Orbitrap velos instrument

Matching of the amino acid sequences with the CE–MS acquired ion peaks was based on mass correlation between CE–MS and LC-tandem MS analysis. The amino acid sequence was determined by MS/MS analysis using either a PACE CE or a Dionex Ultimate 3000 RSLS nanoflow system (Dionex, Camberly UK) coupled to an Orbitrap Velos instrument (Thermo Scientific), as previously described.^{26 (link)} Protein matching and data analysis was based on Proteome Discoverer 1.2 (activation type: HCD; precursor mass tolerance: 5 ppm; fragment mass tolerance: 0.05 Da). No fixed modifications were selected, oxidation of methionine and proline were selected as variable modifications. The data were searched against the UniProt human database²⁷ without enzyme specificity. Further validation of the obtained peptide identifications is based on the assessment of the peptide charge at the working pH of 2.2 and the CE-migration time results.^{28 (link)}

The amino acid composition was acquired by MS/MS analysis using either a PACE CE or a Dionex Ultimate 3000 RSLS nanoflow system (Dionex, Camberly UK) coupled to an Orbitrap Velos instrument (Thermo Scientific), as previously described^{18 (link)}. Protein matching and data analysis was based on Proteome Discoverer 1.2 (activation type: HCD; precursor mass tolerance: 5 ppm; fragment mass tolerance: 0.05 Da). The data were searched against the UniProt human database³⁵ without enzyme specificity. Matching of the amino acid sequences with the CE-MS acquired ion peaks was based on mass correlation between CE-MS and liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS). Further validation of the obtained peptide identifications is based on the assessment of the peptide charge at the working pH of 2.2 and the CE-migration time results^{32 (link)}.

S2 cells were lysed in 0.075% SDS, 1X c0mplete Protease Inhibitor Cocktail (Roche) with three rounds of freeze thawing and clarified. Total protein (1X Tricine Loading buffer, 2.5% vol/vol βME) was run on 10–20% MiniProtean Tris-Tricine Gels (Bio-Rad) and the 5–15 KDa region excised. Mass spectrometry was performed by Cambridge Centre for Proteomics (University of Cambridge, UK) using in-gel trypsin digestion and LC-ESI-MS/MS using an Orbitrap Velos Instrument (Thermo Fisher Scientific) with the following parameters: 2 missed Trypsin cleavages, 25 ppm Precursor mass error, 0.8 Da fragment mass tolerance, carbamidomethylation of cysteine as a fixed and methionine oxidation as a variable modification. Spectra were matched against Drosophila melanogaster (5.55) proteome using generic Mascot algorithm.