MSRC Proteomics Laboratory Platforms Analytical Platforms

Multidimensional LC-MS-MS


​2-D Difference Gel Electrophoresis and Mass Spectrometry (DIGE/MS)

Difference Gel Electrophoresis (DIGE) technology enables quantification with statistical confidence for 2D gel experiments, where thousands of proteins are resolved by charge (using isoelectric focusing) and apparent molecular mass (using SDS-PAGE). DIGE analyses are typically used for differential-display proteomics on a global scale, sometimes testing a specific hypothesis, but often used to generate new hypotheses.

In some cases, it can be used to analyze relative stochiometry and/or post-translational modifications (that resolve into differentially-charged isoforms) of proteins in defined complexes (e.g., immunopurified complexes).

Protein samples are differentially labeled using three cyanine fluorescent dyes (Cy2, Cy3 and Cy5; Amersham Biosciences) prior to gel electrophoresis. Groups of labeled samples are then resolved together on the same gel and imaged separately using mutually-exclusive excitation/emission spectra, allowing for direct quantitative measurements without distortion from gel-to-gel variation.

In most cases, a pooled-sample internal standard is present on each gel, allowing for quantification of multiple (and repetitive) samples across multiple DIGE gels with statistical confidence (Student's t-test, ANOVA). The pooled-sample internal standard represents every protein present across all samples in an experiment. Each protein in the experiment therefore has an unique signal in the internal standard, which is used for direct quantitative comparisons within each gel, and also to match patterns and normalize quantitative abundance values for each protein between gels.

After proteins of interest have been flagged using DIGE, mass spectrometry and database interrogation are used for protein identification. Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) and data-dependent TOF/TOF tandem MS are used to acquire peptide ion masses (peptide mass mapping/fingerprinting) and individual peptide ion fragmentation patterns to provide candidate protein matches with statistical confidence from selected databases.


Multidimensional LC-MS-MS

Multidimensional Liquid Chromatography-Tandem Mass Spectrometry (Multidimensional LC-MS-MS)

MD_LC-MS-MS_Scheme.jpgThis technique is used for analysis of complex proteome samples (e.g., cell lysates, subcellular fractions) or multiprotein complexes (e.g., ribosomes or spliceosomes). This approach is often termed "shotgun" proteome analysis, because it is analogous to the shotgun sequencing approaches used in genome sequencing.

In contrast to 2D gel-based proteome analyses, the protein mixture is not first separated into components. Instead, the proteins are digested together to produce a highly complex mixture of peptides.

These peptides are analyzed by multidimensional LC-MS-MS and the MS-MS spectra are mapped to database peptide and protein sequences. This approach is the most powerful means of identifying the component proteins of a complex sample.

It is not uncommon for a multidimensional LC-MS-MS analysis to generate several thousand peptide identifications from a complex sample. The large data sets can be handled with new Bioinformatics Tools.

The objective of the approach is to obtain MS-MS spectra of as many of the peptides in the mixture as possible. This is accomplished by multidimensional peptide separations, typically combining strong cation exchange chromatography to separate the peptide mixture into 5-20 fractions, which then are individually analyzed by reverse phase LC-MS-MS. The multidimensional separation "spreads out" the complex peptide mixture into ion exchange fractions, which each contain fewer peptides

The sum of the identifications done on all the fractions is much greater than the number of identifications that could be done in a single reverse phase LC-MS-MS run. This approach has been referred to in the literature as MudPIT (Multidimensional Protein Identification Technique) and DALPC (Direct Analysis of Large Protein Complexes) and there are many variations of the basic approach.

While this approach is most commonly applied to identify components of complex protein mixtures or multiprotein complexes, multidimensional LC-MS-MS is also useful for identifying modified proteins. This is particularly true when modifications are of low stoichiometry or low abundance, as the modified peptides would be difficult to detect in complex mixtures. Multidimensional LC-MS-MS can be combined with stable isotope tagging to enable estimation of quantitative changes in complex proteome samples.