Amino Acid Sequencers Of Today: Sensitive, Efficient-And Very Costly

Amino acid sequencing has become a highly automated and efficient process. It plays an invaluable role in protein biochemistry, and is making an increasing contribution to modern molecular biology and to the interpretation of DNA sequences. But things have changed since Frederick Sanger, a pioneer in protein chemistry, first determined the amino acid sequence of insulin more than 30 years ago. The advent in recent years of rapid nucleic acid sequencing techniques has taken much of the drudgery away from direct amino acid sequencing of large proteins, and has instead put more emphasis on the importance of determining at least limited amounts of sequence directly from the protein of interest. The development of amino acid sequencing has depended almost entirely upon the Edman method of chemical degradation that removes the N-terminal amino acid from a protein or peptide chain, leaving a new N-terminus on the chain—now one residue shorter. This procedure can be applied sequentially so that the amino acid sequence beginning from the N-terminus is found by identifying the residues removed at each successive cycle of Edman degradation (usually the phenylthiohydantoin or PTH derivatives of the amino acids). One major requirement for sequencing is that it should go as far as possible into a sequence before the noise overwhelms the signal. This penetration of a sequencing operation depends upon the efficiency with which the Edinan reaction is carried out (the repetitive yield), and the sensitivity of the method for detecting the N-terminal fragments. Automation of the sequencing reaction has made substantial improvements in repeti- tive yield over manual techniques. HPLC analysis of PTH derivatives gives quantitative results for yields, and this has greatly improved the “calling” of sequences even when the background noise is considerable. Immediate analysis of PTH derivatives by “on-line” HPLC has improved the overall sensitivity and the identification of labile residues: the use of computer techniques for chromatogram comparison has further enhanced the performance. The first automated amino acid sequencing machine was the “spinning cup” or “liquid-phase” machine. This machine essentially holds the sample in aqueous solution, helped in recent years by the detergent “polybrene,” and extracts reaction products with nonmiscible organic solvents. This was followed by the “solid-phase” machine, with the sample covalently linked to a solid support, which had the advantage of resisting extraction of hydrophobic peptides, but the disadvantage of requiring an initial covalent coupling that was not always satisfactory. The “gas-phase” machine, in which the sample is absorbed onto a glass-fiber disc and some reagents applied in the gas phase, is the major development of recent years, and has become the industry standard. (It is produced by Applied Biosystems, Foster City, Calif.) In labs concerned mainly with producing results rather than with development of technique, current standards of performance suggest that one should be able to penetrate 30,40, or possibly 50 residues or more into a sample containing 100 picomoles of free N-terminal residue, with a cycle time of less than an hour. Indeed, techniques for direct blotting of sample from polyacrylamide gel have improved greatly in recent months with the introduction of polyvinylidene diflouride (PVDF) membrane, and have opened up a whole new range of proteins to sequence analysis. Proteins with blocked N-termini can still yield useful sequence after the products of chemical or enzymatic digestion have been separated by polyacrylamide gel electrophoresis. The elegantly simple gas-phase method of Amons for reduction and pyridylethylation of disulphide bridges has useful application in this context. The upsurge in activity in amino acid sequencing in recent years has been met by a response from sequencer manufacturers. There are now several alternatives to the Applied Biosystems machine mentioned above, but none has yet been subjected to sustained routine sequencing in a normal laboratory, which is the most ufeful criterion for prospective buyers. Most of the machines produce results comparable to the Applied Biosystems machine, but one must be skeptical of exaggerated advertising claims of “low picomole sensitivity” with “sequences up to 100 residues.” The best efforts of many specialized laboratories have not produced a repetitive yield consistently better than 96%. Technical support available from the manufacturers in machine maintenance and in method development are also important points to be considered before purchasing an amino acid sequencer. Technical development has been expensive, so the price of automatic sequencers is quite high— about $250,000. The cost is now approaching that of the smaller mass spectrometers, which, with recently improved sensitivity, are beginning to become alternative instruments to be considered for amino acid sequencing. John Fothergill is professor of biochemistry at the University of Aberdeen, Scotland and director of the SERC amino acid sequencing facility there.

Amino Acid Sequencers Of Today: Sensitive, Efficient-And Very Costly

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