Ms grade trypsin

Those protein spots that exhibited significantly different (p < 0.05) normalized spot volumes between the Ctrl and Mut groups using either Pro-Q Diamond or RuBPS staining were cut out manually from the mix gel with a pipette tip and were subjected to in-gel digestion. Initially, the gel spots were destained using a 1:1 ratio of 25 mM ammonium bicarbonate pH = 8.5 and 50% acetonitrile, than were reduced by 20 mM DTT for 1 hour at 56°C. Next alkylation was accomplished by 55 mM IAA for 45 min at room temperature in the dark, then an overnight digestion was performed with 100 ng stabilized MS grade trypsin (ABSciex) at 37°C. The reaction was stopped by concentrated formic acid (FA). The tryptic peptides diffused out of the gel pieces were dried in speed-vac concentrator (Thermo Scientific).

The protein bands were excised from the gel and subjected to in-gel trypsin digestion. The bands were destained using a 1:1 ratio of 25 mM ammonium bicarbonate (pH 8.5) and 50% acetonitrile followed by the reduction in the proteins using 20 mM dithiothreitol (Sigma, St. Louis, MO, USA) for one hour at 56 °C. The samples were further alkylated with 55 mM iodoacetamide (Sigma, St. Louis, MO, USA) for 45 min in dark. Overnight trypsin digestion was carried out with 100 ng stabilized MS grade trypsin (ABSciex, Framingham, MA, USA) at 37 °C. The reaction was stopped with the addition of concentrated formic acid. The tryptic peptides were extracted from the gel pieces, dried in a vacuum concentrator (Thermo Scientific, Waltham, MA, USA) and kept at −20 °C until the mass spectrometry analysis conducted.

Protein bands excised from gels were cut into smaller pieces and dried in speed-vac. Gel pieces were destained using 25 mM ammonium bicarbonate pH 8.5:50% acetonitrile (ACN) (1 : 1) solution, followed by reduction with 20 mM dithiothreitol (Bio-Rad) for 1 h at 56 °C. The alkylation was done using 55 mM iodoacetamide (IAA) (Bio-Rad) for 45 min at room temperature in the dark and the digestion was carried out overnight at 37 °C using 100 ng stabilized MS grade trypsin (ABSciex, LLC, Framingham, MA, USA). The reaction was stopped by adding formic acid (FA) (VWR, Radnor, PA, USA) and the tryptic peptides were extracted from the gel pieces, concentrated and re-dissolved in 10 μl 1% FA and used for mass spectrometry-based protein identification.