Proteomics Laboratory | Tutorial

Overview of Protein sequencing and identification by tandem mass spectrometry in the Lerner Research Institute's Proteomics Laboratory.

Proteins are identified by internal sequencing experiments performed by tandem mass spectrometry in the Lerner Research Institute's Proteomics Laboratory.

The proteins are presented for analysis as stained bands in polyacrylamide gels. The protein bands of interest digested with trypsin directly in the polyacrylamide gel bands. For the digestion, 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 m m id capillary columns that we pack a Phenomenex Jupiter C18 stationary phase. Volumes of the digests (between 1 and 15 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 0.25 µL/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. This mode of analysis produces up to 2000 collisionally induced dissociation (CID) spectra of ions ranging in abundance over several orders of magnitude.

The data are analyzed by submitting all of the CID spectra collected in an the LC-MS run to the database search program Mascot, which compares those spectra with spectra expected from calculated tryptic peptides for all proteins in the current non-redundant database. Peptide-spectra matches are scored and ranked in a process that groups the spectra according to the protein sequence that contains the matching peptide thereby identifying the source protein. All matches are verified by manual inspection of the matching spectra. It is possible to identify more that one protein in a given band.