| Popis: |
One-dimensional (1-D) or two-dimensional (2-D) polyacrylamide gel electrophoresis is a convenient technique for purifying small amounts of proteins from very complex mixtures (O’Farrell, 1975; Celis & Bravo, 1984). For structural analysis, proteins are electrotransferred from the gels onto immobilizing membranes for subsequent NH2-terminal sequence analysis (Vandekerckhove et al., 1985; Aebersold et al., 1986; Matsudaira, 1987). Alternatively, proteins can be cleaved either as membrane-bound molecules (Aebersold et al., 1987; Bauw et al., 1988) or when still present in the gel matrix (Rosenfeld et al., 1992). The resulting peptides are then separated for further characterization. Recently, computer searching algorithms have been developed that use peptide mass fingerprinting to identify proteins whose sequences are stored in databases (Maim et al., 1993; Pappin et al., 1993; Yates et al., 1993). Such peptide mass information can be obtained from previously unseparated mixtures using matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) (Mann et al., 1993, Zhang et al., 1994) or from a reversed phase column eluate on-line connected with an electrospray ionization mass spectrometer (ESI-MS). Conventional automated Edman degradation techniques or mass spectrometric-based methods allow sample analysis in the low picomole or even femtomole range. Unfortunately, when only such small amounts are present in the starting mixture it is difficult to purify and digest the protein with high peptide recoveries. This is probably due to adsorption of a fraction of the protein within the pores of the immobilizing membrane by which they are trapped, out of reach of the proteases. In case of in-gel cleavage a fraction of the protein or its fragments stay inside the gel matrix. The problems mentioned above can be reduced by working in small but highly concentrated protein spots, thus reducing the membrane surface or gel matrix volume. When only small amounts of proteins are present in the gels, these conditions are seldomly met and therefore it is necessary to combine several protein spots into a small volume. In this paper we describe a method that allows to reproducibly elute and concentrate proteins from combined gel pieces. The concentration factor can be larger then 50 and the protein is concentrated into an agarose gel. The protein is then melted out of the agarose gel prior to proteolytic cleavage so that the digestion proceeds in a soluble phase. The overall peptide yields are at least 70% of those obtained from direct cleavage in free solution. This approach does not suffer from the problems of adsorption or in-gel trapping and should therefore be a better procedure. In addition the technique is amenable for miniaturization. Here we report our initial experiences with this technique and show that it can serve as an efficient link between polyacrylamide gel purification and protein identification by microsequencing or mass spectrometry in the very low picomole range. |
