2-D LC Tandem Mass Spectrometry Services

Complex mixtures of proteins can be analyzed using a two-dimensional liquid chromatography separation (2-D LC) of proteolytic fragments, coupled to tandem MS analysis (MS/MS) and database searching to identify the original proteins. Samples such as cell lysates or tissue extracts are digested enzymatically into massive numbers of peptide fragments. In contrast to intact proteins, the proteolytic fragments are handled more easily by both LC and MS. Peptide signal is spread out in time using a fractionation strategy that may be an on-line coupled strong-cation exchange, off line cation exchange of peptides, or an SDS-PAGE run on the protein mixture followed by in-gel digests to obtain peptides for analysis.  Each fraction is then loaded on a reversed phase column and resolved with a water-acetonitrile gradient. Peptides are electrosprayed into the mass spectrometer (Agilent ion trap) run in a data dependent mode where each peptide is subjected to MS/MS as it elutes. Database search algorithms are used to identify proteins using the mass spec data.

The Mass Spec Facility has invested in several proteomic software packages for working with large datasets. We have a copy of Mascot, which allows for larger file sizes and more IDs than the publicly available version, and Spectrum Mill MS proteomics workbench, which allows for comparisons of multiple runs and quantitation by ICAT.