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The development and expansion of the core facility concept are

We report the purification and characterization of a novel neuropeptide from Aplysia nervous tissue. The peptide was termed cerebral peptide 2 (CP2) because it was the larger of two peptides predominantly synthesized in the cerebral ganglia and transported to other regions of the central nervous system. The purification of CP2 from extracts of cerebral ganglia using three sequential modes of high-pressure liquid chromatography (HPLC) was followed using the [35S]methionine-labeled peptide obtained from transport experiments. The primary structure of CP2 was determined by automated Edman degradation of native CP2 and its proteolytic fragments in conjunction with mass spectrometry. CP2 is a 41 amino acid peptide with an amidated carboxyl terminal. A peptide with the proposed sequence of CP2 was synthetized and compared by HPLC with the native peptide. Chromatographic properties of the synthetic and native peptide labeled in vivo were found to be identical. CP2 does not appear to be a member of any previously identified peptide family.

In 2007, The Association of Biomolecular Resource Facilities (ABRF) Survey Committee surveyed the ABRF membership and scientists at-large concerning the current state of funding in service-oriented laboratories. Questions pertained to services offered, cost recovery, capital equipment funding, and future outlook. The web-based survey, available for 3 weeks, achieved participation from 209 respondents in 13 countries, 77% of which represented academic laboratories. Most respondents (75%) directed their laboratories. Laboratories depend largely on institutional support and customer recharges to fund operations, but National Institutes of Health and National Science Foundation Shared Instrumentation Grant programs are considered critical to meeting future needs. Source allocations supporting capital equipment acquisitions, operations, and laboratory director salary are presented.

A survey of 124 protein and/or nucleic acid chemistry facilities has provided a basis for estimating the resources needed to establish a facility, the financial support needed to keep it operating, and the technical capabilities it might reasonably be expected to achieve. Based on these data, an average core facility occupied 870 ft2, was staffed by three full-time personnel, and was equipped with 4-5 major instrument systems. Because user fees generated an average of about $101,000/year in income compared with an average operating budget of about $197,000/year, even a facility that charged user fees would, on average, still require an annual subsidy of about $96,000. Although most government and industrial core facilities did not assess user fees, at least 83 of the 124 respondents did have a preestablished schedule of service charges that enabled a compilation to be made of the average cost of providing a number of typical facility analyses and syntheses. The greater than 100-fold range in charges asses...

Introduction History and Activities of the ABRF Amino Acid Analysis Protein Sequencing Chemistry and Sample Preparation ABRF Protein Sequence Studies Instrument Technological Improvement/Design over the Course of ABRF Studies Internal Protein Sequencing Peptide Synthesis Carbohydrate Analysis Nucleic Acids ABRF DNA Synthesis Studies ABRF DNA Sequencing Studies Mass Spectrometry Initial Results of the Survey Preliminary Results of the Analysis of ABRF MS1 and MS2 Test Conclusions Surveys of Core Laboratories Laboratory Quality and Compliance Effective Use of Biomolecular Resource Facilities Before Submission of a Request After Receipt of Your Data or Reagents Future of Core Laboratories Paradigm Shift in the Postgenome Era Automation Future Opportunities Acknowledgments Bibliography

A survey of 128 biotechnology core facilities has provided data on the finances, services, space requirements, and personnel. An average facility had four full-time personnel and 7.5 major instrument systems, and occupied 969 sq. ft. Average total income was $244,000/year, but annual user fee income was only $125,000. Typically, facilities required substantial institutional support or grants. Cost recovery (user fee income divided by total income) averaged 49%. During the last 5 years user fee income, total income, and cost recovery have increased. In-house charges for protein sequencing and peptide synthesis increased approximately 30%, while oligonucleotide synthesis charges decreased by 74%. The costs (charges corrected for subsidy from non-user fee income) for most services did not significantly change, except that oligonucleotide synthesis costs decreased by 25% in 1992. DNA synthesis had the highest throughout per month (116 samples), followed by amino acid analysis (86 samples) and DNA sequencing (67 samples). Other services averaged from 5 to 60 samples. DNA synthesis and purification were the services used by the greatest number of principal investigators. A number of services including DNA sequencing, mass spectrometry, capillary electrophoresis, RNA synthesis, electroblotting, and carbohydrate analysis have been introduced in the last 3 years. Although these services are characterized by high levels of methods development and non-user runs, they are offered by twice the percentage of facilities as in 1989, and are increasingly contributing to facility income.

Publisher Summary A continuing effort to inform investigators about the average capabilities of protein sequencing core laboratories has been mounted by the Association of Biomolecular Resource Facilities (ABRF) sequencing committee for the past several years. Samples with both specific design goals and well-characterized components were distributed to ABRF-member core facilities worldwide. The analysis of these data for STD-1, ABRF-89SEQ, and ABRF-90SEQ hasd provided valuable information to users regarding the performance of the average facility. In continuation of this effort, a test sample designated ABRF-91SEQ was distributed to 198 ABRF-member protein sequencing core facilities. This chapter describes the results from the analysis of the first 90 sets of data. The results of ABRF-91SEQ are consistent with and further support the conclusions reached previously. These results are based on a one time analysis of a sample with little background information provided. There were 19 responses with 100% accuracy and 26 responses with 90–99.9% accuracy.

Publisher Summary This chapter discusses a simple, high sensitivity method to obtain N-terminal and internal protein sequence from the same sample. The cyanogen bromide (CNBr)/ ortho-phthalaldehyde (OPA) method to reuse PVDF samples involves three steps: 1) determination of the N-terminal sequence (if not blocked), 2) location of proline residues within CNBr fragments, and 3) isolation of unique sequence by delivering OPA when a proline becomes N-terminal during sequencing. CNBr in combination with OPA is a high sensitivity method to obtain N-terminal and limited internal sequence data. Methods that purify fragments for internal sequencing would have eliminated the N-terminal sequence determination necessary to verify a full-length clone and reduced the quantity available for sequencing even more. Reusing PVDF electroblotted protein samples allow obtaining N-terminal (when not blocked) and internal protein sequence data, consuming only protein that has been dedicated to N-terminal sequence determination.

Based on analyses and surveys completed by as many as 112 protein chemistry laboratories, an average of nearly 100 picomoles of protein is needed to sequence the first 15 or more residues while a total of about 4 μg protein is sufficient to carry out a triplicate hydrolysis/analysis that will give an amino acid composition with about 86% compositional accuracy. On average, approximately 700 picomoles protein is required in order to isolate and sequence a 15 residue tryptic peptide.

A procollagen C-proteinase was purified about 100-fold from the medium of cultured mouse fibroblasts by a combination of ammonium sulfate precipitation, gel-filtration, and affinity chromatography on a column of Sepharose coupled to the carboxyl propeptide of type I procollagen. The purified enzyme did not exhibit other proteolytic activities, and it cleaved type I, II and III procollagens to produce the corresponding pN alpha chains and carboxyl propeptides as the only products. Amino acid sequencing of the first 14-18 residues at the N-terminus of the carboxyl propeptides generated by the enzyme from human pro alpha 1(I), pro alpha 2(I) and pro alpha 1(III) chains showed that the cleavage occurred at the physiological site, i.e. at the specific Ala-Asp bond in the pro alpha 1(I) and pro alpha 2(I) chains, and at the specific Gly-Asp bond in the pro alpha 1(III) chain. The pH optimum of the enzyme is 8.5 and its molecular weight as estimated by gel-filtration is about 125,000 daltons. The enzyme is inhibited by metal-chelators, various amines, dithiothreitol, N-ethylmaleimide and serum, but it is insensitive to pepstatin, leupeptin and serine proteases inhibitors. The enzyme differs from the C-proteinase described by Njieha et al. (Biochemistry 21:757-764, 1982), and the catheptic activities reported by Davidson et al. (Eur. J. Biochem 100:551-558, 1979) and Helseth and Veis (Proc. Natl. Acad. Sci. USA 81:3302-3306, 1984). The specificity of the enzyme is offered as evidence for a unique, C-proteinase, and its recovery from culture medium supports an extracellular location for procollagen processing.

A survey of 40 protein and nucleic acid chemistry facilities has provided data about the capabilities of core facilities and the cost of the services they provide. Approximately 43% of the +158,000 average annual operating budget for a typical university facility is derived from service charges. After correcting for the various degrees of subsidization of the different facilities, it was found that it costs a typical university facility +65 to carry out an acid hydrolysis and amino acid analysis on a protein. A 25-residue peptide can be synthesized and cleaved for +2078, whereas sequencing the same peptide costs +874. A 25-residue oligonucleotide can be synthesized for +258. The total work output per month of an average facility corresponds to 65 amino acid analyses, 15 amino acid sequencing runs, three peptide syntheses, and 16 oligonucleotide syntheses. Depending on the approach used, from 85 to nearly 200 pmol of protein are required to obtain an accurate amino acid composition. To sequence the first 1...

More than 100 attendees from 13 different countries participated in discussions of problems found in the operation of core laboratories. The meeting was opened by Ron Niece who described how this meeting grew out of the realization at the Symposium of American Protein Chemists in San Diego in 1985 that many protein sequencing service facilities share common problems. A survey of interested facility directors identified topics for discussion at this meeting.

The free amino acid concentrations in cotyledons and axes of soybean ( Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment. Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments. Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.

After hypertransfusion, normal ww and anemic WW(T) mice show the same increase in red-cell volume, decrease in reticulocytes, and temporary cessation of blood formation. The time required for the red cell volume to return to the value observed before treatment is the same for both groucps of inice. The cell volume at which new erythrocytes are again released into the circulation depends upon the genotype of functioning blood-forming tissue. Injections of erythropoietin stimulate red cell formation in polycythemic ww mice, but have much less (if any) effect upon polycythemic WW(T) mice.