This special symposium was held on May 23, 1994 at the Washington Convention Center. It was chaired by Lowell H. Ericsson from the University of Washington and was attended by approximately 500 scientists. The following provides a brief summary of the topics that were covered.
M.R. Bean
SmithKline Beecham Pharmaceuticals
Mass spectrometry has traditionally been a tool used by the petroleum and natural products industries. Recently, the cost of instrumentation has dropped from the million dollar range to a few hundred thousand or less and the range of molecules that can be analyzed has broadened to include large biopolymers. Mass spectrometry helps diagnose problems in peptide synthesis, aids in determination of protein sequence data obtained from Edman degradation, helps confirm peptide sequences, maps disulfide bridges, detects and corrects protein sequences derived from DNA or cDNA data and establishes and characterizes structures of posttranslationally modified proteins. Examples using both electrospray and laser induced ionization mass spectrometry were presented. Analysis of the glycosylation states of recombinant proteins produced under different conditions using time-of- flight mass spectrometer was nearly in real time. Stepped-collision energy LC/MS was used for simultaneous mass determination and identification of posttranslational modifications using a triple quadrupole instrument. Examples of the determination of N-linked and O-linked oligosaccharides and deamidation sites were also presented. Sample preparation requirements and problems due to buffer components were also covered.
K.A. Walsh, L.H. Ericsson, and R.S. Johnson
University of Washington
The answer to the question posed by the title of this talk is an unequivocal yes. Structure determination is not complete without characterizing posttranslational modifications; function may depend on posttranslational changes and in the biotechnology industry recombinant proteins must be compared to naturally produced molecules. Proteins are modified to introduce a covalently attached prosthetic group, to target them to different cell compartments, to stabilize them or to regulate their function. Chemical additions increase mass while cross-linking and proteolytic processing reduce the mass of the original polypeptide. The limit of accuracy in mass spectrometry of biopolymers is about 1/10,000 and heterogeneity enlarges that error. Common sources of heterogeneity include glycosylation, phosphorylation and proteolytic processing. Often, several different types of processing are present in the same molecule. In one example of direct assessment of a posttranslational modification, an unmodified recombinant protein served as an internal standard against which it was possible to predict that an N-myristoyl group on the native protein contained a double bond.
R.L. Niece and L.H. Ericsson
University of Wisconsin Biotechnology Center
The different capabilities of resource facilities were described along with the many names used for this type of laboratory. The goals of ABRF are to promote and facilitate communication and cooperation among facilities, provide research mechanisms for evaluating and improving instrumentation, protocols and operations and to educate staff, users, the scientific community and administrators. Core facilities provide an efficient mechanism for sharing the costs of expensive instrumentation and for saving capital costs and space. One measure of the impact of core facilities on research is the number of research groups that are served. In 1992, the average ABRF core facility synthesized DNA for more than 50 groups, sequenced proteins for 24 researchers, synthesized peptides for 19 groups, carried out amino acid analyses for 17 laboratories, and served as the mass spectrometry resource for 14 principal investigators. With more than 225 ABRF core laboratories, clearly, many hundreds of research programs depend on the critical resources provided by these facilities. Users of core facilities expect technical capabilities with appropriate quality controls along with scientific interactions plus training and education. Cost recovery targets for services varies. Core facilities contribute significantly to methods development in all areas where they provide services. A well-rounded facility provides product or service of quality and quantity appropriate to their user community and is continuously involved in methods development, education, and training to keep up to date.
P. Matsudaira, S. Kron. M. Footer, I. Coull, and M. Egholm
Whitehead Institute and Millipore Corporation
Peptide nucleic acids (PNA) can be used to inhibit gene expression either as antisense or antigene compounds, sequence DNA in hybrid arrays on chips, purify nucleic acids by capture, and for gene mapping. The spacing of bases in PNAs is identical to that in nucleic acid-nucleic acid hybrids because of triple helix formation. Two chemistries for solid phase synthesis are available and provide good yields. Fluorescently labeled PNAs have been used to map genes in a manner similar to fluorescence in situ hybridization. Using laser tweezers, this mapping can be performed on single molecules that are stretched to reduce coiling. Resolution is 10-1000 bases or about 0.34 nm/base.
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