Created: 1st June 2000, last updated: 30th August 2000, © 2000 ABRF
The education committee organized a Poster Competition at the ABRF 2000 meeting. The competition was an informal try out this year to introduce the idea and to sound out opinions on making a poster competition an annual feature of the ABRF meeting.
The winners were picked by a ballot, which also asked for comments on the competition. The winners were P71 Applying Surface Plasmon Resonance Biosensors to Drug Discovery (first author: David Myszka) and P92 Conversion and distribution of DNA sequence electropherograms as portable document files (first author: Genis J. Wiebe). We extend our thanks to Millipore for providing this years prize.
All the returned ballot papers, with one exception, were in favor of holding an annual competition. As a result of this endorsement, the education committee intends to organize a more formal poster competition at ABRF 2001. The committee hopes the competition will encourage presenters to make even better posters and provide a recognition process for the grassroots of our organization.
As a complement to the poster competition, a short tutorial on poster preparation was given by Dr. Dina F. Mandoli at ABRF 2000. For those of you who are interested, a summary of Dr. Mandoli's tips can be found on the Internet (http://www.aspp.org/education/poster.htm). Other useful information on poster presentation also is available on-line (http://darkwing.uoregon.edu/~jhodder/newpage7.htm).
The Fragment Analysis Research Group (FARG) conducted a study consisting of three samples, each with two allele sizes. The samples were sent to interested ABRF members' facilities that perform fragment analysis. The participating facilities returned results and information about instrumentation and run parameters. The FARG compared results for accuracy and reproducibility. Preliminary results were presented during the ABRF 2001 Conference in Seattle Washington. A more complete analysis of the final results will be summarized in a written manuscript and submitted to the Journal of Biomolecular Techniques.
A closely related study is in the planning stages, and samples will be available to any facility interested in participating. The results from this new study will examine problems that are caused by more difficult marker pairs, including multiplexing samples and stuttering. The results will be used as an educational tool to assist laboratories that deal with similar problems. The results from the study will be presented at ABRF 2001.
The FARG will also be participating at Genome Sequencing and Analysis Conference (GSAC) in Miami, Florida, on September 12-15, 2000. This will be a joint presentation with other research groups in the DNA field. A short presentation focusing on the 2000 study presented at ABRF 2000 is planned to familiarize the audience with our activities. The 2001 FARG study will be introduced at this meeting. It is hoped that discussion about these studies will increase interest in research group activities among non-ABRF researchers attending the GSAC meeting.
Qualification of Three Candidate Peptides for Suitability as Standards
The Association of Biomolecular Resource Facilities (ABRF) has been organized in part to provide a mechanism for the self-evaluation and improvement of procedural and operational accuracy, precision, and efficiency in resource facilities and research laboratories. The Quality and Compliance Committee (QCC) of the ABRF has brought a proposal to the ABRF to present a Standard program to help support the needs of the ABRF as presented above.
The committee conducted a survey through the ABRF mailing list and newsletter to assess the interest in standards. As a result of the survey responses and the great interest on the part of the ABRF Peptide Synthesis Committee in peptide standards, a joint working committee was formed to design and test several peptides to serve as candidates as reference standards. The joint working committee was composed of Nick Ambulos, Lisa Bibbs, Eleanor Canova-Davis, Michael Cohrs, Beth Fowler, Ashok Khatri, and Al Smith.
The candidate peptides were designed with several properties in mind, including purity, ability to serve in a variety of analytical techniques, and ease of use, including interpretability of results, ease of handling, ease of synthesis and purification, and ability to fully characterize. This report presents the initial work involved in the characterization of the candidate peptides as a first step in establishing them as working standards to be used by academia and industry to help improve the procedural and operation accuracy, precision, and efficiency of the operational laboratories.
Candidate Peptides
The following candidate peptides were selected for the initial qualification.
| DAEPDILELATGYR | 1563 MW | -3 charge | ||
| KAQYARSVLLEKDAEPDILELATGYR | 2950 MW | -1 charge | ||
| RQAKVLLYSGR | 1290 MW | +3 charge |
These peptides represent a variety of molecular weights, mass/charge ratios, and physical properties. It is hoped that these peptides can be useful in assessing the performance of a variety of techniques, including
| Reversed-phase high-performance liquid chromatography (RP-HPLC) |
| Cation-exchange chromatography |
| Anion-exchange chromatography |
| Capillary electrophoresis (CE) |
| Amino acid analysis |
| UV spectrophotometry |
| N-terminal sequence analysis |
| C-terminal sequence analysis |
| MALDI-TOF and electrospray mass spectrometry |
| Enzymatic digestion |
The candidate peptides were synthesized at the University of Maryland core facility on a Rainin Sonata peptide synthesizer at a 0.25 mM scale using HOBt/HBTU (AnaSpec, Inc.) activation. Amino acids were purchase from PE Biosystems and employed the following side chain protective groups: Boc (Lys), OTBU (Asp, Glu), Pbf (Arg), TBU (Ser, Thr, Tyr), and Trt (Gln). The peptide was cleaved in Reagent K (88% trifluoroacetic acid [TFA], 5% water, 5% phenol, and 2% triisopropylsilane) for 3 hours, filtered, ether precipitated, and washed three times with cold ether. The peptides were purified on a Beckman HPLC with System gold, using a Dynamax 25 X 2.5 cm C18 preparative reversed-phase column with a binary solvent system with buffer A containing water/0.1% TFA and buffer B containing 80% ACN with 0.1% TFA with a gradient of 0% to 100% over 60 minutes.
Analytical Techniques
RP-HPLC was performed using a Beckman system gold. The purity values are reported in Table 1.
TABLE 1
Analytical Data of Three Candidate Peptides Proposed for Use as Reference Standards
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| Assay | Parameter | Candidate Peptide A |
Candidate Peptide B |
Candidate Peptide C |
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| RP-HPLC (lab 1) | Purity (main peak) | 96% | 88% | 97% | ||||
| RP-HPLC (lab 2) | 92% | 87% | ||||||
| CE (lab 1) | Purity (main peak) | 91% | 81% | 81% | ||||
| CE (lab 2) | 82% | 77% | 75% | |||||
| SCX-HPLC | Purity (main peak) | 95% | 95% | 79% | ||||
| % more acidic | 2 | 4 | 17 | |||||
| % more basic | 3 | 1 | 4 | |||||
| Weak anion exchange- | Purity (main peak) | 94 | 74 | Not retained | ||||
| HPLC | % more acidic | 0.5 | 11 | |||||
| % more basic | 6 | 15 | ||||||
| Amino acid analysis | Identity | AA composition confirmed | AA composition confirmed | AA composition confirmed | ||||
| (correct 14 residues) | (correct 26 residues) | (correct 11 residues) | ||||||
| UV spectrophotometry | Identity quantitation | epsilon280 0.7224 | epsilon280 0.7704 | epsilon280 0.9617 | ||||
| epsilon276 0.8111 | epsilon276 0.8575 | epsilon276 1.0819 | ||||||
| N-terminal sequencing | Identity | Correct sequence of 14 residues | Correct sequence of 26 residues | Correct sequence of 11 residues | ||||
| as measured from the N terminus | as measured from the N terminus | as measured from the N terminus | ||||||
| C-terminal sequencing | Identity | Correct sequence of first 4 | Correct sequence of first 4 | Correct sequence of first 4 | ||||
| residues as measured from | residues as measured from | residues as measured from | ||||||
| the C terminus | the C terminus | the C terminus | ||||||
| Mass spectrometry | ||||||||
| Predicted | Molecular mass | 1563 | 2950 | 1290 | ||||
| MALDI (#1) | 1563.5 | 2953 | 1289.7 | |||||
| MALDI (#2) | 1563.3 | 2951.5 | 1290.8 | |||||
| Electrospray | 1561.9 | 2949.9 | 1290.3 | |||||
| Enzymatic hydrolysis | Lys-C; trypsin | All expected fragments observed | All expected fragments observed | All expected fragments observed | ||||
| in MALDI and RP-HPLC | in MALDI and RP-HPLC | in MALDI and RP-HPLC | ||||||
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Cation-exchange chromatography was performed using a HP1090 instrument. Separation was achieved across a LiChrosphere Tentacle SO3- column, measuring 4.6 X 50 mm and 5 µm, with smaller than 800A packing material. The column was run at ambient temperature using a gradient of 20 mM sodium phosphate, pH 2.5, to 20 mM sodium phoshpate, pH 2.5, with 1 M sodium chloride over 100 minutes at a flow rate of 0.5 mL/min and detection at 214 nm. The elution showed single peaks for each of the candidate peptides with a few minor impurities.
Anion-exchange chromatography was performed using HP1090 instrument. Separation was performed across a TSK DEAE-2SW column (4.6 X 250 mm, 5 µm, 125 A) using a sodium chloride gradient in 20 mM sodium phosphate, pH 7.0, from 0 to 500 mM over 50 minutes at ambient temperature with a flow rate of 0.5 mL/min and detection at 214 nm. Each elution showed single peaks for each of the candidate peptides A and B with a few minor impurities. Candidate peptide C was not retained.
Capillary electrophoresis was performed on an HP3DCE instrument. Separation was obtained using a PVA-coated capillary (50 µm X 64.5 cm) under electrophoretic conditions of 20°C and 465 V/cm. The peptides migrated in a buffer of 50 mM sodium phosphate at pH 2.5, detection was performed at 210 nm. Candidate peptide A yielded an electrophoretic profile of three peaks, denoting three uniquely charged species. Candidate peptides B and C each yielded profiles of single peaks. The CZE did show more minor impurities reflecting its sensitivity to charge and or size differences.
Sample amino acid analysis was performed according to the AccQ-Taq method. Samples were hydrolyzed for 24 hours at 110°C in the Waters Pico-Tag Workstation using 6 N HCl with 1% phenol. The samples were derivatized using the Waters AccQ-Fluor reagent kit. All analyses were performed on a Waters 2690 HPLC with a Waters Nova PakR C18 3.9 X 150 mm column. The samples were detected with a Waters 474 Scanning Fluorescence Detector. The excitation wavelength was set to 250 nm. The emission wavelength was set to 395 nm. The candidate peptides were confirmed in their amino acid composition and the concentrations were established.
UV spectrophotometry was performed by scanning the candidate peptides from 200 to 600 nm using a Hewlett-Packard 8452 diode array spectrophotometer. The concentrations as derived from amino acid analysis were used to determine the extinction coefficients. The extinction coefficients were determined for absorbances at 276 and 280 nm. The data are shown in Table 1.
N-terminal sequencing was performed using an Applied Biosystems 475A sequenator. The residues were detected by LC-mass spectrometry. Each of the candidate peptides were confirmed for all residues from the N terminus.
C-terminal sequence analysis was performed by digestion with a carboxypeptidase B and carboxypeptidase Y combination followed by detection using mass spectrometry (MALDI). Each of the candidate peptides was confirmed for four residues from the C terminus.
MALDI TOF mass spectrometry was performed by two different facilities. The results were compared with each other, with predicted values, and with results obtained by electrospray mass spectrometry. All results were comparable and confirm each other.
All of the candidate peptides were digested with Lys-C and separately with trypsin. The released peptides were analyzed by mass spectrometry and compared with predicted cleavages. The results of the digestion to predicted were comparable, confirming their identities. A summary of the results is presented in Table 1.
Conclusions
Each of the three candidate peptides was synthesized as expected. The characterization performed confirmed the identities and established the purity of the candidate peptides. The identities and purity have been measured by a number of orthogonal assays that serve to confirm that the assays were operating as intended. The peptides have been shown to be suitable to serve as references in a number of assays. Each assay has shown them to be of suitable consistency for use in a number of ways, including verifying system suitability or correct operation of the instrument or method.