Acclaim pepmap

The dried peptide samples were re-suspended in resuspension buffer (0.05%v/v trifluoroacetic acid in water) to a final concentration of 1 μg/μl. These samples were filtered using a Millex filter before subjecting to HPLC-MS analysis. Nano-HPLC-MS/MS analysis was performed using an on-line system consisting of a nano-pump (Dionex Ultimate 3000, Thermo-Fisher, UK) coupled to a QExactive instrument (Thermo-Fisher, UK) with a pre-column of 300 μm x 5 mm (Acclaim Pepmap, 5 μm particle size) connected to a column of 75 μm x 50 cm (Acclaim Pepmap, 3 μm particle size). Samples were analyzed on a 90 min gradient in data dependent analysis (1 survey scan at 70k resolution followed by the top 10 MS/MS).

Nano-LC-MS/MS was performed on an LTQ-Orbitrap Elite mass spectrometer coupled to an Ultimate 3000 RSLC (both Thermo Fisher Scientific). Briefly, peptides were preconcentrated on a C18 trapping column (Acclaim PepMap, 100 μm × 2 cm, 5 μm particle size, 100 Å pore size, Thermo Fisher Scientific) in 0.1% TFA and separated on a C18 main column (Acclaim PepMap, 75 μm × 50 cm, 2 μm particle size, 100 Å pore size, Thermo Fisher Scientific) using a binary gradient (solvent A: 0.1% formic acid (FA); solvent B: 0.1% FA, 84% ACN) ranging from 3 to 42% B in 185 min, at a flow rate of 250 nL min⁻¹. MS survey scans were acquired in the Orbitrap from 300 to 2000 m/z at a resolution of 60,000 using the polysiloxane m/z 371.1012 as a lock mass. The 20 most intense signals above an intensity threshold of 10⁴ and with charge states 2–5 were subjected to collision induced dissociation in the ion trap with a normalized collision energy of 35%, taking into account a dynamic exclusion of 10 s. Automatic gain control (AGC) target values and maximum injection times were set to 10⁶ and 50 ms for MS and 10⁴ and 100 ms for MS^{2 (link)}.

Mass spectrometry was performed and analysed as previously described [34 (link)]. High performance liquid chromatography was conducted using a 2 cm pre-column (Acclaim PepMap 50 mm × 100 um inner diameter (ID)), and 50 cm analytical column (Acclaim PepMap, 500 mm × 75 um diameter; C18; 2 um; 100 Å, Thermo Fisher Scientific, Waltham, MA), running a 120 min reversed-phase buffer gradient at 225 nl/min on a Proxeon EASY-nLC 1000 pump in-line with a Thermo Q-Exactive HF quadrupole-Orbitrap mass spectrometer. A parent ion scan was performed using a resolving power of 60,000, then up to the twenty most intense peaks were selected for MS/MS (minimum ion count of 1,000 for activation) using higher energy collision induced dissociation (HCD) fragmentation. Dynamic exclusion was activated such that MS/MS of the same m/z (within a range of 10 ppm; exclusion list size = 500) detected twice within 5 s were excluded from analysis for 15 s.

Samples were analysed with a nano-LC (Ultimate RSLC Nano, Thermo Scientific) equipped with a C18 trap column (PepMap Acclaim 300 µm mm * 5 mm, Thermo Scientific), and a C18 separation column (PepMap Acclaim 75 µm * 500 mm, Thermo Scientific), coupled to a Fusion Tribrid Orbitrap mass spectrometer (Thermo Scientific), fitted with an Easy Spray ion source. Loading buffer was 0.05% TFA; Buffer A was 0.1% FA; and Buffer B was 84% ACN, 0.1% FA. The following gradient was used: 0 min, B 0%; 50 min, B 55%; 60 min, B 100%. The mass spectrometer was operated in the positive ion mode. A full Orbitrap MS scan (R = 120 k, AGC target = Standard, max injection time = 50 ms) was followed by data dependent Orbitrap MS/MS scans (isolation window = 1.5, activation type = HCD, R = 50 k, AGC target = 300%, max. injection time = 90 ms) with 3 s cycle time. For SPS-MS3, the following parameters were used: a full Orbitrap MS scan (R = 120 k, AGC target = standard, max injection time = 50 ms) was acquired, followed by Ion Trap MS/MS scans (isolation window = 0.7, activation type = CID, AGC target = Standard, max. injection time = 50 ms) and Orbitrap MS3 scans (MS2 isolation window (m/z) = 2, number of SPS precursors: 5, Activation type = HCD, AGC target = 200%, max. injection time = 105 ms).

An online reversed-phase chromatography was used to separate the sample, through a Thermo Scientific Proxeon easy nLC1000 equipped with a Proxeon trap column (100 μm ID x 2 cm, Thermo Scientific) and a C18 packed-tip column (Acclaim PepMap, 75 µm ID x 15 cm, Thermo Scientific). Peptides were separated with an increasing amount of ACN (2%-40% over 60 min) at a flow rate of 300 nL/min. The peptides were electrosprayed directly from the analytical column and a voltage of 1.7 kV was applied via the liquid junction of the nanospray source. The chromatography system was coupled to the mass spectrometer Thermo Scientific Q-exactive programmed with a top 10 data-dependent mode for all the samples. The resolving power was 70,000 FWHM (m/z 400), in a positive mode and using an AGC target of 3^e6. Default charge state was set at 2, unassigned and +1 charge states were rejected and dynamic exclusion was enabled for 25 s. The scan range was set to 300-1600 m/z. For ddMS², the scan range was between 200 and 2000 m/z, 1 microscan was acquired at 17,500 FWHM and an isolation window of 4.0 m/z was used.

Pooled TMT-labeled peptides were separated on a 150 mm, 300 μm OD, 2 μm C18, Acclaim PepMap (Thermo Fisher Scientific) column using an Ultimate 3000 (Thermo Fisher Scientific). The column was maintained at 30°C. Separation was performed with a flow of 4 μl using a segmented gradient of buffer B from 1–50% for 85 min and 50–95% for 20 min. Buffer A was 5% acetonitrile 0.01 M ammonium bicarbonate, buffer B was 80% acetonitrile 0.01 M ammonium bicarbonate. Fractions were collected every 150 s and combined into nine fractions by pooling every ninth fraction. Pooled fractions were dried in Concentrator plus (Eppendorf), and resuspended in 5 μl 0.1% formic acid, from which 2 μl were analyzed by LC-MS/MS.

All liquid chromatography with tandem MS (MS/MS) analyses were conducted using an Ultimate 3000 UHPLC system (Dionex) coupled with a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific), as previously described, with some modifications (17 ). Peptides were separated on a two-column system equipped with a trap column (Thermo Fisher Scientific, Acclaim PepMap, C18 5 μm, 100 Å, 300 μm I.D. × 5 mm) and an analytical column (Thermo Fisher Scientific, EASY-Spray column, C18 1.9 μm, 100 Å, 75 μm I.D. × 50 cm) using 90-min gradients from 7% to 30% acetonitrile at a flow rate of 300 nl/min. Column temperature was maintained at 60 °C using a column heater. MaxQuant.Live version 1.2 was used for BoxCar acquisition (18 ). The MS1 resolution was set to 120,000 at m/z 200 for BoxCar, and the acquisition cycle comprised two BoxCar scans at 12 boxes (scaled width, 1 Th overlap) with a maximum ion injection time of 20.8 per box, with the individual AGC target set to 250,000. MS/MS spectra were acquired at a higher-energy collisional dissociation-normalized collision energy of 30, with a resolution of 17,500 at m/z 200. The maximum ion injection durations for the full and MS/MS scans were 20 ms and 100 ms, respectively.