To analyze a complex protein sample by LC/MS, so called "shotgun sequencing" approach is often used.  The  sample is digested with a suitable protease, commonly trypsin, and the resulting peptides are separated by HPLC and then characterized by tandem MS.  Proteins are identified by matching the MS fragmentation patterns with predicted information from genomics or proteomics databases.  While many studies have used one dimensional reversed phase HPLC before the MS measurement, such an approach is inherently limited by the number of peptides that can successfully be loaded and resolved on the column and detected by the mass spectrometer.  If we assume an average protein MW of 50,000 that yields about 40 peptides upon digestion by trypsin, then a sample mixture of 1,000 proteins - many fewer than are present in many proteomics samples - will have 40,000 peptides that we might potentially identify.  It is clearly not possible to resolve all of these peptides using a single HPLC column.

To increase the resolving capability of the HPLC separation, 2D HPLC techniques are employed using a combination of ion exchange chromatography, where the peptides are separated based on  their charge, followed by RP HPLC, which separates the peptides based on their hydrophobicity.  This approach greatly reduces the complexity of the peptide mixture being analyzed by the mass spectrometer at any given time point, improving the odds that a particular peptide will be detected.  This is especially true for less abundant or poorly ionized peptides.

• Identify proteins rapidly with High Throughput Protein ID using fast chromatographic separations and automated Data Dependent MS and MS/MS techniques.
• Analyze proteins comprehensively with Maximum Sequence Coverage using chromatographic separation and in-depth MS analysis.
• Identify phosphopeptides and phosphorylation sites with Phosphorylation Site Mapping using automated Data Dependent Neutral Loss MS³ analysis.
• Maximize productivity with MudPIT (multidimensional protein identification technology) analysis of highly complex samples using multidimensional LC/MS to separate and identify a myriad of proteins

You can now also order additional proteomics resources, or learn about other mass spectrometry or proteomics solutions.