294
biotopics N-termini are, on average, one rum deeper into the membrane than the C-termi:fi. This may be due to the interaction of the helix dipoles with the charged lipid headgroups. KINEMAGE is not intended to compete with the sophisticated graphics packages now available, but instead to provide an easy-touse tool for the improved communication of structural information. The ~St fruits are awaited with considerable interest. 'It's a big one, the carboxyl terminus is somewhere over there.' enzyme. Another example showed the NMR NOE (nuclear Overhanser ett'ec0 distance measurements on a peptide model of a folding intermediate of pancreatic trypsin inhibitor. The programme and the accruing data promise not only to get away from the tiring stereo pair images of journals, but
References
also to represent a significant educational tool. The reader may even be able to contribute to the discovery process by spotting some feature unnoticed by others. An example of this is the Richardsons' finding that the termini of transmembrane helices are not lined up, but are offset with respect to each other. The
t Geisow, M. J. (1990) Trends Biotechnol. 8, 312-313 2 Richardson, J. S. and Richardson, D. C. (1989) Trends Biochem. Sd. 14, 304-309 $ Kussu, I. M. (1991) Trends Bioteehnol. 9, 96--104
Michael J. Geisow Biodigm, 81 Kneeton Road, East Bridgford, Nottingham NG13 8PH, UK.
Peptide synthesis- theme and variations Peptides are used extensively by the bioscience community for their intrinsic biological activity (e.g. hormones and neurotransmitters), for the production of peptide antisera and for analysis of structure-function relationships. The latter is exemplified by the use of synthetic peptides containing natural or unnatural amino acids as antagonists of biological response. More recently, the role of synthetic peptides for investigating a-helix formation t and MHC antigen function2 has been prominent. As a result, peptide synthesis has become a large and economically significant industry. Remarkable progress has been made in the degree to which peptide synthesis has become accessible to the community at large since the initial breakthrough in 1963 when Ik. B. Merrifield showed how long-chain peptide synthesis could be automated. Since that time, the Merrifield concept has been much refined with changes in the solidphase supports (hydrophobic to hydrophilic) and in the nature of the amino acid amino-function protecting groups (tBOC to FMOC) (see Glossary). What about the quality of TIBTECHSEPTEMBER1991 (VOL9)
the peptides that underpin so much of our fundamental research? At a recent ABR.F meeting*, the response appears to be: 'much room for improvement'. The ABRF exists to promote communication, evaluation and improvement in expertise of the core facilities which increasingly carry out protein and nucleic acid synthesis and characterization for their corporate or academic colleagues. ABRF numbers 185 core facilities in 12 countries amongst its membership. The evaluation function is an important aspect of ABKF. Each year a series of test samples for amino acid composition analysis and sequence determination are sent to ABR.F members and the results compiled. More recently, however, ABR.F has requested synthesis of a specified test peptide by its members for evaluation in a few 'well found' *The Association of Biomolecular Resource Facilities (ABRF) held an open meeting, on 22 June 1991 at the Baltimore Conference Centre, as a satellite to the Protein Society meeting. ABRF Secretary: Dr E. Fowler, Ciba-Geigy Biotechnology, PO Box 12257, Research Triangle Park, hlC 27709, USA.
centres. The recent report concemed a test peptide made in 38 different core facilities. The crude isolates as well as the purified synthetic peptides were analysed. The results may be taken as an indicator of the 'state of the art' in core facility peptide synthesis, according m the degree to which you comider the 38 facilities to be representative of the several hundred that exist. The resuits were rather revealing. A variety o f methods Contributing facilities had used tBOC and FMOC chemistries about equally. The peptides were analysed by amino acid analysis, reversed phase liquid chromatography (HPLC), capillary electrophoresis (CE), two mass spectrometries (electrospray and plasma desorption) and, finally, by bioassay, since the test peptide had been designed to act as a substrate for different protein kinases. (For readers still unfamiliar with the scope of capillary electrophoresis and electrospray mass spectrometry these were both recently reviewed in TIBTECH3,4.) Detailed analysis and statistics on the contributed peptides are available at cost from ABRF (who would be glad to wel-
© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00
295
biotopics Glossary
come more members- both associate and core facility). A general picture o ~ y can be given here. Caveat
The best peptides generally came from facilities employing the FMOC chemistry, particularly where morereactive coupling reagents had been used. The amino acid composition analyses were very similar on all samples but, and here is the note of caution, this was a very poor indicator of sample homogeneity. Also disturbing was the observation that a number of heterogeneous samples appeared homogeneous either on CE or HPLC or even by both methods. Despite the general media 'hype', CE was no more advantageous than HPLC in revealing problems. Mass spectrometry turned out to be the single most important technique for evaluating synthetic peptide quality. No small contributing factor was that not only could heterogeneities be detected, but the nature of the impurities could usually be inferred from the known masses of side chain protecting groups. The synthetic peptides received had amino acid deletions, oxidized residues and a range of incom-
tBOC - tertiary butyloxycarbonyl group [(CH3)3C-O-CO-] used as blocking agent for the
amino group of amino acids in solid phase protein synthesis. FIMOC - 9-fluorenyl methyl oxycarbonyl group used as a blocking agent for the amino
function. It is stable to acid but removeable in mild base. pletely removed protecting groups and synthesis reagent adducts. Electrospray gave a more representative picture than plasma desorption, which tended to over-emphasize some of the contaminating products, possibly because of the hydrophobic nature of blocking groups retained in these. HPLC linked to dectrospray was the most discriminating form of analysis. It was then revealed that only around 10% of core facilities have routine access to mass spectrometry - a surprising and, in view of the results, worrying statistic. Why worrying? Well, the final assessment- bioassay of the contributed peptides showed that the contributed peptides (which had passed the amino acid composition test) varied very widely in quality. It is upon the resuits of a bioassay, that conclusions will be drawn and the scientific paper based. In summary, although current methods for peptide synthesis are
very good and there were excellent products (>95% homogeneous) among the ABRF peptides, the survey points out weaknesses in custom synthesis. It also demonstrates that core facilities are, at present, not as well equipped as they could be to recognize problems when they do occur. The importance is that many conclusions will be drawn and published on the assumption that the custom peptide is precisely what it should be. References 1 Chakrabartty, A., Schellman, J. A. and Baldwin, R. L. (1991) Nature351,586-588 2 Elliot, T., Cerundolo, V., Elvin, J. and Townsend, A. (1991) Nature351,402-406 3 Frenz,J. and Hancock, W. S. (1991) Trends Biotechnol. 9, 243-250 4 Geisow, M.J. (1990) Trends Biotechnol. 8, 301-303
Michael J. Geisow Biodigm, 81 Kneeton Road, East Bridgford, NottinghamNG13 8PH, UK.
Targeting peptides and proteins: NATO ASI Proteins and polypeptides, because of their size, complex physical properties and diverse functions and sites of action, present unique challenges in their use as therapeutic agents. A recent NATO Advanced Studies Institute (ASI) meeting* was concerned not only with the problems of targeting in vivo natural, synthetic and engineered proteins and peptides, but also with the crafting ofpolypeptides as carriers of drugs or toxins. Molecules such as single chain ribosome inactivating protein, or peptides of the extracellular V1 domain of the CD4 *A NATO AS! on 'Targeting of Drugs: the Challenge of Peptides and Proteins', directed by G. Gregoriadis, A. T. Florence and G. Poste was held at Cape Sounion Beach, Greece fiom 24June to 5 July, 1991.
cell-surface receptor of HIV, represent some of the highly specific agents that were discussed. While these may act with high degrees of precision in vitro, the problem of administration and subsequent targeting of the proteins and peptide constructs to their sites of action in the whole organism provided the main thrust of the meeting. Language and other vehicles for deliver}" Participants were drawn from two main camps, the molecular manipuhtors and immunologists forming one rehtively homogeneous grouping, and drug targeters the other. Placed in close contact for 12 days, in lectures and the vital tutorial sessions, there was considerable intellectual cross-fertilization, but
© 1991, Elsevier Science PublishersLtd (UK) 0167 - 9430/91/$2.00
there were two almost distinct (sometimes coded) languages to be learned (as often happens in science), which hinders the facility of transitions between disciplines. It is essential that they do fuse and work together, as George Poste (SmithKline-Beecham, Philadelphia USA) pointed out forcibly, since progress in our ability to generate complex molecules has, he maintained, outstripped our capacity to develop suitable delivery systems to optimize their applicability. The greatest challenge is the development of systems for the delivery of autocrine/ paracrine molecules which act at extravascular sites, and to deliver a wider range of endo~nous mob ecules (such as gonadotropin) and their analogues in a manner which reflects the natural periodicity of delivery. Intravenous (IV) administration of autocrine/paracrine
meeting report
TIBTECHSEPTEMBER1991 (VOL9)
biotopics N-termini are, on average, one rum deeper into the membrane than the C-termi:fi. This may be due to the interaction of the helix dipoles with the charged lipid headgroups. KINEMAGE is not intended to compete with the sophisticated graphics packages now available, but instead to provide an easy-touse tool for the improved communication of structural information. The ~St fruits are awaited with considerable interest. 'It's a big one, the carboxyl terminus is somewhere over there.' enzyme. Another example showed the NMR NOE (nuclear Overhanser ett'ec0 distance measurements on a peptide model of a folding intermediate of pancreatic trypsin inhibitor. The programme and the accruing data promise not only to get away from the tiring stereo pair images of journals, but
References
also to represent a significant educational tool. The reader may even be able to contribute to the discovery process by spotting some feature unnoticed by others. An example of this is the Richardsons' finding that the termini of transmembrane helices are not lined up, but are offset with respect to each other. The
t Geisow, M. J. (1990) Trends Biotechnol. 8, 312-313 2 Richardson, J. S. and Richardson, D. C. (1989) Trends Biochem. Sd. 14, 304-309 $ Kussu, I. M. (1991) Trends Bioteehnol. 9, 96--104
Michael J. Geisow Biodigm, 81 Kneeton Road, East Bridgford, Nottingham NG13 8PH, UK.
Peptide synthesis- theme and variations Peptides are used extensively by the bioscience community for their intrinsic biological activity (e.g. hormones and neurotransmitters), for the production of peptide antisera and for analysis of structure-function relationships. The latter is exemplified by the use of synthetic peptides containing natural or unnatural amino acids as antagonists of biological response. More recently, the role of synthetic peptides for investigating a-helix formation t and MHC antigen function2 has been prominent. As a result, peptide synthesis has become a large and economically significant industry. Remarkable progress has been made in the degree to which peptide synthesis has become accessible to the community at large since the initial breakthrough in 1963 when Ik. B. Merrifield showed how long-chain peptide synthesis could be automated. Since that time, the Merrifield concept has been much refined with changes in the solidphase supports (hydrophobic to hydrophilic) and in the nature of the amino acid amino-function protecting groups (tBOC to FMOC) (see Glossary). What about the quality of TIBTECHSEPTEMBER1991 (VOL9)
the peptides that underpin so much of our fundamental research? At a recent ABR.F meeting*, the response appears to be: 'much room for improvement'. The ABRF exists to promote communication, evaluation and improvement in expertise of the core facilities which increasingly carry out protein and nucleic acid synthesis and characterization for their corporate or academic colleagues. ABRF numbers 185 core facilities in 12 countries amongst its membership. The evaluation function is an important aspect of ABKF. Each year a series of test samples for amino acid composition analysis and sequence determination are sent to ABR.F members and the results compiled. More recently, however, ABR.F has requested synthesis of a specified test peptide by its members for evaluation in a few 'well found' *The Association of Biomolecular Resource Facilities (ABRF) held an open meeting, on 22 June 1991 at the Baltimore Conference Centre, as a satellite to the Protein Society meeting. ABRF Secretary: Dr E. Fowler, Ciba-Geigy Biotechnology, PO Box 12257, Research Triangle Park, hlC 27709, USA.
centres. The recent report concemed a test peptide made in 38 different core facilities. The crude isolates as well as the purified synthetic peptides were analysed. The results may be taken as an indicator of the 'state of the art' in core facility peptide synthesis, according m the degree to which you comider the 38 facilities to be representative of the several hundred that exist. The resuits were rather revealing. A variety o f methods Contributing facilities had used tBOC and FMOC chemistries about equally. The peptides were analysed by amino acid analysis, reversed phase liquid chromatography (HPLC), capillary electrophoresis (CE), two mass spectrometries (electrospray and plasma desorption) and, finally, by bioassay, since the test peptide had been designed to act as a substrate for different protein kinases. (For readers still unfamiliar with the scope of capillary electrophoresis and electrospray mass spectrometry these were both recently reviewed in TIBTECH3,4.) Detailed analysis and statistics on the contributed peptides are available at cost from ABRF (who would be glad to wel-
© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91/$2.00
295
biotopics Glossary
come more members- both associate and core facility). A general picture o ~ y can be given here. Caveat
The best peptides generally came from facilities employing the FMOC chemistry, particularly where morereactive coupling reagents had been used. The amino acid composition analyses were very similar on all samples but, and here is the note of caution, this was a very poor indicator of sample homogeneity. Also disturbing was the observation that a number of heterogeneous samples appeared homogeneous either on CE or HPLC or even by both methods. Despite the general media 'hype', CE was no more advantageous than HPLC in revealing problems. Mass spectrometry turned out to be the single most important technique for evaluating synthetic peptide quality. No small contributing factor was that not only could heterogeneities be detected, but the nature of the impurities could usually be inferred from the known masses of side chain protecting groups. The synthetic peptides received had amino acid deletions, oxidized residues and a range of incom-
tBOC - tertiary butyloxycarbonyl group [(CH3)3C-O-CO-] used as blocking agent for the
amino group of amino acids in solid phase protein synthesis. FIMOC - 9-fluorenyl methyl oxycarbonyl group used as a blocking agent for the amino
function. It is stable to acid but removeable in mild base. pletely removed protecting groups and synthesis reagent adducts. Electrospray gave a more representative picture than plasma desorption, which tended to over-emphasize some of the contaminating products, possibly because of the hydrophobic nature of blocking groups retained in these. HPLC linked to dectrospray was the most discriminating form of analysis. It was then revealed that only around 10% of core facilities have routine access to mass spectrometry - a surprising and, in view of the results, worrying statistic. Why worrying? Well, the final assessment- bioassay of the contributed peptides showed that the contributed peptides (which had passed the amino acid composition test) varied very widely in quality. It is upon the resuits of a bioassay, that conclusions will be drawn and the scientific paper based. In summary, although current methods for peptide synthesis are
very good and there were excellent products (>95% homogeneous) among the ABRF peptides, the survey points out weaknesses in custom synthesis. It also demonstrates that core facilities are, at present, not as well equipped as they could be to recognize problems when they do occur. The importance is that many conclusions will be drawn and published on the assumption that the custom peptide is precisely what it should be. References 1 Chakrabartty, A., Schellman, J. A. and Baldwin, R. L. (1991) Nature351,586-588 2 Elliot, T., Cerundolo, V., Elvin, J. and Townsend, A. (1991) Nature351,402-406 3 Frenz,J. and Hancock, W. S. (1991) Trends Biotechnol. 9, 243-250 4 Geisow, M.J. (1990) Trends Biotechnol. 8, 301-303
Michael J. Geisow Biodigm, 81 Kneeton Road, East Bridgford, NottinghamNG13 8PH, UK.
Targeting peptides and proteins: NATO ASI Proteins and polypeptides, because of their size, complex physical properties and diverse functions and sites of action, present unique challenges in their use as therapeutic agents. A recent NATO Advanced Studies Institute (ASI) meeting* was concerned not only with the problems of targeting in vivo natural, synthetic and engineered proteins and peptides, but also with the crafting ofpolypeptides as carriers of drugs or toxins. Molecules such as single chain ribosome inactivating protein, or peptides of the extracellular V1 domain of the CD4 *A NATO AS! on 'Targeting of Drugs: the Challenge of Peptides and Proteins', directed by G. Gregoriadis, A. T. Florence and G. Poste was held at Cape Sounion Beach, Greece fiom 24June to 5 July, 1991.
cell-surface receptor of HIV, represent some of the highly specific agents that were discussed. While these may act with high degrees of precision in vitro, the problem of administration and subsequent targeting of the proteins and peptide constructs to their sites of action in the whole organism provided the main thrust of the meeting. Language and other vehicles for deliver}" Participants were drawn from two main camps, the molecular manipuhtors and immunologists forming one rehtively homogeneous grouping, and drug targeters the other. Placed in close contact for 12 days, in lectures and the vital tutorial sessions, there was considerable intellectual cross-fertilization, but
© 1991, Elsevier Science PublishersLtd (UK) 0167 - 9430/91/$2.00
there were two almost distinct (sometimes coded) languages to be learned (as often happens in science), which hinders the facility of transitions between disciplines. It is essential that they do fuse and work together, as George Poste (SmithKline-Beecham, Philadelphia USA) pointed out forcibly, since progress in our ability to generate complex molecules has, he maintained, outstripped our capacity to develop suitable delivery systems to optimize their applicability. The greatest challenge is the development of systems for the delivery of autocrine/ paracrine molecules which act at extravascular sites, and to deliver a wider range of endo~nous mob ecules (such as gonadotropin) and their analogues in a manner which reflects the natural periodicity of delivery. Intravenous (IV) administration of autocrine/paracrine
meeting report
TIBTECHSEPTEMBER1991 (VOL9)
