In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis. These identifications were then linked with corresponding ESI-TOF-MS intact protein mass data to permit full protein characterization. This methodology was applied to an E. coli cytosolic protein fraction, and enabled the identification and characterization of proteins exhibiting co-translational processing, post-translational modification, and proteolytic processing events. The approach also provided the ability to distinguish between closely related protein isoforms. The summary of results from this study indicated that roughly one-third of all detected components generated corresponding data from both top-down and bottom-up analyses, and that significant and novel information can be derived from this application of the hybrid analytical methodology.

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A review of liquid chromatography (LC) techniques for the subfractionation of complex protein mixtures into ''subproteomes'' is presented. Fractionation of protein mixtures into general subclasses based on physical, chemical, and biological attributes of proteins is an effective way of classifying and characterizing complex proteomes. Classical chromatographic techniques, such as reverse-phase, ion-exchange, and size exclusion chromatography (SEC) are discussed, as well as a variety of affinity chromatography techniques such as immobilized metal affinity chromatography (IMAC) and lectin affinity chromatography. Applications of these methods to proteomic endeavors are highlighted. Multidimensional chromatography methodologies are examined for combining orthogonal chromatographic formats interfaced with mass spectrometric (MS) detection. The versatility of liquid chromatography for proteomic analysis makes LC a valuable tool for the protein characterization of living systems.