Ltq orbitrap xl ms

45 μl purified protein complex (4.5 mg/ml for Tpz1-Ccq1; 4 mg/ml for Tpz1-Poz1; 1.25 mg/ml for Tpz1-Pot1) was mixed with 5 μl DSSO (dissolved in DMSO) to the final concentration as indicated in results. Crosslinking was performed for 30 min and quenched with 2 μl 1 M Tris-HCl (pH 8.0) for 15 min. The cysteine residues were reduced with 4 mM TCEP and alkylated with 20 mM iodoacetamide in dark, followed by terminating alkylation reaction with 20 mM cysteine for 30 min. The crosslinked proteins were digested overnight at 37°C with trypsin (2%, w/w) and chymotrypsin (5%, w/w), separately. Crosslinked peptides were analyzed by LC MSⁿ utilizing an LTQ-Orbitrap XL MS (Thermo Fisher Scientific) coupled online with an Easy-nLC 1000 (Thermo Fisher Scientific) as described (Kao et al., 2011 ). Each MSⁿ experiment consists of one MS scan in FT mode (350–1,400 m/z, resolution of 60,000 at m/z 400) followed by two data-dependent MS² scans in FT mode (resolution of 7,500) with normalized collision energy at 20% on the top two MS peaks with charges 3⁺ or up, and three MS³ scans in the LTQ with normalized collision energy at 35% on the top three peaks from each MS².

Cytochrome C and lysozyme were purchased from Sigma. Cross-linking of these proteins followed the same procedure as described previously.^{30 (link)}Cross-linked peptides were analyzed by LC MSⁿ utilizing an LTQ-Orbitrap XL MS (Thermo Fisher, San Jose, CA) coupled on-line with an Easy-nLC 1000 (Thermo Fisher, San Jose, CA) as previously described.^{3 (link),7 (link)} Each MSⁿ experiment consists of one MS scan in FT mode (350–1400 m/z, resolution of 60,000 at m/z 400) followed by two data-dependent MS² scans in FT mode (resolution of 7500) with normalized collision energy at 20% on the top two MS peaks with charges at 3+ or up, and three MS³ scans in the LTQ with normalized collision energy at 35% on the top three peaks from each MS².