Easy spray nanosource

For data base analysis, STK19 detected peptides were downloaded from peptide Atlas (Deutsch et al., 2008 (link)) and Proteomics DB (Schmidt et al., 2018 (link)) latest versions. To minimize unspecific mapping, peptides between 7 and 20, found in more than one experiment and with a maximum of one missed cut site were analyzed. Peptides with the same start or end position were merged for plotting. For the analysis of overexpressed mis-annotated STK19, eluted proteins from immunoprecipitations were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), until the running front had migrated approximately 1–2 cm into the gel (10% NuPAGE, Invitrogen, NP0301), and stained with colloidal Coomassie (InstantBlue, Expedeon). After excision of 8 horizontal gel slices per lane, proteins were in-gel digested with trypsin (Promega/Pierce) using a Janus liquid handling system (Perkin Elmer). Tryptic peptides were analyzed by liquid chromatography-mass spectrometry (LC–MS) using an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 uHPLC equipped with an EASY-Spray nanosource (Thermo Fisher Scientific) and acquired in data-dependent mode. The data were searched against the human Uniprot database using the Andromeda search engine. Raw data were processed using MaxQuant v1.6.0.1 (Cox and Mann, 2008 (link)). Peptide intensities were log2 transformed.

Samples were resuspended in 2% (v/v) Acetonitrile/0.1% (v/v) TFA supplemented with iRT standards (Biognosys) and loaded on a 50 cm Easy Spray PepMap column (75 μm inner diameter, 2 μm particle size, Thermo Fisher Scientific) equipped with an integrated electrospray emitter. Reverse phase chromatography was performed using the RSLC nano U3000 (Thermo Fisher Scientific) with a binary buffer system (solvent A: 0.1% v/v Formic acid, 5% v/v DMSO; solvent B: 80% v/v Acetonitrile, 0.1% v/v Formic acid, 5% v/v DMSO) at a flow rate of 275 nL/min. The samples were run on a linear gradient of 2-8% B in 5.5 min, 8-25%B in 54.5 min, 25-40% B in 31 min, and 40-50% B in 7 min with a total run time of 120 min including column conditioning. The nanoLC was coupled to an Orbitrap Eclipse mass spectrometer using an EasySpray nano source (Thermo Fisher Scientific). The Orbitrap Eclipse was operated in data-independent mode (DIA) with following settings: MS1 data acquired in the Orbitrap with a resolution of 120,000, max injection time of 20 ms, AGC target of 1e6, in positive ion mode and profile mode, over the mass range 375-1,500 m/z. DIA segments over this mass range (variable size windows, 34 in total) were acquired in the Orbitrap following fragmentation in the HCD cell (30%), with 30,000 resolution over the mass range 200-2000 m/z and with a max injection time of 70 ms and AGC target of 1e6.

Protein bands were excised and in-gel–digested using trypsin. The peptides were analyzed with an Orbitrap Fusion Lumos mass spectrometer coupled to an UltiMate 3000 HPLC equipped with an EASY-Spray nanosource (Thermo Fisher Scientific). Raw data were processed using MaxQuant v1.6.0.1 and searched against a UniProt-extracted S. pombe FASTA file amended to include common contaminants, with phosphorylation (STY), acetylation (K), and methylation (KR) being selected as variable modifications. The proteingroup.txt and phosphoSTY.txt output tables were imported into Perseus software for further processing. All intensity values were log₂-transformed.

Eluted proteins were separated by SDS-PAGE until the running front had migrated ~2 cm into the gel and stained with colloidal Coomassie (InstantBlue; Expedeon). After the excision of eight horizontal gel slices per lane, proteins were in-gel digested with trypsin (Promega/Pierce) using a Janus liquid-handling system (PerkinElmer). Tryptic peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS) using an Orbitrap Velos mass spectrometer coupled to an Ultimate 3000 ultrahigh-performance liquid chromatography (uHPLC) instrument equipped with an Easy-Spray nanosource (Thermo Fisher Scientific) and acquired in data-dependent mode. The RAW data were searched using the MaxQuant (v1.6.1.0) tool against the T. gondii ME49 databases from ToxoDB (https://toxodb.org)^{69 (link)}. Peptide and protein identifications were filtered to a 1% false discovery rate (FDR), and reversed proteins, contaminants, and proteins identified by only a single modification site were removed from the data set.

Purified γTuRC was separated by SDS-PAGE and stained using InstantBlue (Expedeon). Protein bands were excised from the gel and analysed by the Francis Crick Institute Proteomics facility. Briefly, Tryptic peptides were analysed using a Q Exactive orbitrap mass spectrometer coupled to an Ultimate 3000 HPLC equipped with an EasySpray nano-source (Thermo Fisher Scientific). A one-hour method of MS1 orbitrap (60k resolution) followed by top 10 HCD MS2 (35k resolution) produced raw data files. Raw files were analysed in MaxQuant (v1.6.0.13) against the SwissProt Homo sapiens protein database (downloaded June 2019) using the iBAQ algorithm. The canonical GCP2 sequence was replaced with the construct sequence (GCP2-5xGly-TEV-5xGly-mBFP-5xGly-AviTag). Variable modifications of methionine oxidation and protein N-terminal acetylation along with a fixed modification of cysteine carbamidomethylation were selected. The proteingroups.txt file was imported in Perseus (v1.4.0.2) for data analysis. Potential contaminants, reverse sequences and proteins identified by site were removed. iBAQ intensities were log₂ transformed.