As in the past three years, the ABRF has been invited by the Educational Affairs Committee of the ASBMB to organize a special session covering state-of-the-art biotechnology. This session will be chaired by Lowell H. Ericsson and Ronald L. Niece. The session will be held on Sunday, May 22, 1994 from 3:15 - 5:30 PM in Room 15 of the Washington Convention Center. The program and abstracts are given below with the first author being the speaker.
A molecule's mass is probably the single most useful physical property that we can measure. Although very approximate means of mass measurement are available cheaply, increases in mass accuracy become exponentially more expensive. It is a testimony to how important this accuracy has become, that access to mass spectrometers has become more generally available in this country. However, instrumentation in mass spectrometry, after nearly 80 years of development, continues to be in a state of active transformation, and it behooves those who will work with this technique to have some understanding of the following questions. When is spectral resolution important? What is the best choice of instrumentation? What kind of information can I obtain and how reliable is it? In this presentation I will touch each important question and discuss the last one at some length. Examples of some of the newer and more innovative modes of mass analysis as applied to polypeptide and protein structure will be drawn from our own laboratory's work in the last two years. These will include methods for automated, rapid measurement of molecular weights, specific detection of glycopeptides and phosphopeptides in protein digests by LC/MS, and ancillary techniques to structure determination by tandem mass spectrometry.
Physical maps of genomes record the relative positions and distances separating sequence landmarks (STS's) along chromosomes. In some simple organisms, a high density of landmarks is mapped resulting in intervals the size of single genes. In contrast, the human genome is mapped at low density; only two or three STS's are mapped with a precision of 200 kb within any 1 mb segment. The large error in measurement leads to uncertainty in the number of genes within each interval. The order of landmarks on human maps is known but their positions remain poorly defined. We are developing instrumentation and biochemical methods to map landmarks at 0.1 kb precision. Our approach uses optical tweezers to grab and straighten strands of genomic DNA so that fluorescent peptide nucleic acid (PNA) probes, hybridized to their target sites, can be imaged. In model studies, fluorescent DNA probes are mapped on a linear lambda DNA with 1 kb precision. Concurrent studies are characterizing PNA's as hybridization probes and developing novel variants. With further improvements to optical tweezers, digital imaging systems, and PNA probes, our goal is to refine physical maps at 0.1 kb precision and to map locations of genes, repetitive sequences, and restriction sites.
R.L. NIECE AND L.H. ERICSSON
University of Wisconsin Biotechnology Center, Madison,
WI 53705 and University of Washington , Seattle, WA 98195.
Centralized facilities frequently provide the technical capabilities for specialized synthetic products. Because of the technical demands of the protocols and the expensive instruments, shared instrumentation laboratories represent an economical approach to providing specialized services. The increasing complexity of modern biochemical sciences portends a continuation of the centralization. Resource facilities provide economies due to scale and experience and allow the users and principal investigators of research projects to concentrate their efforts, time, space and funds on experimental design and interpretation of the data. The variety of technologies offered by shared resource facilities continues to increase and often encompasses automated DNA sequencing, capillary electrophoresis, mass spectrometry and carbohydrate analysis as well as the more traditional services of protein and nucleic acid chemistry. Expert scientists and technicians in shared instrumentation laboratories provide a significant component to the improvement of protocols and to the development of new applications, especially for the techniques recently moving into service laboratories.
K.A. WALSH, L.H. ERICSSON AND R.S. JOHNSON
University of Washington, Seattle, WA 98195
All known eukaryotic proteins appear to be posttranslationally modified during their normal maturation or during their regulation, either by removal of amino acid residues or by covalent attachments. These covalent "editing" events serve diverse biological functions, including cellular targeting, stabilization of structure, introduction of novel chemical prostheses, covalent regulation, and turnover initiation. Often, identification of such covalent modifications is critical to understanding a structure/function relationship. Moreover, if cDNA clones are expressed in heterologous cells, the metabolic machinery may lack the capacity for fidelity of normal posttranslational editing; such differences in recombinant proteins must be detected, and defined or avoided. Currently, the most powerful method to detect posttranslational modifications, and to compare natural and recombinant proteins, is mass spectrometry (MS) in the electrospray ionization (ESI) or matrix-assisted laser desorption mode. A single measurement of mass with an accuracy of 1 part in 10,000 gives the first indication whether mass has been added to that predicted by the DNA, or whether residues have been removed by cellular processes. Coupled HPLC/ESI/MS of an enzymatic digest detects modified peptides, and tandem MS/MS may provide specific detail. The diversity of natural covalent modifications will be summarized from a biological perspective, with examples of detection and placement by ESI Mass Spectrometry.
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