Multidimensional Chromatography Methods

Alternative to 2DE, proteins and peptides can be separated by multidimensional (micro) liquid chromatography methods and/or capillary electrophoresis on-line coupled to multidimensional mass spectrometry (MSn) (Shen and Smith, 2002). Protein mixtures are proteolytically digested, and a large number of the resulting peptides are resolved and identified in a single run, a strategy termed ''multidimensional protein identification technology'' (MudPIT) (Washburn et al., 2001; Wolters et al., 2001). Orthogonal separation modes of any choice can be coupled, provided that the eluate composition of an earlier separation mode is compatible with the following separation mode. Prefractionation of protein families (e.g., through affinity purification, solution isoelectric focusing, etc.) (Righetti et al., 2005), followed by one- or multidimensional separation, increases the dynamic range of the analytical method allowing for the monitoring of even low abundance proteins (Gygi et al., 2002). This is necessitated by the MS detector, where large amounts of high-abundance peptides can suppress the ionization of lower abundant peptides.

To achieve relative quantitation of proteins during 2DE or chromatographic runs, Aebersold and coworkers have developed the isotope-coded affinity tag (ICAT) methodology (Gygi et al., 1999): in the original approach proteins/peptides are derivatized with a bifunctional reagent (structure 1, as shown in Figure 9.1) which contains an electrophile on one end and biotin on the other end.

The electrophile affords covalent derivatization of Cys, and the biotin moiety enables affinity purification of the labeled proteins/peptides. An organic linker between the functional groups of the reagent contains

either normal (H) or heavy (D) hydrogen isotopes. When two pools of proteins are labeled with the normal or heavy isotope-containing ICAT reagent, their relative abundance can be determined by quantitative MS analysis of the ratio of normal to heavy ICAT-derivatized peptides of specific proteins. During 2DE, normal and heavy isotope ICAT-derivatized proteins comigrate, ensuring an accurate relative quantification of the proteins from different sources (Smolka et al., 2002). However, slight differences in the elution profiles of normal and heavy isotope ICAT-labeled peptides have been noted during reverse-phase chromatography (Zhang et al., 2001). Therefore, an accurate relative quantification of ICAT-labeled peptides requires MS analysis at the respective peak maxima of the individual chromatographic peaks of normal and heavy isotope ICAT-labeled peptides. Complications have also been noted with the recovery of ICAT-labeled peptides during biotin-avidin affinity purification (Gygi et al., 2002), and with tandem MS sequencing of ICAT-labeled peptides.

Recent improvements of ICAT reagents include the design of a solid-phase isotope tagging method, avoiding biotin, and incorporation of a photocleavable linker (structure 2, as shown in Figure 9.2); after solid-phase purification of ICAT-labeled peptides and photolysis, the remaining peptides essentially contain an additional normal or heavy isotope-containing Leu residue (Zhou et al., 2002b).

Using the ICAT methodology, complex mixtures of peptides from digested proteins can be reduced to one or a few peptides per protein (essentially as many peptides as the protein contains reduced or reducible Cys residues). Such a reduction in sample size significantly enhances the dynamic range of the proteomic analysis. For example, using the codon bias value as a measure for protein expression, Gygi et al. (2000) showed that traditional 2D gel electrophoresis was not able to indicate low abundant proteins with codon bias values of <0.1, whereas many of these proteins were detected with a multidimensional separation strategy including ICAT-labeling and affinity purification (Gygi et al., 2002). We note that irreversibly oxidized Cys residues (to cysteic acid) will not be amenable to ICAT labeling.

Surface-enhanced laser desorption/ionization (SELDI) represents a concept with great promise for sample size reduction and enrichment of specific classes of analytes (Merchant and Weinberger, 2000). Protein-chips with specifically modified surfaces allow the affinity purification of analytes prior to on-chip digestion and MALDI-TOF MS analysis. Various different chemical and biochemical surfaces have been designed containing immobilized hydrophobic, ionic, or mixed-mode phases or antibodies; IMAC supports; polynucleotides; or specific enzymes or receptors.

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