Protein micro- and macroarrays: digitizing the proteome.

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry.

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.