Omix tips

For each replicate, the volume of protein extracts corresponding to 100 µg of protein was adjusted to 20 µL using 6 M urea in 50 mM ammonium bicarbonate (AB). Two micrograms of recombinant green fluorescent protein (MBP-GFP) was added to each protein sample as an internal standard for retention time adjustment and intensity normalization. The tryptic digestion was performed following a previously published protocol (Gomes et al. 2019 (link)). It started with the reduction of protein disulfide bonds, followed by their alkylation before the incubation with trypsin. Vacuum-dried (Scientific Speed-Vacuum, Thermo) tryptic digests re-suspended in 2% (v/v) acetonitrile (ACN) 1% (v/v) formic acid were desalted and concentrated by C18 microcolumns (OMIX tips, Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instructions.

Fifty micrograms of soluble proteins were used for the enzymatic digestion, where the final volume of the reaction was adjusted with a buffer containing 6 M urea and 50 mM ammonia bicarbonate (AB). Green fluorescent protein (MBP-GFP, 2 µg/100 µg total protein) was added to each protein sample as an internal standard for retention time determination and intensity normalization. Reduction of disulfide bounds was performed with ditiotreitol (DTT) (10 mM, 56 °C for 1 h) followed by alkylation of thiol groups with iodoacetamide (30 mM, 25 °C, 30 min in the dark) and at the end a quenching with N-acetil-L-cysteine (37.5 mM, 25 °C, 15 min) was performed. Subsequently, 50 mM AB was added to dilute urea before adding trypsin (1 mg/mL, Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI, USA) for protein digestion, followed by incubation at 37 °C for 13 h. The trypsin lysis was interrupted by the addition of 0.5% of formic acid. Tryptic peptides mixtures were dried using a SpeedVac Savant SPD131DDA (Thermo Electron Co., Hopkinton, MA, USA) and resuspended in acetonitrile 2% containing 1% formic acid. Peptide solutions were desalted by C18 microcolumns (OMIX tips, Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s instruction, vacuum-dried and maintained at −20 °C for liquid chromatography tandem mass spectrometry (LC-MS/MS) assays.

Denatured samples were alkylated with acrylamide and subjected to in-gel digestion following the short-GeLC approach [30 (link)]. Briefly, samples were loaded onto a “4–20% TGX Stain-Free Gel” (Bio-Rad, Amadora, Portugal), followed by partial electrophoretic separation (SDS-PAGE). Proteins were subsequently visualized with Colloidal Coomassie Blue staining [70 (link)]. Gel lanes were sliced into seven bands of equal size and further sliced into small pieces for independent processing. Gel pieces were destained, dehydrated, and rehydrated with 75 μL of trypsin solution (0.01 μg μL⁻¹ trypsin in 10 mM of ammonium bicarbonate). Protein digestion was performed overnight at room temperature, and digested peptides were extracted from the gel by sequential incubation with acetonitrile (ACN) solutions (30%, 50%, and 98%) in 1% formic acid (FA). Peptides extracted from different bands were pooled together in two peptide mixtures per sample for subsequent liquid chromatography (LC)-MS/MS analysis. Peptide mixtures were dried and desalted using OMIX tips with a C18 stationary phase (Agilent Technologies, Lisbon, Portugal). To monitor losses during sample preparation, samples were spiked with 1 μg of recombinant green fluorescent protein (GFP) before digestion.