Q exactive hf mass spectrometry

The fractions were resolved in 0.1% formic acid and loaded on an in-house packed analytical column (75 μm × 20 cm x 1.9 μm) on an EASY-1200 coupled to Q-Exactive HF mass spectrometry (Thermo Fisher Scientific). Peptides were eluted in a 130 min linear gradient (5–35% solvent B, 0.1% formic acid in 80% acetonitrile) at 300 nl/min. The DDA acquisition settings were as follows: MS1 survey scan was 300 to 1650 m/z with resolution at 60,000 with automatic gain control (AGC) target 3 × 10⁶ with maximum injection time 20 ms. Top 15 precursors were selected for MS2 data acquisition. MS2 resolution was 15,000 with an AGC target set to 1 × 10⁵ with a maximum injection time of 25 ms and 1.4 m/z isolation window, and 27% NCE.

The sample was loaded to the column equilibrated with buffers A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, Dionex) was equilibrated with liquid A by Ultimate 3000 nanoUPLC (Dionex), and the sample was eluted onto an Acclaim PepMap RSLC C18 analytical column (75 μm × 25 cm, Dionex) at a flow rate of 300 nl/min. The liquid-phase gradient was given as follows: 0–6 min, where the linear gradient of buffer B was from 2 to 10%; 7–51 min, where the linear gradient of buffer B was from 10 to 20%; 51–53 min, where the linear gradient of buffer B was from 20 to 80%; 53–57 min, where the linear gradient of buffer B solution was held at 80%.
Mass analysis was performed by nano-spray ionization-mass spectrometry (NSI-MS) and Q-Exactive HF mass spectrometry (Thermo Scientific). The intact peptide was detected by the Orbitrap, the scanning range was set to 250–1,500 m/z, the automatic gain control (AGC) target was 3E6, the resolution was 70,000, the max injection time (IT) was 250 ms, and the dynamic exclusion time was 15 s. The peptide was selected and fragmented for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap, the resolution was 17,500, AGC was 1E5 or 5E4, and the maximum IT was 100 or 200 ms. LC-MS was performed by Micrometer Biotech (Hangzhou, China).

The digested peptides were subjected to LC-MS/MS in a Q-Exactive HF mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). The conditions of LC-MS/MS are furnished in the Supplementary File S2. The protein spectra thus obtained were analyzed using the software Proteome Discoverer Version 2.2.0.388 (Thermo Fisher Scientific, Waltham, MA, USA) with the SEQUEST HT engine. The MS/MS spectra of the peptides were analyzed against a Universal Protein Resource (UniProt) database organism: Staphylococcus pasteuri; enzyme: trypsin; search parameters for identification of sequences were a precursor mass tolerance of 5 ppm and a fragment mass tolerance of 0.05 Da. The target FDR (strict) and target FDR (relaxed) were set at 0.01, and the validation was based on the q-value. Peptides sequenced by MS/MS were searched and highlighted on the aligned protein sequences using a macro code snippet. The profiled protein mass spectrometric data have been deposited at the ProteomeXchange database (Accession number: PXD023616).