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ESRG 2005: Edman Chemistry Sequence of a Synthetic Peptide Containing Seven Modified Amino Acids—Survey on the Current State of Edman Sequencing
The Edman Sequencing Reserch Group (ESRG) 2005 study will have two components, one an Edman chemistry laboratory challenge and the other a survey to obtain information about how Edman chemistry is being used by the ABRF community. Questions for the survey are being defined by the ESRG group and will be posted using a web-based service (www.surveymonkey.com). The laboratory challenge will build on the ESRG 2004 study to identify seven additional modified amino acids that are routinely found in proteins or that might be generated during sample preparation. The modified amino acids will be incorporated into a synthetic peptide interspersed between normal amino acids, using a design similar to last year’s challenge peptide, so that we can compare results. Some of the normal amino acids will be repeated in the sequence to allow calculation of initial yield (IY) and repetitive yield (RY). A member of the committee will synthesize, purify, and characterize the peptide. Members of the ESRG group will sequence the peptide in their own laboratories before sending it out to participating labs. The main goal is to obtain information from participating laboratories as to how the modified amino acids behave during Edman degradation chemistry and to obtain their high-performance liquid chromatography (HPLC) elution patterns. This information will be collated in our final report. We will also report on the instruments and protocols used by the participating laboratories. Participant laboratories will be asked to fill out a detailed survey on the sequencer and HPLC conditions used for the run, so a consensus may be reached on peak patterns and relative retention times for each of the modified amino acids under a given set of conditions. There will be several components to the challenge: (1) identification of the modified amino acids; (2) the reproducibility of results from laboratories using the same instrumentation; and (3) the ability of laboratories to determine premature termination of sequence. The ESRG will compile the results from multiple runs using similar conditions and instrumentation to help define the patterns that are likely to be seen when these modified amino acids are found in proteins. The ESRG expects to have enough of the peptide to distribute aliquots that will allow for numerous runs and which can be used as a reference for future use. In addition we will post the retention times for the modified PTH amino acids on the ESRG web page along with the results from last year’s study. We expect this study to increase the confidence level of participating laboratories to identify modified amino acids in the future and to begin to build an on-line resource for the evaluation of these amino acids by Edman chemistry.
Is the ABRF doing the best possible job in serving the interests of its members? To help address this question, the ABRF Membership Committee polled those who did not renew their memberships for the year 2003 or 2004. These nonrenewing individuals were contacted by phone or by e-mail and asked to complete a very simple survey. The goal of the survey was to learn if members are leaving ABRF because their needs and interests are indeed not being addressed. Additionally, we hoped to give these ex-members a forum to express concerns or make suggestions to improve the ABRF.
Initially, the committee members attempted to contact by phone those who had not renewed in the year 2003. We found that it was very difficult to actually connect with the desired persons by phone, but did learn that 3 ex-members had retired and that 20 were no longer employed in their previous positions. We changed tactics and e-mailed the persons and asked them to complete our attached survey. At that time we included those who had not renewed in 2004; a total of 361 persons were asked to complete the survey.
Thirty-eight responses were received. Not all questions were answered by all respondents. The length of time that the respondents had been members of the ABRF varied from 1 to 15 years, and 14 answered that they had been members for 5 or more years. Fifty-two percent stated that they were employed by a not-for-profit organization, the remainder by industry. When asked why they joined the ABRF, the strongest responses were for networking and technical help. The newsletter and test samples were only about half as much incentive. Thirty out of 31 believed that membership was productive and 84% thought that the ABRF was a community that was professional, supportive, and open. When asked to rate the various aspects of the ABRF, the most enthusiasm was expressed for the ABRF list and the annual meeting, with about half of the respondents rating them as “very useful.” The research groups, web site, and opportunities for networking were rated as “useful” by most, and JBT received a generally “useful” to “OK” rating.
When asked if they had not renewed intentionally, 73% responded that they had. Of the 31 who responded, 12 stated that their research interest had changed and that the discussions of the bulletin board were no longer relevant to their work. Two persons told us that they had retired and one was on maternity leave. Six said that they had just forgotten and intended to renew. Five stated that they could not afford the membership fee.
Seventy-four percent of the ex-members responding said that they had attended an annual meeting. The vast majority rated the relevance of the sessions at the meetings as “sometimes” or “not often” relevant to their current work. All but 2 expressed satisfaction with the electronic bulletin board; the majority stated that its discussions were “sometimes” relevant to their work.
Many of the respondents provided us with enlightening general comments. Several ex-members employed by vendors expressed their frustration with the ABRF and the meetings. Opinions were expressed that the meetings seemed controlled by the large companies, that booths set up at the meetings did not generate sufficient business and that some vendors are allowed to freely endorse their products on the bulletin board or publish in the JBT, while others are not. A couple of persons encouraged the ABRF to give more focus to industry and provide an outlet for advertising. Another area of frustration seemed to come from those who live outside of the United States. Comments were made that the ABRF is an American organization that reflects American needs, that meetings are difficult to attend because of prohibitive travel expenses and that even transfer of payment to the ABRF can be difficult. Some felt the ABRF did not have a sufficiently tight focus or was not at the forefront of technical strategies, while others believed it was too focused or too high tech. Two respondents stressed a lack of DNA emphasis. In general, most felt that the ABRF was an important organization that they would recommend to their colleagues and that it still provided the practical details needed to run small laboratories.
A data summary is posted on the ABRF web site: http://www.abrf.org/other/survey_of_past_members.htm
The Nucleic Acid Research Group (Greg Shipley as new Chair) initiated a survey and research project in 2003 under the capable guidance of our past Chair, Scottie Adams. The survey was designed to determine the status of PCR technology in laboratories around the world and consisted of 58 questions. We received responses from over 100 individuals. The results should be of interest to anyone performing real-time qPCR. The survey results, raw data, and an excellent summary poster made by Brian Holloway and Tony Yeung can be seen at http://www.abrf.org under Research Groups/Nucleic Acid. We also presented data from last year’s research project, which focused on the many different parameters that effect the efficiency of a Taqman probe-based qPCR assay. We had over 20 laboratories send in designs for more than 32 assays for the mouse IFNγ− gene. A number of rules used to design probe-based assays were tested. Some were validated and some were found not to be as important as originally thought. Tony Yeung devised a formula for comparing multiple assays and determining the best one that goes beyond just looking at the slope of the assay. Our poster containing the results of this study can be seen at the above web site. Deborah Grove went from Portland, OR, to the First International Meeting on PCR held in Munich, Germany, where she presented both NARG posters. She also made a presentation entitled “Real-Time PCR in Core Facilities.”
We are now working on this year’s research project. We plan to send reagents for a probe-based human β-actin qPCR assay along with matched synthetic RNA and oligo DNA templates. Each participating lab will run the assay utilizing their chemistry preference and real-time platform. Data will be collected by the NARG and analyzed. Both templates should result in identical 6-log standard curves. The data will give each lab a chance to compare their RT-PCR verses PCR-alone technique on this control assay. We will obtain data from many chemistries, probe-based and/or SYBR green I, as well as some idea of how the many different real-time platforms compare in performance. This information should be very useful to both the participants and the real-time PCR community as a whole.
The Peptide Standards Project is being conducted by the Peptide Standards Project Committee (PSPC) in collaboration with the National Institute of Standards and Technology (NIST). The goal of this project is to produce and characterize three synthetic peptides to serve as registered and certified peptide reference standards. The Peptide Standards Project was originally initiated by interactions between the Quality Compliance Committee (QCC) and Peptide Synthesis Research Group (PSRG). The two committees designed, synthesized, analyzed, and characterized three synthetic peptides and found them to be useful as peptide reference standards. The details and results of this study are published in the Journal of Biomolecular Techniques [11;102–105:2000)].
The first step in the Peptide Standards Project was the large-scale synthesis of the three peptides in high purity, followed by packaging in 1-mg quantities for storage at NIST. Next, the peptides will be tested for stability and shelf life by the PSPC and analyzed for physical and chemical properties by the ABRF membership in order to generate a Certificate of Analysis. Analysis by the ABRF membership will provide statistical validation for certification.
The peptide sequences are:
Peptide A: DAEPDILELATGYR
Peptide B: KAQYARSVLLEKDAEPDILELATGYR
Peptide C: RQAKVLLYSGR
Peptides A, B, C have been successfully synthesized in an academic core facility at either the 7.5- or 10-millimole scale. Purity of 93% or higher was achieved using either ion-exchange chromatography followed by reversed-phase HPLC or two-step reversed-phase HPLC. Orthogonal analytical techniques were used to confirm the identity and purity of the peptides. Purification yielded 1–2 g and the peptides were packaged at 1-mg per vial. Sets containing one vial of each peptide have been prepared and have been designated “NIST Standard Reference Material SRM 2397.” Because of a shortage of peptide B, 983 peptide sets were obtained. The sets are stored at NIST with 80 peptide sets to be distributed for the collaborative analysis in order to generate the Certificate of Analysis.
The peptides will be analyzed by both the PSPC and the ABRF membership. The ABRF membership will be asked to perform their analyses between July and September 2004. All ABRF member laboratories are invited to participate; however, as only 80 sets will be available for distribution, the number of laboratories could potentially be limited. Eighty peptide sets will be distributed to a maximum of 40 laboratories, as NIST requires that analyses be performed on two peptide sets per laboratory. The minimum requirements by NIST are that at least four different laboratories provide data from at least four replicate measurements for each analysis technique used. Participating laboratories will be asked to use their established protocols for analysis and will be asked to provide information on the types of analyses they can perform. The number of replicate measurements will be specified for each analysis technique. Required analysis techniques for certification are reversed-phase HPLC, electrospray MS, MALDI-MS, quantitative amino acid analysis, and UV-spectroscopy. Additional techniques that could be performed are N-terminal sequence analysis, enzymatic hydrolysis, capillary zone electrophoresis, ion exchange HPLC, LC-MS, and MS/MS.
The PSPC is composed of the following members: Henriette Remmer (chair, The University of Michigan); Nick Ambulos (ad hoc, University of Maryland); Lynda Bonewald (EB liaison, University of Missouri); John Dougherty, Jr. (Eli Lilly and Company); Ed Eisenstein (ex officio, National Institute of Standards and Technology); Elisabeth Fowler (Millennium Pharmaceuticals, Inc), Jinny Johnson (Texas A&M University); Ashok Khatri (Massachusetts General Hospital); Nadine Ritter (Biologics Consulting Group); and Susan Weintraub (University of Texas Health Science Center at San Antonio).