Velos pro mass spectrometer

Samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on either a Dionex Ultimate 3000 RSLCnano ultra-high performance liquid chromatography (UHPLC) system (Thermo Fisher Scientific) or an Easy-nLC 1200 (Thermo Fisher Scientific) coupled to an Orbitrap Fusion Tribrid or Velos pro mass spectrometer (Thermo Fisher Scientific). Peptides were first loaded onto either an Acclaim PepMap RSLC nano trap column (Dionex; Thermo Fisher Scientific) or, prior to separation, on a 75-μm by 15-cm Acclaim PepMap rapid-separation liquid chromatography (RSLC) C18 column (Dionex; Thermo Fisher Scientific) using either a 5-to-40% (vol/vol) or 5-to-32% (vol/vol) acetonitrile gradient over 95 or 120 min at 300 nL/min. The eluting peptides were injected directly into the mass spectrometer using a nano-electrospray source. The instruments were operated in a data-dependent mode with parent ion scans (MS1) collected at 120,000 or 60,000 resolution. Monoisotopic precursor selection and charge state screening were enabled. Ions with a charge of +2 or greater were selected for collision-induced dissociation fragmentation spectrum acquisition (MS2) in the ion trap. Sample were run three times to generate technical-replicate data sets.

Proteins were identified using a Thermo Scientific Velos Pro Mass Spectrometer. The digestion enzyme was Trypsin (cleaves on R and K except for C-terminal P obstructing it; not independent; not semi-specific, i.e., fully specific). The Max # accepted missed cleavages: 1; Fixed modifications: Carbamidomethyl (C) (+57), Variable modifications: Oxidation (M) (+16); Precursor tolerance: 0.6 Da; Fragmentation tolerance: 0.5 Da. The software generating the peaklist was Thermo’s ExtractMSN (version 5.0) and the search engine was Mascot v2.4.1. The database searched was SGD (version 2015-01-13; 6713 entries (or proteins) searched). The estimation of false discovery rate (FDR) for the protein level was 0.1% with an FDR of 8.7% to 10.3% at the peptide level calculated using Protein Prophet and Peptide Prophet. The version of Scaffold used to generate the report was 4.11.0. Processed data are shown in Table S1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (78 (link)) partner repository with the dataset identifier PXD023668.

The samples were treated with SDS–PAGE loading buffer supplied with 10 mM DTT for 5 min at 85 °C. The proteins were alkylated by the addition of iodoacetamide to the final concentration of 15 mM. The samples were subjected to SDS–PAGE and the whole lanes were cut out and digested with trypsin in-gel for 2 h. The resulting peptides were extracted, dried, and resuspended in 0.1% formic acid with 5% acetonitrile prior to loading onto a trap EASY-column (Thermo Scientific) coupled to an in-house-made nano HPLC column (20 cm × 75 μm) packed with LUNA C18 media. Analysis was performed on a Velos Pro mass spectrometer (Thermo Scientific) operated in data-dependent mode using 90-min gradients in an EASY-LC system (Proxeon) with 95% water, 5% acetonitrile (ACN), 0.1% formic acid (FA) (solvent A), and 95% ACN, 5% water, 0.1% FA (solvent B) at a flow rate of 220 nl/min. The acquisition cycle consisted of a survey MS scan in the normal mode followed by twelve data-dependent MS/MS scans acquired in the rapid mode. Dynamic exclusion was used with the following parameters: exclusion size 500, repeat count 1, repeat duration 10 s, and exclusion time 45 s. Target value was set at 10⁴ for tandem MS scan. The precursor isolation window was set at 2 m/z. The complete analysis comprised two independent biological replicates.