Acclaim pepmap 100 c18 column

Tryptic peptides were separated by AcclaimTM PepMapTM 100 C18 column (Thermo, 164,941) using a 140 min of total data collection (100 min of 2–22%, 20 min 22–28% and 12 min of 28–36% gradient of B buffer (80% acetonitrile and 0.1% formic acid in H2O) for peptide separation, following with two steps washes: 2 min of 36–100% and 6 min of 100% B buffer) with an Easy-nLC 1200 connected online to a Fusion Lumos mass spectrometer (Thermo). Scans were collected in data-dependent top-speed mode with dynamic exclusion at 90 s. Raw data were analyzed using MaxQuant version 1.6.0.1 search against Mouse Fasta database, with label free quantification and match between runs functions enabled. The output protein group was analyzed and visualized using DEP package as described before. Processed IP-mass date are provided in Additional file 10: Table S3 IP-MS.

The samples were resuspended in 20 µL of 0.1% FA for a final concentration of 0.25 µg/µL. An aliquot of 4 µL was injected into a Dionex Ultimate 3000 UPLC system. Peptides were purified and desalted using an Thermo Scientific^TM Acclaim^TM PepMap^TM 100 C18 Nano Trap pre-column (100 µm × 2 cm, 100 Å pore size, 5 µm particle size) and transferred to a Thermo Scientific^TM Acclaim^TM PepMap^TM 100 C18 column (75 µm × 50 cm, 100 Å pore size, 2 µm particle size) for chromatographic separation. Peptide elution was achieved by applying an 80-minute run for each sample with a flow rate of 0.3 µL/min at 45 °C using a gradient with Eluent A consisting of 0.1% FA and eluent B of 0.1% FA in 90% acetonitrile starting with 2% solvent B. Solvent B was increased to 30% in 65 min followed by a linear gradient elevating the concentration to 90% in 70 min. Finally, the concentration of buffer B was reduced to 2% after 70.1 min. The eluting peptides were analyzed on a Quadrupole Orbitrap hybrid mass spectrometer (QExactive, Thermo Fisher Scientific, Waltham, MA, USA). Here, the ions that were responsible for the 15 highest signal intensities per precursor scan (1 × 10⁶ ions, 70,000 resolution, 240 ms fill time) were analyzed by MS/MS (HCD at 25 normalized collision energy, 1 × 10⁵ ions, 17,500 resolution, 50 ms fill time) in a range of 400–1200 m/z. A dynamic precursor exclusion time of 20 s was used.

Peptides after digestion were separated by AcclaimTM PepMapTM 100 C18 column (Thermo, 164941) using a 140 min of total data collection (100 min of 2–22%, 20 min 22–28% and 12 min of 28–36% gradient of B buffer (which containing 80% acetonitrile and 0.1% formic acid in H2O) for peptide separation, following with two steps washes: 2 min of 36–100% and 6 min of 100% B buffer) with an Easy-nLC 1200 connected online to a Fusion Lumos mass spectrometer (Thermo). Scans were collected in data-dependent top-speed mode with dynamic exclusion at 90 s. MaxQuant version 1.6.0.1 search against Mouse Fasta database was used to analyze raw data, with label free quantification and match between runs functions enabled. DEP package was used to analyze and visualize the output protein group.

Tryptic peptides were separated by AcclaimTM PepMapTM 100 C18 column (Thermo, 164941) using a 140 min of total data collection (100 min of 2–22%, 20 min 22–28% and 12 min of 28–36% gradient of B buffer (80% acetonitrile and 0.1% formic acid in H2O) for peptide separation, following with two steps washes: 2 min of 36–100% and 6 min of 100% B buffer) with an Easy-nLC 1200 connected online to a Fusion Lumos mass spectrometer (Thermo). Scans were collected in data-dependent top-speed mode with dynamic exclusion at 90 s. Raw data were analyzed using MaxQuant version 1.6.0.1 search against Mouse Fasta database, with label free quantification and match between runs functions enabled. The output protein group was analyzed and visualized using DEP package as described before^{44 (link)}.

A total of 12 μl of peptide mixture was loaded onto a trap column (Acclaim PepMap100 C18 column, 0.3 mm id × 50 mm, Thermo Fisher Scientific) at a constant flow rate of 4 μl/min. Peptides were separated in a PepMap C18 nano column (75 μm × 50 cm, Thermo Fisher Scientific) using a 0–35% gradient (0–215 min) of 90% acetonitrile, 0.1% formic acid at a flow rate of 200 nL/min followed by acetonitrile wash and column re-equilibration for a total gradient duration of 4 h with a RSLC Ultimate 3000 (Thermo Fisher Scientific, Dionex). Peptides were sprayed using an EASYSpray source (Thermo Fisher Scientific) at 2 kV coupled to a quadrupole-Orbitrap (QExactive, Thermo Fisher Scientific) mass spectrometer.

Gel pieces were processed as described previously [28 (link)]. The bands were then reduced with 5 mM DTT, alkylated with 15 mM iodoacetamide, and digested with sequencing-grade trypsin (Promega) in 40 mM Tris pH 8.0. Peptides were extracted using 50% ACN and 5% formic acid followed by separation by reversed-phase chromatography on Acclaim PepMap100 C18 column (Thermo Scientific). The separated peptides were ionized with the Nanospray Flex Ion Source (Thermo Scientific) and introduced into a Fusion mass spectrometer (Thermo Scientific).
Data were analyzed using Proteome Discoverer 2.1 (Thermo Scientific). The Uniprot_Hum_Compl_20170714 database was used, and a reverse decoy protein database was run to eliminate false discovery. The results were introduced into Scaffold 4.8 (Proteome Software) for distribution. Lowest protein identification odds were set at ≥99.0% with 2 unique peptides at ≥99.0% minimum peptide identification probability.