Acclaim pepmap rslc c18

Mtb-HAg digest peptide sequences were detected by LC–MS using a Q Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) and Dionex Ultimate 3000 RSLCnano liquid chromatography (Thermo Fisher Scientific, MA, USA). The injection volume is 6 μL in LC–MS analysis. Column information: 300 µm idx 5 mm, Acclaim PepMap RSLC C18, 5 µm, 100 A (Thermo, 160,454); Acclaim PepMap 75 µm × 150 mm, C18, 3 µm, 100 A (Thermo Fisher Scientific, MA, USA). The elution conditions for LC were as follows: eluent A consisted of 0.1% formic acid in Milli-Q water, eluent B consisted of 0.1% formic acid and 80% acetonitrile in Milli-Q water, and the flow rate was set to 300 nL/min. The elution procedure was as follows: 0–5 min, 5% B; 5–45 min, 50% B; 45–55 min, 90% B; 55–65 min, 5% B. The MS parameters for Mtb-HAg analysis were set as follows: resolution = 70 k; scan range (m/z) = 350–1800; maximum injection time = 40 ms; AGC target = 300, 0000. The MS/MS parameters for Mtb-HAg analysis were set as follows: resolution = 17.5 k; AGC target = 100,000; maximum injection time = 60 ms; NCE/stepped NCE = 27. The mass spectrometry raw files were processed and converted by Proteome Discoverer 1.4 software to obtain MGF format files, and then the uniprot database (Mtb H37Ra (https://www.uniprot.org/taxonomy/419947) was searched using MASCOT (http://www.matrixscience.com/).

One µg total of sample was analysed by a Dionex Ultimate 3000 nano-LC system (Sunnyvale, CA, USA) combined with an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nano electrospray ion source. Peptide mixtures were pre-concentrated onto an Acclaim PepMap 100—100 μm × 2 cm C18 (Thermo Fisher Scientific, Waltham, MA, USA) and separated on EASY-Spray column, 25 cm × 75 μm ID packed with Thermo Scientific Acclaim PepMap RSLC C18, 3 μm, 100 Å, at 35 °C and flow rate 300 nL/min. Mobile phases were the following: 0.1% formic acid (FA) in water (Buffer A); 0.1% FA in water/acetonitrile with 2/8 ratio (Buffer B). The elution gradient was from 96% Buffer A to 95% Buffer B for 110 min. MS spectra were collected over an m/z range of 375–1500 Da at 120,000 resolutions, operating in the data dependent mode, cycle time 3 s between master scans. Higher-energy collision dissociation was carried out with collision energy set at 35 eV and a positive polarity. The experiment was repeated three times and each sample was run in three technical replicates.

Tryptic peptide samples were prepared for injection into an Ultimate3000 Nano/Capillary LC System (Thermo Scientific, UK) coupled to a Hybrid quadrupole Q-Tof impact II™ (Bruker Daltonics) equipped with a Nano-captive spray ion source. Briefly, 1µL of peptide digest was enriched on a µ-Precolumn 300 µm i.d. X 5 mm C18 Pepmap 100, 5 µm, 100 A (Thermo Scientific, UK), separated on a 75-µm I.D. × 15 cm column packed with Acclaim PepMap RSLC C18, 2 μm, 100 Å, nanoViper (Thermo Scientific, UK). The C18 column was enclosed in a thermostatic oven set to 60 °C. Solvent A was 0.1% formic acid in water, solvent B was 0.1% formic acid in 80% acetonitrile. A gradient of 5–55% solvent B was used to elute the peptides at a constant flow rate of 0.30 µL/min over 30 min. Electrospray ionization was carried out at 1.6 kV using CaptiveSpray. Nitrogen was used as drying gas (flow rate approximately 50 L/h). Collision-induced-dissociation (CID) product ion mass spectra were obtained using nitrogen gas as the collision gas. Mass spectra [30 (link)] and MS/MS spectra were obtained in the positive-ion mode at 2 Hz over the range (m/z) 150–2200. The collision energy was adjusted to 10 eV as a function of the m/z value. The LC–MS analysis of each sample was performed in triplicate. The protein spectral data obtained in this study has been deposited at ProteomeXchange: PXD035792.

Quantity of 20 μg of whole-protein extracts prepared using Illustra TriplePrep kit (GE Healthcare) were diluted 10-fold in 8 M urea in 0.1 M Tris-HCl, pH 8.5, filtered into the Microcon Ultracel YM-30 devices (Millipore), and centrifuged at 14,000×g for 15 min. Samples were then further diluted in 8 M urea, centrifuged again, reduced in 10 mM DTT for 30 min, and then alkylated in 50 mM IAM for 20 min. After four washes (2 in 8 M urea and 2 in 50 mM NH₄HCO₃), trypsin solution was added in an enzyme-to-protein ratio of 1:100 w/w, and samples were maintained at 37 °C for 16 h. Peptides were centrifuged and acidified by trifluoroacetic acid, desalted-concentrated on C-18 ZipTip (Millipore), dried under vacuum and then resuspended in 20 µl of ACN/H₂O (FA 0.1%) (2:98, v/v). Separation was obtained using an EASY-nLC 1000 UPLC (Thermo Scientific) through 75 mm × 2 cm pre-column with nanoViper fittings (Acclaim pepMap 100, C18, 2 µm, Thermo Scientific) and 50 mm ID × 150 mm analytical column with nanoViper fittings (Acclaim PepMap RSLC, C18, 2 µm, Thermo Scientific). Elution was carried out over 120 min by using a 2-h gradient of ACN. The Q-Exactive instrument (Thermo Scientific) was set up to a spray voltage of 1.6 kV and the survey scans were taken at 70,000 FWHM (at m/z 400) resolving power in positive ion mode with a scan range from 300 to 1600 m/z.

Samples of tryptic peptide were examined using the Thermo Scientific Ultimate3000 Nano/Capillary LC system (Thermo Scientific, UK) coupled to a hybrid quadrupole Q-ToF Impact II (Bruker Daltonics Ltd; Hamburg, Germany) with a nano captive-spray ion source. Peptide digests were enhanced on a μ-precolumn 300 μm i. d. X 5 mm C18 Pepmap 100, 5 μm, 100 Å (Thermo Scientific, UK) and separated on a 75 μm I.D. x 15 cm column and packed with Acclaim PepMap RSLC C18, 2 μm, 100 Å, nanoViper (Thermo Scientific, UK). The C18 column was surrounded with a column oven set at 60 °C. Solvents A and B were supplied with 0.1% formic acid in water and 0.1% formic acid in 80% acetonitrile, respectively, for the analytical column. Peptides were eluted for 30 min at an unchanged flow rate of 0.30 μl/min using a gradient of 5–55% solvent B. At 1.6 kV, electrospray ionization was accomplished using CaptiveSpray. Nitrogen was used as the drying gas at a rate of around 50 l/h. Collision-induced dissociation (CID) product ion mass spectra were received using nitrogen gas as the collision gas. Over the (m/z) range of 150–2200, positive ion mode mass spectra (MS) as well as MS/MS spectra were recorded at 2 Hz. According to the m/z value, the collision energy was adjusted to 10 eV. LC-MS was used to evaluate each sample three times.

MS analysis was carried out using an LTQ-Orbitrap Velos (Thermo Scientific) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA, now part of Thermo Scientific). Before loading, peptide mixtures were purified using ZipTip Pipette Tips (Millipore), according to the manufacturer’s recommendations. After loading, peptide mixtures (4 μg per run) were concentrated and desalted on a trapping pre-column (Acclaim PepMap C18, 75 μm × 2 cm nanoViper, 3 μm, 100 Å, Thermo Scientific), using 0.2% formic acid at a flow rate of 5 μl/min. The peptide separation was performed at 35°C using a C18 column (Acclaim PepMap RSLC C18, 75 μm × 15 cm nanoViper, 2 μm, 100 Å, Thermo Scientific) at a flow rate of 300 nL/min, using a 485 min gradient from 1 to 50% eluent B (0.2% formic acid in 95% ACN) in eluent A (0.2% formic acid in 5% ACN).
The mass spectrometer LTQ-Orbitrap Velos was set up in a data dependent MS/MS mode, as described previously [48 (link)]. Briefly, the lock mass option was enabled on a protonated polydimethylsiloxane background ion for internal recalibration, peptide ions were selected as the ten most intense peaks of the previous scan, and Higher Energy Collisional Dissociation (HCD) was chosen as the fragmentation method.

The nanoLC–MS/MS analysis was performed in duplicate on a Q-Exactive mass spectrometer (Thermo Fisher Scientific) coupled with a nanoHPLC (UltiMate 3000 RSLCnano System, Thermo Fisher Scientific). The peptide samples were loaded onto a trap column (Acclaim PepMap 100 C18, 75 μm × 20 mm, 3 μm particle, 100 Å pore size, Thermo Fisher Scientific) in 2% acetonitrile with 0.05% TFA at a flow rate of 5 μl/min and further separated on analytical column (Acclaim PepMap RSLC C18, 75 µm × 500 mm, 2 µm particle, 100 Å pore size, Thermo Fisher Scientific) with a 240 min gradient from 2 to 40% acetonitrile in 0.05% formic acid at a flow rate of 300 nl/min. Eluting peptides were ionised using a Digital PicoView 550 nanospray source (New Objective, Woburn, MA, USA) and acquired in an MS data dependent mode using the top twelve method with 30 s of dynamic exclusion. Full scan MS spectra were acquired with a resolution of 70,000 over a mass range of 300 to 2000 m/z with an automatic gain control (AGC) target of 1e6. The MS/MS spectra were acquired with a resolution of 17,500 with an AGC target of 5e5. The maximum ion accumulation times for the full MS and the MS/MS scans were 120 ms and 60 ms, respectively. The lock mass option was used to perform internal calibration.