T3 hss nano column

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nanoUltra Performance Liquid Chromatography (10 kpsi nano-Acquity; Waters, Milford, MA, USA). The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Dry peptides were dissolved in 97:3 water:acetonitrile (v/v) + 0.1% formic acid solution. Desalting of the samples was performed online using a reversed-phase C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters) at 0.35 μL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 20%B in 140 min, 20% to 90%B in 25 min, maintained at 90% for 5 min and then back to initial conditions.

ULC/MS‐grade solvents were used for all chromatographic steps. Each sample was loaded using split‐less nano‐Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: (A) H₂O + 0.1% formic acid, and (B) acetonitrile +0.1% formic acid. Desalting of the samples was performed online using a reversed‐phase Symmetry C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). The peptides were then separated using a T3 HSS nano‐column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters) at 0.35 μl/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4–30% B over 55 min, 30–90% B over 5 min, maintained at 90% for 5 min and then back to the initial conditions. The nanoUPLC was coupled online through a nanoESI emitter (10 μm tip; New Objective, Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive HF; Thermo Scientific) using a FlexIon nanospray apparatus (Proxeon). Data were acquired in data‐dependent acquisition (DDA) mode, using a Top10 method. MS1 resolution was set to 120,000 (at 200 m/z), mass range of 375–1,650 m/z and AGC of 3e6, and maximum injection time was set to 60 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.7 m/z, AGC of 1e5, dynamic exclusion of 20 s and maximum injection time of 60 ms.

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-ultra performance liquid chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: A) H₂O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 27%B in 105 min, 27% to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split‐less nano‐ultra performance liquid chromatography (10 kpsi MClass; Waters, Milford, MA, USA). The mobile phase was: a) H₂O + 0.1% formic acid and b) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed‐phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nanocolumn (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µL min⁻¹. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 30%B in 155 min, 30% to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA). The mobile phase was: A: H₂O + 0.1% formic acid and B: acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; waters). The peptides were then separated using a T3 HSS nano-column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; waters) at 0.35 μl/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 20% B in 155 min, 20% to 90% B in 5 min, maintained at 90% B for 5 min and then back to initial conditions.

Sorted cell pellets were lysed in 50 mM Tris-HCl pH 7.4, 5% SDS, and sonicated (Bioruptor Pico, Diagenode, USA). Protein concentration was measured using the BCA assay (Thermo Scientific, USA). From each sample, 20 μg of total protein was subjected to in-solution tryptic digestion using the suspension trapping (S-trap) method as previously described [49 (link)]. Liquid chromatography and Mass Spectrometry was performed as described previously [24 (link)], using split-less nano-Ultra Performance Liquid Chromatography, reversed-phase Symmetry C18 trapping column (Waters) and a T3 HSS nano-column (Waters) for desalting and separation, coupled to a quadrupole orbitrap mass spectrometer (Q Exactive HFX, Thermo Scientific). Data acquisition, processing and analysis was performed as described [20 (link)]. Differential protein abundance was tested using the glht R function by ANOVA followed by Tukey analysis on the intensity values using a logarithmic scale. Changes in protein abundance of at least 1.5 between conditions, with p.value < 0.05 were considered significant.

ULC/MS grade solvents were used for all chromatographic steps. Dry digested samples were dissolved in 97:3% H₂O/acetonitrile containing 0.1% formic acid. Each sample was loaded using split-less nano-ultra performance liquid chromatography (nanoUPLC; 10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: (A) H₂O with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid. Samples were desalted online using a reversed-phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µl/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4–20% B in 155 min, 20–90% B in 5 min, maintained at 90% for 5 min and then back to initial conditions.