Mass spectrometry accounts for essentially all de novo protein identification. For the most part, applications fall into one of two categories, either systems biology or standard identification of proteins isolated by traditional means such as multicolumn chromatography. The latter purification schemes can be very costly and time consuming, yet they typically yield only one or a few purified proteins, which can be identified within a day or so by a proteomics facility. Molecular and cellular biologists could take better advantage of the much improved protein identifying capabilities and throughput if robust, facile methods were available to scale down and accelerate the purification process, a bridge,

From: Methods in Molecular Biology, vol. 338: Gene Mapping, Discovery, and Expression: Methods and Protocols Edited by: M. Bina © Humana Press Inc., Totowa, NJ

as it were, between traditional life sciences research and the analytical power of proteomics. This has been particularly the case in the field of nucleic acid biochemistry, including the study of DNA binding proteins that function in transcriptional regulation, replication, and maintenance of chromosomal integrity.

We describe an efficient, accelerated method for affinity capture of transcription factors on specific DNA-magnetic particles, to yield final preparations in a form and amounts that are compatible with standard MALDI-TOF MS-based protein identification. A major obstacle to developing this approach into a widely applicable protocol is the inadvertent, extensive binding of nonspecific proteins to the bait. This problem can be addressed at two levels. First, a single batch-fractionation on P11 serves to increase the relative concentration of the transcription factors of interest, removes several of the abundant nonspecific DNA binding proteins, and may resolve multiple factors in different fractions that can subsequently be used for parallel affinity capture. Second, a number of counteracting measures can be implemented. Protein fractions are incubated with magnetic beads carrying concatamerized DNA binding sites, and in the presence of short oligo(dI:dC) competitor, resulting in higher specific-ligand density and displacement of proteins that preferentially bind to DNA nicks and ends. The beads are easily collected from larger volumes using high-powered magnets. Preclearance with mutant DNA-beads (negative selection) greatly reduces the background of nonspecific proteins in the final preparation. The number of negative selections depends on the abundance and binding kinetics of the respective transcription factors and is determined experimentally. In the case of low-affinity binders, negative selection is preceded by a single positive selection, which results in much reduced protein mass and volume. Affinity capture of high-affinity binding transcription factors, on the other hand, can be performed in high salt, increasing the stringency of the procedure.

A targeted proteomic analysis of this nature should always be preceded by a thorough molecular biological analysis of the system under study. Proteomics is not a stand-alone science, and the field would benefit greatly if more projects were initiated by the traditional, hypothesis-driven research groups. The best way to popularize it among biologists and clinical scientists is by bringing a large portion of proteomic research activities into their own laboratories, leaving only the final readout (i.e., protein identifications) to specialized facilities. The approach and protocol that are presented here should be considered in that context.

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