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Separation of proteins by 2D SDS-PAGE

Protein mixtures are resolved by 2S-SDS-PAGE on large format gels. Isoelectric focusing in various pH ranges is available. Gels are stained with Sypro Ruby and an image is recorded. Protein spots are identified by MALDI-MS-MS or LC-MS-MS as described below.

2D Difference Gel Electrophoresis (2D-DIGE)

This technique is used to detect proteome changes between pairs or groups of samples. The two protein samples to be compared are labeled with proprietary cyanine dyes (Cy2, Cy3, or Cy5) and mixed before undergoing separation by 2D SDS-PAGE.

Quantitative fluorescence imaging is done with selective excitation and emission wavelengths to identify those proteins changing during an experiment. Proteins of interest may be selected for subsequent identification by MS.

Simple Protein Identification

This service provides identification of proteins from 2D gel spots, from 1D gel bands of simple mixtures and from nominally pure proteins.

Core personnel do the in-gel or in-solution digestion with trypsin or other proteases, elute the peptides from the gel, perform a additional cleanup as necessary, and analyze the peptide mixture by MALDI-MS-MS and identify the protein(s) from MS-MS spectra with the aid of database search software (Mascot).

Shotgun Proteome Analysis by Multidimensional LC-MS-MS

Multidimensional LC (ion exchange/reverse phase) coupled to MS-MS is the most powerful technique available to proteome mining of complex mixtures. This approach is ideal for identifying components of complex proteomes and multiprotein complexes.

In multidimensional LC analyses, peptide mixtures are resolved into 5-20 fractions on a strong cation exchange (SCX) column using a salt gradient. Each ion exchange fraction is then analyzed by reverse phase capillary LC-MS-MS using a reverse phase column.

In some cases, eluent from the reverse phase column is spotted to MALDI targets with a micro fraction collector and the samples are analyzed by MALDI-MS-MS. Peptide identification from MS-MS spectra is done by database search software (Sequest, X!Tandem or Mascot).

Identification of Protein Post-Translational Modifications or Protein Adducts

Proteins are analyzed by any of the approaches described above and MS-MS data are analyzed either with database search software with specified modifications (e.g., phosphorylation at Ser/Thr/Tyr).

Application of specialized bioinformatics tools, such as P-Mod and SALSA software enables discovery of unanticipated modifications. In most cases, detection of low abundance modifications is facilitated by selective enrichment of the protein of interest or by affinity capture of modified protein or peptide forms.

Quantitative Analyses of Differential Protein Expression and Modification

2D-DIGE provides relative quantitation of changes in protein expression and/or modification, provided that the proteins of interest are detected by staining and are resolved by the 2D-SDS-PAGE separation.

Use of mixed standards and Cy2/Cy3/Cy5 dyes allows reliable quantitation across multiple gels in larger experiments. Relative quantitation is also done with stable isotope tags. In this approach, protein extracts from two samples to be compared are chemically tagged with light and heavy (unlabeled vs. deuterium or 13C-labeled) reagents, then combined, digested and the tagged peptides are then analyzed by LC-MS-MS.

Signal intensities for the differently tagged isotopomers indicate relative levels of specific proteins of protein forms in the samples. A variety of isotope tagging chemistries are used, depending on the problem to be studied. Absolute quantitation of specific proteins or modified protein forms is done by stable isotope dilution with 13C/15N-labeled peptide internal standards and selected reaction monitoring LC-MS-MS.