Ultimate 3000 nano uhplc system

Approximate 1 μg of each dried peptide sample was dissolved in 10.5 μL of 0.05% trifluoroacetic acid with 3% (vol/vol) acetonitrile containing spiked-in indexed Retention Time Standard containing 11 artificially synthetic peptides (Biognosys). The spiked-in 11-peptides standard mixture was used to account for any variation in retention times and to normalize abundance levels among samples. 10 μL of each sample was injected into an Ultimate 3000 nano UHPLC system (Thermo Fisher Scientific). Peptides were captured on a 2-cm Acclaim PepMap trap column and separated on a heated 50-cm Acclaim PepMap column (Thermo Fisher Scientific) containing C18 resin. The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (vol/vol) acetonitrile (buffer B) run with a linear 60-min gradient of 6–30% buffer B at a flow rate of 300 nL/min. The UHPLC was coupled online with a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 375 to 1,500 with the resolution of 60,000) was followed by MS/MS of the 15 most intense ions (30,000 resolution; normalized collision energy - 28%; automatic gain control target (AGC) - 2E4, maximum injection time - 200 ms; 60sec exclusion).

DL-Dithiothreitol (DTT), iodoacetamide (IAA), formic acid (FA), and acetonitrile (ACN) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Trypsin from bovine pancreas was purchased from Promega (Madison, WI, United States). Ultrapure water was prepared from a Millipore purification system (Billerica, MA, United States). An Ultimate 3000 nano-UHPLC system coupled with a Q Exactive^TM HF mass spectrometer (Thermo Fisher Scientific) with an ESI nanospray source.

Protein concentrations of inner and OM fractions were determined by Pierce™ bicinchoninic acid (BCA) Protein Assay Kit (ThermoFisher Scientific, Waltham, MA, USA). For liquid chromatography-mass spectrometry (LC/MS) analysis, all samples were adjusted to the protein concentration of 0.4 mg mL⁻¹ in 10 mmol L⁻¹ HEPES of pH 7.4. The samples were digested with 10 μg trypsin (sequencing grade) (Promega, Madison, WI, USA) overnight at 37 °C. The digestion was stopped by adding 5 μL 50% formic acid and the generated peptides were purified using a ZipTip C18 (Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions and dried using a Speed Vac concentrator Plus (Eppendorf, Hamburg, Germany). The tryptic peptides were analyzed using an Ultimate 3000 nano-UHPLC system connected to a Q Exactive mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) equipped with a nano electrospray ion source.

DL-dithiothreitol (DTT), iodoacetamide (IAA), trifluoroacetic Acid (TFA), acetonitrile (ACN), and methanol were purchased from Sigma. Trypsin was purchased from Promega. C18 SPE Cartridge was purchased from Waters (Milford, MA, USA). Anti acetyl-Lysine beads were purchased from Cell Signaling Technology. Ultrapure water was prepared from a Millipore purification system. An Ultimate 3000 nano UHPLC system coupled with a Q Exactive HF mass spectrometer (Thermo Fisher Scientific) with an ESI nanospray source was used.

The full phosphopeptide sample and 1 μg of the peptide sample each was dissolved in 10.5 μl of 0.05% trifluoroacetic acid with 3% (v/v) acetonitrile containing spiked-in indexed Retention Time Standard artificially synthetized peptides (Biognosys). The spiked-in 11-peptides standard mixture was used to account for any variation in retention times and to normalize abundance levels among samples. Ten microliters of each sample were injected into an Ultimate 3000 nano UHPLC system (Thermo Fisher Scientific). Peptides were captured on a 2-cm Acclaim PepMap trap column and separated on a heated 50-cm Acclaim PepMap column (Thermo Fisher Scientific) containing C18 resin. The mobile phase buffer consisted of 0.1% formic acid in ultrapure water (buffer A) with an eluting buffer of 0.1% formic acid in 80% (v/v) acetonitrile (buffer B) run with a linear 90-min gradient of 6–30% buffer B at flow rate of 300 nL/min. The UHPLC was coupled online with a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode, in which a full-scan MS (from m/z 375 to 1,500 with the resolution of 60,000) was followed by MS/MS of the 15 most intense ions (30,000 resolution; normalized collision energy—28%; automatic gain control target [AGC] - 2E4, maximum injection time—200 ms; 60 sec exclusion).