Nanolc ultra system

The proteins identified in IMS were actually detected in the data of three brain tumor samples by LC/MS, and included in the higher coverage (%) (>5.0) group (Supplementary Table S2–4. The LC/MS measurement method at that time is shown below this section^{32 (link)}. Samples were homogenized and suspended in 20 μL of 0.1 M NH₄HCO₃, containing 30% (v/v) CH₃CN. After centrifuge, incubation, and cooling, samples were incubated in trypsin solution at 37 °C overnight. We added 10 mM DTT, and heated the digests at 95 °C for 5 min. After drying, we re-suspended them in 0.1% TFA containing 2% CH₃CN. The samples were separated, using a nano-flow reverse-phase LC (NanoLC-Ultra System; Eksigent, Dublin, CA). We injected an aliquot of 5 μL of each sample into a trap column, and washed it for 10 min, using 0.1% formic acid. Peptides were eluted for further analyses, using Triple TOF 5600 system (AB SCIEX, Framingham, MA) with a nano-electrospray ionization source (NanoSpray; AB SCIEX, Framingham, MA). We performed MS/MS scans, using a collision energy of 35 kV with unit-resolution.

Sixty millions of NoORF and GCNT3 SW620 cells (2 replicates) were lysed and 5 mg of total protein were incubated for 1 h at 4 °C with 200 µl protein A beads (Roche, Basel Switzerland) as a pre-clear step. Pre-cleared lysates were incubated overnight at 4 °C with 2 µg of anti V5 antibody followed by 2 h incubation with 100 µl of protein A beads. After incubation, beads were washed and transferred to Proteomic Core Unit of the Spanish National Cancer Research Centre (CNIO) for analysis. Peptides were analyzed by LC-MS/MS, Impact (Bruker Daltonics) coupled online to a nanoLC Ultra system (Eksigent), equipped with nanoelectrospray ion source supplemented with a CaptiveSpray nanoBooster operated at 0.2 bar/minute with isopropanol as dopant. For protein identification and quantification raw data were analyzed by MaxQuant (1.5.1.2) using a Human Uniprot Canonical database plus the most common contaminants (20584 entries in total).
We used STRING (http://string-db.org/) to illustrate protein-protein interaction networks. Functional analysis of Gene Ontology (GO) terms and protein set enrichment analysis were carried out using the GeneCodis3 tool^{46 (link)–48 (link)}. REViGO tool was used to summarize Gene Ontology terms by removing redundant GO terms^{49 (link)}.

The fractions prepared in the previous step were analyzed with nanoHPLC-MS/MS. The NanoLC-Ultra system (Eksigent, Dublin, CA) was used coupled to an Orbitrap Velos hybrid mass spectrometer (Thermo-Finnigan, San Jose, CA). The separation was performed on a tip column packed with Magic RP C18 AQ, 200A, 3 μm beads (Bischoff GmbH, Leonberg, Germany), with the previously described gradient [16 (link)]. The mass spectrometer was operated in data-dependent mode with the previously reported ion scanning parameters [16 (link)]. Fragmented peptide masses were set in dynamic exclusion for 60 s and singly charged ions were excluded from MS/MS analysis. To improve sensitivity of the MS/MS analysis for peptides of low-abundance proteins, each fraction was run a second time excluding previously fragmented precursors.

Cardiac proteins were analysed using our previously described method^{16 (link)}. Briefly, samples were homogenized and suspended in 20 μL of 0.1 M NH₄HCO₃ that contained 30% (v/v) CH₃CN, and then centrifuged at 10,000 × g for 1 min. Samples were incubated at 95 °C for 90 min, centrifuged at 10,000 × g for 1 min, and cooled on ice. Next, 1 μL of 1 μg/μL trypsin solution was added. Samples were incubated at 37 °C overnight. The next day, 10 mM DTT was added and the digests were heated at 95 °C for 5 min, dried, and re-suspended in 0.1% TFA containing 2% CH₃CN to obtain a final protein concentration of 0.2 μg/μL. Samples were separated using a nano-flow reverse-phase LC (NanoLC-Ultra System; Eksigent, Dublin, CA). An aliquot of each sample (5 μL) was injected into a trap column and washed for 10 min using 0.1% formic acid. Peptides were eluted for further analyses using a quadrupole time-of-flight hybrid mass spectrometer (Triple TOF 5600 system; AB SCIEX, Framingham, MA), equipped with a nano-electrospray ionization source (NanoSpray; AB SCIEX, Framingham, MA). Tandem MS (MS/MS) scans were performed using a collision energy of 35 kV with unit-resolution.

Metabolic proteins were extracted from whole grains as described in Bonnot et al. (2017) (link). Briefly, proteins were extracted for 2 h at 4°C in 10 mM sodium phosphate, 10 mM NaCl, pH 7.8, supplemented with a cocktail of plant protease inhibitors (Sigma, St. Louis, MO, United States). After centrifugation at 8,000 × g for 20 min at 4°C, proteins in the supernatant were precipitated with ice-cold acetone for 2 h at -20°C. After centrifugation at 10,000 × g for 5 min at 4°C, the resulting pellets were washed three times in ice-cold acetone then dried at room temperature and afterward stored at -20°C. The precipitated AG proteins were suspended in a solubilization buffer (0.1% ZALS I, 6 M urea, 2 M thiourea, 10 mM DTT, 30 mM Tris–Hcl pH 8.8, 50 mM NH₄HCO₃). Proteins were digested in-solution by trypsin and resulting peptides were analyzed by LC-MS/MS using a nanoLC Ultra system (Eksigent) and a Q-Exactive mass spectrometer (Thermo Electron).

LC-MS/MS experiments were performed using a TripleTOF 5600 System (SCIEX, Canada) and a NanoLC Ultra System (Eksigent, USA), equipped with a trap column (150 μm i.d. × 1 cm long; PGC, 5 μm; Proteomics Front, China) and a separation column (75 μm i.d. × 10 cm long; PGC, 5 μm; Proteomics Front, China). MS was operated in the positive-ion mode with a mass range of 500–3000 m/z, and MS/MS was acquired in the information dependent acquisition (IDA) mode with a mass range of 100–2000 m/z. The 20 most abundant precursor ions with charge numbers from 2 to 5 were scanned in the IDA mode. Each cycle consisted of a MS acquisition for 0.25 s and a total of 20 MS/MS scans for 2 s.