Methods for determining the presence of mutations and modified amino acids were the focus of this wide ranging symposium, chaired by Ron Niece and Ken Williams. This ABRF/ASBMB sponsored symposium was attended by more than 400 participants at the recent ASBMB/Biophysical Society Meeting in Houston. Since total sequence analysis provides one approach to detecting mutations, an underlying theme of this symposium was an appraisal of automated protein and DNA sequencing.
Finn Wold (University of Texas) surveyed the more than 200 modified amino acids that have been found in proteins. He predicts that as the methodology is further improved and as approaches are found to stabilize labile modifications, many more will be discovered. Although the specificity of co-and post-translational modification is exquisite, very little is yet known about the determinants which account for this specificity. He noted the need for a standardized compilation of the elution positions of modified amino acid derivatives as observed during protein sequencing. Note, in response to this suggestion, Greg Grant will soon begin collecting these data (see page 3).
Paul Tempst (Memorial Sloan-Kettering Cancer Center) described techniques for improving proteolytic cleavages and for utilizing comparative HPLC peptide mapping to identify peptides containing minor structural alterations. By supplementing the digest buffer with denaturing (urea or guanidine hydrochloride) and/or solubility promoting (detergents) agents, even intransigent proteins can be reproducibly cleaved. Under optimum conditions, even sub-picomole amounts of peptides isolated by HPLC can be partially sequenced with the limiting factor being the recovery of pyridylethylcysteine, arginine, serine, and threonine.
A.L. Burlingame (University of California) emphasized that mass spectrometry (MS) is intrinsically better suited than most established methodologies in biological chemistry for dealing with unanticipated substances including the myriad of covalent modifications that are found in biopolymers. Recent advances in multi-channel array detection have vastly increased the sensitivity of tandem MS and new ionization techniques such as electrospray and matrix-assisted laser desorption now enable accurate molecular weights to be obtained on intact proteins.
Greg Grant (Washington University) provided a retrospective evaluation of four years of ABRF studies involving the distribution and analysis of "unknown" proteins and peptides by as many as 90 member laboratories. These studies help establish realistic expectations regarding accuracy of sequence calling and the length of sequence that is likely to be obtained from a given amount of sample. Another conclusion is that experienced protein sequence operators continue to maintain a lower error rate and call longer sequences than available "automatic sequence calling" software.
Steve Carr (SmithKline Beecham) emphasized the benefits of a combined mass spectrometric/protein chemistry approach to structural analysis and the fact that even relatively complex mixtures are amenable to MS analysis. He described the results of a recent ABRF study in which the same peptide was synthesized by 40 different laboratories and then extensively characterized by the ABRF. Amino acid analysis proved to be a poor predictor of the relative abundance of the desired product. Rather, a quantitative separation technique (such as HPLC) coupled with MS proved essential for detecting the numerous subtle alterations (such as dehydration, oxidation, and incomplete deprotection) that occur during the cleavage and deprotection of synthetic peptides.
C. Thomas Caskey (Baylor College and HHMI) described molecular scanning methods for detecting mutations. The polymerase chain reaction (PCR) provides a facile means for quickly generating material for analysis. Multiplex amplification utilizing two different PCR "cocktails" is sufficiently sensitive to detect 98% of the mutations that give rise to muscular dystrophy. Chemical mismatch cleavage and DNA sequencing is useful for detecting point mutations.
Lloyd Smith (University of Wisconsin) described the various steps involved in DNA sequencing and their limitations. He estimated that the overall cost of DNA sequencing is still in the range of $5-10 per finished base. Potential developments which may increase throughput and reduce the cost of large scale DNA sequencing were discussed. Sequencing by hybridization represents a viable alternative for obtaining short sequences. Capillary electrophoresis in a slab gel format using fluorescence detection may also significantly increase throughput. Mass spectrometry is being actively investigated as an analytical system for the detection of Sanger sequencing reaction products.
Ron Niece and Ken Williams
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