Analyte Detection and Subsequent Analysis

A 2D system coupled with a TOF-MS detector provides not only resolution for a large number of protein components, but also yields accurate intact molecular weight information (e.g., Opiteck et al., 1997 Liu et al., 2002 Millea et al., 2005). Moreover, by splitting the effluent just prior to the MS interface, a small portion can be diverted for MS analysis, whereas the bulk of the sample can be collected for subsequent analysis, following enzymatic digestion, to provide positive identification and characterization of the proteins present in the fraction. 

The ESI-MS of an intact protein yields a series of ions with mfc values corresponding to sequentially charged species (Fenn et al., 1989). Algorithms and software for the deconvolution of these peaks into a single neutral mass have been available for many 

The combination of this top-down proteomics approach, which generates information on the structure of the intact protein, with a bottom-up approach for protein identification (using MS/MS data of tryptic peptides from the collected fractions) has been particularly useful for identifying posttranslational modifications, cotransla-tional processing, and proteolytic modifications in a number of proteins. Examples from our work will be shown to illustrate this hybrid methodology for proteomics analysis. 

---