LC-MS analysis of acetylated peptides and crosslinked peptides

Peptide samples were analyzed by LC–MS using an Easy-nLC (Thermo Fisher Scientific) coupled to a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto a 3 cm × 100 μm inner diameter–fused silica trap column packed with a stationary phase consisting of 5 μm Reprosil C8 particles with 120 Å pores (Dr. Maisch GmbH) with a flow rate of 2 μL/min of mobile phase consisting of solvent A (H₂O containing 0.1% formic acid) for 10 minutes. Peptides were then fractionated over a 60 cm × 75 μm inner diameter fused silica analytical column packed with 5 μm Reprosil C8 particles with 120 Å pores by applying a linear gradient from 95% solvent A, 5% solvent B to 60% solvent A, and 40% solvent B over 120 minutes at a flow rate of 300 nL/min. Eluting peptides were ionized by electrospray ionization by applying a positive 2.2 kV potential to a laser pulled spray tip at the end of the analytical column. The mass spectrometer was operated using a top 20 data-dependent acquisition method with a resolving power setting of 70,000 for MS1 and 17,500 for MS2 scans. Additional settings include an AGC target value of 1 × 10⁶ with a maximum ion time of 100 ms for the MS1 scans and an AGC value of 5 × 10⁴ with a maximum ion time of 100 ms for the MS2 scans. Charge state exclusion parameters were set to only allow ions with charge states from 2+ to 8+ to be selected for MS2. Ions selected for MS2 were isolated with a 3 m/z window and fragmented by HCD using a normalized collision energy setting of 27. Ions for which MS2 was performed were then dynamically excluded from further selection for MS2 for 30 seconds.

Identification and quantification of acetylated peptide and protein Raw MS data were processed using MaxQuant software (ver.1.6.17.0). Acetylated peptides were identified using the integrated Andromeda search algorithm (35). Mouse samples were searched against Uniprot mouse protein sequence database (downloaded on April 28, 2018). Human samples were searched against Uniprot human protein sequence database (downloaded on April 28, 2018). rCKM data were searched against Uniprot Ecoli FASTA database (downloaded on August 6, 2019) spiked with rCKM sequence. The following parameters were used for searches: trypsin specificity with a maximum of 4 missed cleavages; carbamidomethylation of cysteine as fixed modification; oxidation of methionine, acetylation of protein N-term, and acetylation of lysine as variable modifications; instrument and MS/MS analyzer parameters used the default settings for Orbitrap; protein quantitation used only unmodified peptides and oxidized methionine; and all peptide and protein identifications filtered using 1% FDR cut-off from reversed sequence decoy database. Acetylated peptides were further filtered with localization probability score greater than 90%.

Crosslinked peptide samples were analyzed with the same LC-MS system described above using the same LC gradient conditions and mass spectrometer parameters with the following differences. The mass spectrometer was operated with a resolving power setting of 70,000 for MS1 and MS2 scans. Charge state exclusion parameters were set to only allow ions with charge states from 4+ to 8+ to be selected for MS2. Ions selected for MS2 were isolated with a 3 m/z window and fragmented by HCD using a normalized collision energy setting of 30.