Current approaches to proteome analysis and mass spectrometry-based protein characterizations include the “bottom-up” and “top-down” approaches. A more-traditional approach sorts proteins by size and isoelectric point using 2D-PAGE and enzymatically digests the proteins within the gel matrix. Eluted protein fragments are analyzed by mass spectrometry and MS/MS to measure their size and sequence. An alternative but complementary bottom-up philosophy advocates first enzymatically or chemically cleaving a complex mixture of cellular proteins, and then sorting the pieces by one or more steps of chromatography. MS analyzes the recovered fragments as in the previous approach, and computers match the fragments to the proteins from which they are derived. However, there is value in the direct measurement and fragmentation of intact proteins, or “top-down” sequencing. The molecular mass of an intact protein defines the native covalent state of a gene’s product including the effects of post-transcriptional/translational modifications, and associated heterogeneity, that are modulated by the actions of other gene products. Moreover, the fragmentation pattern from large proteins can generate sufficient information for identification from sequence databases, particularly when combined with accurate mass measurements of both the intact molecule and its product ions.
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