Proteomics using 2-D SDS Polyacrylamide Gel Electrophoresis
2D gel electrophoresis: full details at http://proteomics.missouri.edu/
Trypsin digestion: Trypsin digestion is done before the mass spectrometer analysis, the gel plugs are washed 4 times 15 min. in wash solution (50% acetonitrile, 50 %, 50 mM ammonium bicarbonate). Gel plugs are then briefly dried in 100 % acetonitrile, incubated for 20 min. in 100 % acetonitrile, and completely air dried. They are rehydrated with a trypsin solution (Promega, 20 U) and incubated for 2 hours at 4°C. The trypsin solution is removed and replaced by 15 μl of 40 mM ammonium bicarbonate and 10 % acetonitrile. Trypsin digestion is performed overnight at 37°C. The samples are centrifuged for 30 sec. at 12000 g. The supernatant containing the peptides is transferred to a new tube containing 4 μl of extraction solution (60 % acetonitrile, 1% trifluoroacetic acid). Gel plugs are washed two times with extraction solution for 10 min. and the supernatants, after a centrifugation of 30 sec. at 12000 g, are collected and transferred to the tubes described above. Samples are then iophylized, reconstituted in 5 μl of 990/10 water/88% formic acid (V/V), ziptiped on a C18 column for purification and reconstituted in 10 μl of elution buffer (700/290/10 (V/V/V) acetonitrile/water/formic acid 88% elution buffer), frozen in liquid nitrogen.
Analysis of tryptic peptides: 0.5 μl digested purified peptides were mixed with the same volume of alpha-cyano-4-hydroxycinnamic acid (Fluka MS grade, Sigma-Aldrich, Saint Louis, USA) solution (5mg/ml in 500/380/20/100 acetonitrile/water/ 10% trifluoroacetic acid/100 mM ammonium dihydrogen phosphate). The sample/matrix (0.3 μl) mix was deposited on a stainless steel plate (ABI01-192-6-AB). The tryptic peptides were analyzed on an Applied Biosystems Inc. 4700 MALDI TOF/TOF MS in positive ion delayed extraction reflector mode with a 355 nm (200 Hz) laser. The instrument was calibrated with ABI peptide standards (4700 Mass standards kit, 4333604). Spectra were analyzed using the GPS Explorer software (v. 3.0) (Applied Biosystems) and the Matrix Science’s Mascot search engine (www.matrixscience.com) against the NCBI Viridiplantae protein database. Search parameters included, a maximum of 150 ions per MS spectrum with a signal to noise ratio >20, a mass error of 0.1 Da for the monoisotopic precursor ions, a maximum of one allowed misscleavage by trypsin, an exclusion of peptide masses corresponding to the autolysis of the trypsin, carbamidomethylation of cysteines and methionine oxidation respectively as fixed and variable modifications.