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Peptide Standard Project

The Peptide Standards Project Committee (PSPC) was formally instituted as an ABRF committee at the annual ABRF meeting in San Diego in February 2001.

The peptide standards project is the production and establishment of three synthetic peptides as registered and certified peptide reference standards in collaboration with the National Institute of Standards and Technology (NIST). The committee’s mission is to provide active support and advice to NIST in conducting this project. Therefore the PSPC focuses on the following:
  1. Large scale synthesis of the standard peptides in high (>98%) purity.
  2. Packaging of the peptides in small quantities for distribution and analysis.

Current Membership
Dr. Henriette A. Remmer (Chair) - University of Michigan 
Dr. Elizabeth Fowler - CMC Consulting Services 
Ms. Virginia J. Johnson - Texas A&M University 
Mr. Ashok Khatri - Massachusetts General Hospital 
Dr. Susan T. Weintraub - Univ. of Texas Health Science Center at San Antonio 
Dr. Nicholas P. Ambulos (Ad hoc) - Univ of Maryland Sch of Med 
Dr. Lynda F. Bonewald (Ad hoc) (Advisor) - Univ. of Missouri at Kansas City 
Dr. Mark O. Lively (EB liaison) - Wake Forest Univ. Sch. of Med. 

Activities
1)

Design of Standard Peptides

The Peptide Standards Project was originally initiated by the joint interactions between the Quality Compliance Committee (QCC) and Peptide Synthesis Research Group (PSRG). They designed, synthesized and qualified 3 synthetic peptide sequences as suitable peptide reference standards. The sequences were designed according to the following considerations:

  • Coverage of a range of molecular weights, charge and hydrophobicity
  • Avoidance of Met, Cys or Trp to facilitate synthesis and improve stability
  • Incorporation of Tyr to allow quantitation by UV spectrophotometry
  • Incorporation of protease cleavage sites
  • Avoidance of acid labile amino acids and sequences resistant to acid hydrolysis
  • Susceptibility of C-terminal sequence to carboxypeptidases

In order to initially qualify these peptides as suitable reference compounds, extensive analysis was performed using mass spectrometry, reversed phase and ion exchange HPLC, amino acid composition analysis, UV spectrophotometry, N- and C-terminal sequence analysis, and enzymatic hydrolysis. The details and results of this study are published in J. Biomolecular Techniques (2000), 11(2), 103-105.

2)

Peptide Sequences

  • Peptide A:
    DAEPDILELATGYR	
  • Peptide B:
    KAQYARSVLLEKDAEPDILELATGYR	
  • Peptide C:
    RQAKVLLYSGR	
3)

Funding and Collaboration

The Peptide Standards Project is the first ABRF project funded by NIST. Collaboration with NIST provides additional recognition and visibility for the ABRF. Vice versa, the ABRF membership is a valuable resource of information, data and data validation for NIST. For the Peptide Standards Project, the collaborative analysis of the peptides by the ABRF membership will yield the required certificate of analysis.

4)

Importance and Potential Applications for the Peptide Standards

The Peptide Standards Project is the first ABRF project funded by NIST. Collaboration with NIST provides additional recognition and visibility for the ABRF. Vice versa, the ABRF membership is a valuable resource of information, data and data validation for NIST. For the Peptide Standards Project, the collaborative analysis of the peptides by the ABRF membership will yield the required certificate of analysis.

5)

Project Status and Results/b>

Peptides A, B and C were successfully synthesized on 7.5 mmole and 10 mmole scale in an academic core facility setting. The purity of 96% (Peptide A), 95% (Peptide B) and 97% (Peptide C) was achieved in a two-step purification using reversed phase HPLC ( Peptide B) or the combination of ion-exchange chromatography and reversed -phase HPLC (Peptides A and C). Orthogonal analytical techniques (mass spectrometry, reversed phase HPLC, capillary zone electrophoresis (CZE), Edman sequence analysis) were use to confirm identity and purity of the peptides. The purified peptides were obtained in 1-2 g quantities, sufficient to provide 1000 vials of the standard reference material packaged at 1 mg per vial. The peptides have been vialed and packaged in sets. A set contains one vial of each peptide.

The set of Peptides A, B, and C is NIST Standard Reference Material (SRM) 2397. SRM 2397 is called the "Peptide Set for Biomolecular Measurements". The peptides set will be certified for three peptide properties: molecular weight (by MALDI-TOF and ESI mass spectrometry) purity (by HPLC and CZE) and Peptide Content ( by AAA and UV). Analyses have been performed by multiple laboratories to validate the certified peptide properties. The Certificate of Analysis is currently being developed. SRM 2397 is expected to be available from NIST in fall of 2005.


Membership History
Member NameOrganizationDetails
Dr. Lynda F. BonewaldUniv. of Missouri at Kansas City EB liaison: 01/01 - 01/02
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