ACN, 0.1% formic acid. Fractions were collected, lyophilized, and resuspended in 0.1% formic acid for LC–MS/MS analysis.