Self pack picofrit column

Samples were subjected to nanoLC-nanoESI MS/MS analysis in an Ultimate 3000 (Dionex) chromatographic system coupled to the Q Exactive Plus mass spectrometer (Thermo). About 1 μg of peptides was initially applied to a 2 cm guard column, followed by fractionation on a 40 cm PicoFritTM Self-Pack column (New Objective) packed with 1.9 μm silica, ReproSil-Pur 120 Å C18-AQ (Dr. Maisch, Germany). Samples were loaded in 0.1% (v/v) formic acid in water (mobile phase A) on the trap column at 2 μL/min, while chromatographic separation occurred at 200 nL/min. Mobile phase B consisted of 0.1% (v/v) formic acid in acetonitrile. Peptides were eluted with a gradient of 2 to 45% B over 32 min, followed by up to 80% B in 4 min. Lens voltage was set to 60 V. Full scan MS mode was acquired with a resolution of 70,000 (FWHM for m/z 200 and AGC set to 1 × 10⁶). Up to 12 most abundant precursor ions from each MS scan (m/z 300 to 1500) were sequentially subjected to fragmentation by HCD. Fragment ions were analyzed (MS2 scan) at a resolution of 17,500 and AGC set to 5 × 10⁴. Samples were analyzed in technical triplicate and data acquired using Xcalibur software (version 3.0.63). The mass spectrometry data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository^{35 (link)} under the identifier PXD023938.

The tryptic digests were analyzed by reversed-phase nanochromatography coupled to high-resolution nanoelectrospray ionization mass spectrometry. Chromatography was performed using a Dionex Ultimate 3000 RSLCnano system coupled to the HF-X Orbitrap mass spectrometry (Thermo Fischer Scientific, CA, EUA). Samples (1 μg per run) were initially applied to a 2 cm guard column, followed by fractionation on a 25.5 cm PicoFritTM Self-Pack column (New Objective) packed with 1.9 μm silica, ReproSil-684 Pur 120 Å C18-AQ (Dr. Maisch, Germany). Samples were loaded in 0.1% (v/v) formic acid (FA) and 2% acetonitrile (ACN) onto the trap column at 2 μL/min, while chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% (v/v) FA in water, while mobile phase B consisted of 0.1% (v/v) FA in ACN. Peptides were eluted with a linear gradient from 2 to 40% eluent B over 32 min, followed by up to 80% B in 4 min. The lens voltage was set to 60 V. Full-scan MS mode was acquired with a resolution of 60,000 (FWHM at m/z 200 and AGC set to 3 × 10⁶). Up to 20 of the most abundant precursor ions from each scan (m/z 350–1400) were sequentially subjected to fragmentation by HCD. Fragment ions were analyzed at a resolution of 15,000 using an AGC set to 1 × 10⁵. Data were acquired using Xcalibur software (version 4.2.47).

The tryptic digests were analyzed in three technical replicates by reversed-phase nanochromatography coupled to high-resolution nanoelectrospray ionization mass spectrometry. Chromatography was performed using a Dionex Ultimate 3000 RSLCnano system coupled to the HF-X Orbitrap mass spectrometer (Thermo Fischer, Waltham, MA, USA). Samples (4 µL per run) were initially applied to a 2 cm guard column, followed by fractionation on a 25.5 cm PicoFrit^TM Self-Pack column (New Objective, Inc., Woburn, MA, USA) packed with 1.9 μm silica, ReproSil-Pur 120 Å C18-AQ (Dr. Maisch/Germany). Samples were loaded in 0.1% (v/v) formic acid (FA) and 2% acetonitrile (ACN) onto the trap column at 2 μL/min, while chromatographic separation occurred at 200 nL/min. Mobile phase A consisted of 0.1% (v/v) FA in water, while mobile phase B consisted of 0.1% (v/v) FA in ACN. Peptides were eluted with a linear gradient from 2 to 40% eluent B over 32 min, followed by up to 80% B in 4 min. Lens voltage was set to 60 V. Full scan MS mode was acquired with a resolution of 60,000 (FWHM for m/z 200 and AGC set to 3 × 10⁶). Up to 20 most abundant precursor ions from each scan (m/z 350–1400) were sequentially subjected to fragmentation by HCD. Fragment ions were analyzed at a resolution of 15,000 using an AGC set to 1 × 10⁵. Data were acquired using Xcalibur software (version 4.2.47).