The Section on Macromolecular Analysis is attempting to upgrade the common practice of gel electrophoresis by undertaking the following projects: the development of theory and computer programs for deriving information from mobility regarding physical properties of analytes and polymer networks and for predicting resolution, resolution of presently unresolved or poorly resolved analyte types such as subcellular-sized particles and the size- and charge-isomeric forms of peptides and their complexes, the development of detergent and miniaturized gel methods suitable for native membrane proteins and their complexes of a low level of occurrence, and the development of automated analytic and preparative gel electrophoresis methods.

A significant portion of current biological research focuses on an account of the cellular proteins responsible for the activities and functions of the cell. One aspect of that research, designated proteomics, is working, by means of mass spectrometry, toward the identification of proteins on the basis of their mass. Conventional practice calls for separating the mixture of proteins in the cellular extract of interest by two-dimensional gel electrophoresis and detecting the proteins as "spots" by specific staining methods. The spot of interest is then excised from the gel, the gel slice is dehydrated and reswollen in the presence of the proteolytic enzyme trypsin, and the tryptic peptides derived from the protein are eluted from the gel slice for measurement of their mass by mass spectrometry. The mass of the peptides is then compared with that of aminoacid sequences in databases stored in computer memory and derived from the corresponding DNA sequence. Such identification therefore depends on a previous genetic analysis of the cellular material in question.

Our laboratory developed an alternative procedure for collecting protein identified as a gel band (or spot); it replaces band excision with direct electroelution of the intact protein by placing electrodes across the band at a direction orthogonal to that of electrophoretic migration in the separating gel (Figure 10). The alternative procedure offers several advantages over conventional practice. First, it is applicable to those cases in which no earlier DNA sequence analysis has been performed and for which, therefore, no sequence exists in the database. Second, even where a sequence has been determined, the procedure adds the mass of the intact protein to that of its constituent peptides for purposes of identification. Third, it allows for electrofocusing (IEF) and critical re-electrophoresis analysis of the eluate before mass spectrometry. Fourth, it avoids the presence of gel during tryptic digestion, thus increasing the rate of digestion and avoiding adsorption of protein, peptides, and enzyme. Fifth, it appears more adaptable than an excision procedure to automation of sequential spot elution. We were able to verify the first three advantages while the last two remain hypothetical. The primary potential importance of such an approach, compared with conventional practice, lies in the fact that the electroelution of the intact protein, when applied to Immobiline IEF gel strips of high load capacity, allows for an accumulation of large protein loads for a subsequent two-dimensional electrophoretic separation with a number of narrow pH gradients in the first dimension and subsequent peptide analysis of the bands. The alternative approach will allow for a significant increase in the number of proteins detected by two-dimensional gel electrophoresis, which currently yields only 10 to 20 percent of proteins, i.e., those that are most abundant. Of further interest for the future of proteomics is the fact that we have achieved a direct electroelution of a native protein from a nondetergent gel. This finding foreshadows the possibility of identifying those post-translationally modified proteins that on SDS-PAGE migrate together.

Figure 10

Apparatus for a direct orthogonal electroelution of gel electrophoretic protein bands.

Sequential electroelution of protein bands on the two-dimensional gels remains to be solved. A possible solution may be to polymerize on a net, which is permeable to the orthogonally directed current of electroelution. A suitable net support has been designed and constructed. We intend to position the gel by moving the net support under micromanipulator control, thereby preventing gel stretching as we move sequentially from spot to spot. Once a suitable frame for the net-supported gel is constructed, the positioning of the net will have to be interfaced with the image analysis system to arrive at a computer-controlled automated protein elution method without any gel sectioning.