( ere two separation techniques are ...)Protein Depletion for Plasma and Serum Proteomic Analysis

ere two separation techniques are achieved in a single column packed with two different separation matrices [91]. Typically MudPIT uses a strong cation exchange and a reversed phase resin in single column that can be interfaced directly with the mass spectrometer. This technology allows a higher level of automation in sample handling, analysis and data processing.

Different combinations of these multidimensional separation technologies are used in both top down and bottom up proteomic analysis. In the top down approach [92] a mixture of proteins in a sample are separated into individual spots or fractions using different separation techniques, and the individual proteins are then analyzed by mass spectrometry to establish their identity. This is accomplished by determining the mass of the whole protein ion and then fragmenting the ionized protein to yield relatively large segments whose masses can then be deconvoluted and compared against known proteins in protein databases. On the other hand, the bottom up approach can be performed by using either of two strategies: In one strategy, samples containing a mixture of different proteins are subjected to multidimensional separation techniques and the individual protein spots or fractions are digested with trypsin to yield peptide fragments. With or without another separation step, the tryptic peptides are analyzed by mass spectrometry to establish their identify, either based on their peptide mass fingerprints or by further mass fragmentation to obtain sequence information. Recently, most bottom up proteomics employ the shotgun strategy [91,93- 99] where, without prior separation, entire samples containing a mixture of a large number of different proteins,
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