Nanoacquity uplc column

A mixture of peptides was obtained from Model D by standard tryptic digestion (omitting reduction and alkylation steps). Tryptic peptides were applied to an RP-18 precolumn (Waters nanoAcquity 20 mm × 180 μm) using aqueous 0.1% trifluoroacetic acid as a mobile phase and then transferred to a HPLC RP-18 column (Waters nanoAcquity UPLC Column 250 mm × 75 μm). Chromatographic separation was performed at a flow rate of 150 nL/min using an acetonitrile gradient (0–60% in 120 min) in the presence of 0.05% formic acid. The column outlet was directly coupled to the ion source of an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bergen) spectrometer working in the regime of data-dependent acquisition to register CID and HCD fragmentation spectra. The normalized collision energy was set to 30%. Dynamic exclusion was disabled. A blank run ensuring a lack of cross-contamination from previous samples preceded each analysis.

The purified proteins were analyzed by LC-MS/MS. The proteins were trypsin digested by a standard in-gel digestion protocol, and analyzed by LC-MS/MS on an LTQ Orbitrap XL (Thermo Scientific) equipped with a nanoACQUITY UPLC system (Waters). A Symmetry C18 trap column (180 μm x 20 mm; Waters) and a nanoACQUITY UPLC column (1.7 μm, 100 μm x 250 mm, 35°C) were used for peptide separation. Trapping was done at 15 μl min⁻¹, 99% buffer A (water with formic acid (0.1%)) for 1 min. Peptide separation was performed at 300 nl min⁻¹ with buffer A and buffer B (CH₃CN containing 0.1% formic acid). The linear gradient was from 5% buffer B to 50% B at 50 min, to 85% B at 51 min. MS data were acquired in the Orbitrap with one microscan, and a maximum inject time of 900 ms followed by data-dependent MS/MS acquisitions in the ion trap (through collision induced dissociation, CID). The Mascot search algorithm was used to search for the appropriate noncanonical substitution (Matrix Science, Boston, MA).

The 10 fractions obtained by high pH fractionation were analyzed using a nanoAcquity UPLC system (Waters GmbH) connected online to a LTQ-Orbitrap Velos Pro instrument (Thermo Fisher Scientific GmbH). Peptides were separated on a BEH300 C18 (75 μm × 250 mm, 1.7 μm) nanoAcquity UPLC column (Waters GmbH) using a stepwise 145 min gradient between 3% and 85% (vol/vol) ACN in 0.1% (vol/vol) FA. Data acquisition was performed using a TOP-20 strategy where survey MS scans (m/z range 375–1,600) were acquired in the Orbitrap (R = 30,000 FWHM) and up to 20 of the most abundant ions per full scan were fragmented by collision-induced dissociation (normalized collision energy = 35, activation Q = 0.250) and analyzed in the LTQ. Ion target values were 1 × 10⁶ (or 500 ms maximum fill time) for full scans and 1 × 10⁵ (or 50 ms maximum fill time) for MS/MS scans. Charge states 1 and unknown were rejected. Dynamic exclusion was enabled with repeat count = 1, exclusion duration = 60 s, list size = 500 and mass window ±15 ppm.

HeLa cells were transfected with empty vector, MARCH1-Myc, or MARCH1ΔRING-Myc along with INSR-GFP and HA-Ubiquitin. Twenty-hour after transfection, cells were serum starved overnight. MG132 was added 4 h before lysis in Co-IP Buffer (Pierce). INSR-GFP was immunoprecipitated using anti-GFP antibody and eluted in 200 μl 0.2 M glycine, pH 2.5. The eluate was partially dried to ∼100 μl and precipitated with MeOH:CHCl₃:Water (4:1:3 ratio). The pellet was dried and reconstituted in 8 M urea, 0.4 M ammonium bicarbonate, reduced with DTT, and alkylated with iodoacetamide in the dark. The sample was then trypsin-digested overnight at 37 °C. The digested sample was injected onto a Q-Exactive Plus (Thermo Fisher) liquid chromatography–tandem mass spectrometry system equipped with a Waters Symmetry C18 180 μm × 20 mm trap column and a 1.7 μm, 75 μm × 250 mm nanoACQUITY UPLC column (37 °C) for peptide separation. Trapping was performed at 5 μl min⁻¹, 99% Buffer A (100% water, 0.1% formic acid) for 3 min. Peptide separation was performed with a linear gradient over 140 min at a flow rate of 300 nl min⁻¹. Collected data were processed using MASCOT Distiller and Search Engine (v.2.4), and modification sites were manually verified.