Proteomics and Metabolomics Laboratory

Tutorials

Proteomics

Proteins are identified by sequencing experiments performed by tandem mass spectrometry in the Lerner Research Institute's Proteomics Laboratory.
The proteins are presented for analysis as either in solution or as stained bands in polyacrylamide gels. The proteins are digested with a protease, usually trypsin, either directly in the polyacrylamide gel or in-solution. For the in-solution digestion, we typically start by protein precipitation followed by solubilization in 6M urea, reduction with 100mM DTT, alkylation with 150 mM iodoacetamide, reduction of urea concentration to less than 2M, and digestion with a protein to trypsin ratio of 50:1 overnight at room temperature. For the digestion of proteins in a gel band, each band is cut from the gel as closely as possible to minimize excess polyacrylamide, divided into a number of smaller pieces and washed and destained in 50% ethanol/5% acetic acid overnight. For 1D gel bands, the gel pieces are then reduced and alkylated in 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and 50 mM iodoacetamide in 0.1 M ammonium bicarbonate, respectively, at room temperature for 0.5 h. We skip these steps for 2D bands because reduction and alkylation is a part of the electrophoresis procedure. The reduced-alkylated bands are then successively, dehydrated in acetonitrile, rehydrated and washed in 0.1 M ammonium bicarbonate, and dehydrated in acetonitrile. The protease trypsin (20 ng/µL trypsin in 50 mM ammonium bicarbonate) is added and driven into the gel pieces by the rehydration. The sample is digested overnight at room temperature and the peptides that are formed are extracted from the polyacrylamide with successive washes with aliquots of 50% acetonitrile/5% formic acid. These extracts are combined and evaporated, and the final sample reconstitute in 1% acetic acid.
Our LC-tandem MS systems use reversed-phase HPLC with 50 or 75 µm id capillary columns packed with either Phenomenex Jupiter C18 or Acclaim Pepmap C18. Volumes of the digests (between 1 and 10 µL) are injected and the peptides are eluted from the column by a linear gradient of acetonitrile in 0.1% formic acid gradient at a flow rate of approximately 0.3 nL/min. The digest is analyzed using the data dependent multitask capability of the instruments in which full scan mass spectra are acquired to determine peptide molecular weights and product ion spectra acquired to determine amino acid sequence in successive instrument scans.