This Committee is planning a study to follow up on last year's study where participants tried to identify a protein directly from its composition. In last year's study, 90% of the participants were able to identify the test sample, rabbit triose phosphate isomerase, a soluble globular protein. This year we will again ask participants to identify a test sample, but this time the protein will be gel-purified and electroblotted to a PVDF membrane. We expect this study will be more difficult than last year's due to the higher errors that are inherent in amino acid analysis of blotted proteins.
This study will have a two-pronged goal: first, to determine whether participants have improved their overall error rate for amino acid analysis of blotted proteins compared to the ABRF-95AAA study; and second, to ascertain the degree of success resource laboratories experience when attempting to identify proteins from PVDF membranes. We will optimize the search parameters used by the protein identification software for identifying proteins analyzed from PVDF blots.
We plan to distribute four samples to each laboratory, all blotted to PVDF membranes: a protein whose identity will be disclosed that can be used as a calibrant for the search programs, an test protein at two different concentrations, and a piece cut from a blank region of the electroblotted membrane that will serve as a negative control. We will also distribute a picture of an SDS-PAGE gel, so participants can estimate each sample's molecular weight. As in previous studies, we will suggest procedures for hydrolysis and extraction.
Because it will require considerable work for us to prepare for this project, we will only send samples to those laboratories who have responded that they would like to participate in the study. If you have not received a request card but would like to participate, contact Alan Mahrenholz (E-mail: mahrenholz@biochem.purdue.edu). Responses are due September 15.
The Internal Protein Sequencing Research Committee invites you to participate in its second collaborative study involving enzymatic digestion of an SDS-PAGE separated protein and HPLC separation of the resulting peptides. The primary goals of this study are:
·to provide our members with a mechanism to anonymously evaluate their ability to generate and isolate peptides for further characterization from an SDS-PAGE separated protein,
·to provide an introduction to these technologies for those laboratories that do not currently offer internal sequence analysis,
·to obtain data that would allow comparison of internal sequencing strategies and aid in optimization of these techniques, and
·to compile data on a common "unknown" that might help establish realistic expectations for internal sequence analysis.
Representative procedures and references will be included with the samples for laboratories that do not have an established protocol but are interested in trying these procedures.
The protein sample for this study (about 50 pmol) will be subjected to SDS-PAGE by the Committee and then distributed as either a Coomassie Blue-stained gel slice or, after electroblotting, as a small section of Amido Black -stained PVDF membrane. Participants will be asked to digest the provided protein and control (gel or PVDF) sample with trypsin and to then subject the resulting digests to reversed- phase HPLC. An external standard peptide will also be supplied. The materials for this year's study will include an important control not included in last year's study: a related protein already digested with trypsin by the Committee.
The Executive Board has approved the formation of a Mass Spectrometry Research Committee. The Committee members are Kristine Swiderek (Beckman Research Institute, City of Hope), Lowell Ericsson (Univ. of Washington), Farzin Gharahdaghi (Rockefeller Univ.), Murray Hackett (Univ. of Washington), and Gary Hathaway (California Institute of Technology). A survey of laboratories and their mass spectrometry equipment will be conducted in September, and a collaborative study involving analysis of one sample is tentatively scheduled for October. The Committee plans to present these survey and study results at ABRF '97.
Thus, analyzing all the components in this year's study will give four HPLC chromatograms: sample, control, pre -digested sample, and an external standard peptide.
For those laboratories that wish to proceed further, a specific tryptic peptide will be designated by the Committee for isolation and further analysis by mass spectrometry and peptide sequencing. Because the study participants' work and the Committee's preparation of the study materials represent substantial effort, a letter was distributed to all facility directors to identify those members who wished to participate. We hope to distribute the study materials in late September. We request that participants send their results to the Committee by Wednesday, November 27, 1996 to allow sufficient time to tabulate and present the results at the ABRF '97. The Committee would like to encourage as much participation as possible. As with past ABRF studies, the resulting data will be returned via a third party to protect the anonymity of the respondents. Summaries of the results of this study will be provided to all participants. We thank you for your support of the ABRF, and we look forward to your participation in this study. Additional inquiries may be directed to William S. Lane (Fax: (617) 495-1374, E-mail: wlane@harvard.edu).
This Committee recently completed a study of the quality of more than 200 25-base and 50-base oligonucleotides synthesized in ABRF member laboratories. The vast majority of syntheses, both 25-mers and 50-mers, exceeded the "unofficial" industry standard of 98% coupling efficiency for instruments and reagents. When the 25-mer oligo -nucleotides were tested as primers for automated DNA sequencing, we found that unpurified oligonucleotides from syntheses exceeding 98.5% coupling efficiency (at least 70% pure) provided sequencing results nearly indistin -guishable from those obtained with purified primers. Together, these observations suggest that raising the industry standard for coupling efficiency from 98% to 98.5% would allow use of unpurified synthetic oligonucleotides as sequencing primers with a high degree of confidence. A manuscript based on this study has been accepted for publication in BioTechniques.
The Committee has recently initiated a new study designed to evaluate primer design practices. Although "rules of thumb" for DNA sequencing primers are widespread, they have not been systematically or empirically verified. This study will attempt to address two questions. The first is "How are sequencing primers selected?" We are interested in determining what parameters are perceived as important in primer design and how the primers are actually selected; i.e.,
by software or "by eye"? The second question is "Which primer selection strategy is most successful?" We hope to document that primers with certain biophysical characteristics are more likely to generate high-quality sequencing data.
Participating laboratories will be provided with a known DNA sequence to be treated as if it were part of a sequencing project. Study participants will be surveyed for the parameters they believe are important in selection of a primer: for example, length, annealing temperature, lack of secondary structure, and self-complementarity. Study participants will then select, synthesize, and forward to us the one primer they consider best for further sequencing. The Committee will analyze the responses of the participants to assess the most common strategies and parameters used by the participants for primer selection. We will also purify all submitted primers and evaluate them by automated sequencing.
Comparing the sequencing results to the parameters and methods used for primer selection will hopefully confirm or refute the current dogmas of primer design. Based on responses to previous studies, we expect 80 to 100 primers will be submitted. Thus, we expect a broad spectrum of primer selection strategies will be used and a reasonably good sampling of primers will be submitted.
For its 1996 study, the Committee sought to assess the extent to which racemization occurs during peptide assembly in peptides synthesized by our member resource laboratories. Laboratories were asked to synthesize a peptide with the sequence RERHAY (ABRF96) by their most frequently used method. The His residue was included, because it is known to be susceptible to racemization during coupling. Coded samples of unpurified peptides were characterized by amino acid analysis (with and without prior reaction with Marfey's reagent), HPLC, ESI-MS, MALDI-MS, and enzymatic digestion by carboxypeptidase A followed by MALDI-MS. Forty-eight laboratories participated in this study and submitted 53 samples for analysis.
Racemization during peptide assembly was not a serious problem in most participating laboratories. However, a few very pure peptides were observed to have unacceptable levels of racemization. There was no correlation between the extent of racemization and the length of coupling time, the type of base, or the source of the His derivative; it is possible that the purity of other components in the coupling cocktail may be important. Because the D-His and L-His forms of the test peptide are easily separated by HPLC, synthesis of this peptide may help determine whether coupling conditions are optimized for minimizing racemization.
In most studies conducted by this Committee, a few peptides stood apart from the majority of high-quality peptides as samples whose problems originate from human error. However, in this study it was clear that these errors were not limited to the participating peptide resource laboratories, but extended to reagent suppliers. Examples of human error include one peptide that had a Gly substituted for the Glu residue, an error that apparently occurred when loading amino acid cartridges with bulk reagent. Another sample contained a large quantity of peptide missing the carboxyl -terminal Tyr, for a laboratory that had prepared its own Tyr -resin. However, purchase of preloaded resins provided no assurance of quality. At least 10 samples that were prepared with resins purchased preloaded with Tyr contained peptides with one or more additional Tyr residues, with as many as eight additional Tyr residues detected. For three of these samples, these contaminants accounted for 10-50% of the product. Thus, better quality control of resins sold by manufacturers should be implemented.
The critical role of peptides in research programs require both suppliers and synthesis laboratories to examine their quality control procedures. While strict application of GLP or ISO9002 protocols may not be necessary for many laboratories, it may be worthwhile to consider incorporating some of these procedures into general laboratory operations. They could provide additional assurance that high laboratory standards are maintained and that when errors occur, they are detected sufficiently early to prevent unnecessary expenditure of time and effort, as well as loss of good will.
Copies of the manuscript describing this study in more detail will be sent to all ABRF members. For its 1997 study, the Peptide Synthesis Research Committee is planning to examine synthesis of a phosphorylated peptide.
The 1997 study of the Protein Sequence Research Committee marks the 10th in a series designed to help participant laboratories in evaluate their abilities to obtain amino acid sequence data, as well as to provide assistance with problematic residues, such as tryptophan and cysteine. This year's sample, ABRF-97SEQ, has been designed to be compatible with both Edman and mass spectrometric sequence analysis.
Because member labs are continually faced with the need to sequence low-level samples, ABRF-97SEQ will be a mixture of two peptides at the 2 and 10 pmol level designed to resemble peptides from a tryptic digest. To aid in cysteine determination, the Committee has already modified the sample's cysteine residues with acrylamide to form Cys-S -
propionamide (Cys-S-PAM) and will provide the PTH-Cys -S-PAM derivative as a control. In addition, an internal sequencing standard (see Anal. Biochem. (1993) 211, 94 -101) will be provided. The purpose of the internal sequencing standard, which would be co-sequenced with the ABRF -97SEQ, is to give an independent monitoring of sequencer performance. Also enclosed with this year's sample will be a matrix-assisted laser desorption/ionization mass spectrum (MALDI-MS) of the sample. Participants will be encouraged to use this spectrum to help determine the approximate lengths of the peptides and to help verify that the correct sequences have been assigned.
We plan to distribute ABRF-97SEQ towards the end of September or the beginning of October, and we request that results be returned by October 31. The sample will be sent automatically to all facility directors who have indicated in the ABRF membership directory that they perform protein sequencing (PSQ). Other facility directors who would like to participate in this year's study should contact Laurey Steinke (Univ. of Nebraska Med. Center, 600 South 42nd St., Omaha NE, 68198-4525; Tel: (402) 559-6647, Fax: (402) 559-6650, E-mail: lsteinke@molbio.unmc.edu
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