S trap micro spin column digestion protocol

Eluents from the ABE assay were dried for approximately 30 min and digested using the S-trap Micro Spin Column Digestion Protocol (Protifi, Huntington, NY) with minor changes. Briefly, 30 μL of 10% sodium dodecyl sulfate (SDS) and 100 mM triethylammonium bicarbonate (TEAB) with Pierce protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA) and phosphatase inhibitors (10 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium orthovanidate, and 1 mM β-glycerolphosphate). Proteins were reduced with a final concentration of 20 mM dithiothreitol (DDT) at 95°C for 10 min, followed by alkylation in the dark at room temperature with 40 mM of iodoacetamide. Next, phosphoric acid was added for a final concentration of 1.2%. Samples were briefly vortexed to mix before 300 μL of S-trap binding buffer (90% MeOH, 100 mM TEAB) was added. Samples were vortexed again prior to loading onto the S-Trap Micro Spin Columns. After four washes with 150 μL of S-trap binding buffer with centrifugation at 1,000×g, 40 μL of 50 mM TEAB containing 0.75 μg of trypsin was added and incubated overnight at 37°C.
Peptides were eluted with 40 μL of each of the following solutions: 50 mM TEAB, 0.2% formic acid (FA), 50% acetonitrile (ACN), and 0.1% FA. The spin column was spun at 4,000×g after adding each solution. Pooled eluents were dried down prior to resuspension in 100 μL of 3% ACN and 0.1% FA.

The samples were stored at −80 °C until preparation was initiated. By the beginning of the preparation, the samples were thawed and Schirmer strips incubated for 20 min in lysis buffer (5% SDS, 50 mM triethylammonium bicarbonate [TEAB]). Samples were prepared according to the S-Trap™ Micro spin column digestion protocol from ProtiFi (ProtiFi, Huntington, NY, USA) as previously described [8 (link)].
The reduction in disulphide bonds, alkylation of cysteines, and digestion in S-Trap micro columns were performed as described in a recent article [8 (link)]. The elution of peptides, recovery of hydrophobic peptides, and measurement of peptide concentration were performed as previously described [8 (link),30 (link)]. Each sample was dried in a vacuum centrifuge and stored at −80 °C until further use.

Sample preparation for LC–MS was carried out using S-Trap micro spin column digestion protocol (Protifi, Huntington, NY). Samples containing 5–10 μg of protein, were diluted to 50 μL with lysis buffer (5% SDS in 50-mM TEAB) and reduced with 20 mM Dithiothreitol at 95 °C for 10 min. After cooling to room temperature, samples were alkylated in the dark for 30 min with 40 mM of iodoacetamide. This mix was then acidified to 1% phosphoric acid and proteins were precipitated with 350 μl of binding buffer [90% methanol, 100-mM Triethyl ammonium bicarbonate (TEAB)]. This protein suspension was loaded onto S-Trap column columns and centrifuged at 4000×g for 30 s. After four washes with 125 μl of 50-mM TEAB, trypsin (0.5 μg in 50 mM TEAB) was added to the trap and incubated overnight. Peptides were then eluted with 80 μl of each of the following: 50-mM TEAB, 0.2% formic acid (FA), and 50% acetonitrile in 0.2% FA with centrifugation at 1000 g after each elution step. All elution fractions were pooled and dried under vacuum and cleaned up by stage-tipping^{51 (link)}.