The 9th International Conference on Methods in Protein Sequence Analysis was held in Otsu, Japan, September 20-24, 1992. ABRF sponsored a symposium on the opening night that was entitled. "Protein Characterization And Synthesis: Results From Analysis Of Unknown ABRF Test Peptides". More than I ()0 protein chemists heard the results of ABRF research on the status of protein sequencing, peptide synthesis, and amino acid analysis in core facilities.
Conclusions drawn from five years of "unknown" amino acid analysis samples were presented by Ray Paxton in the opening talk. "Current State Of Amino Acid Analysis: Methodology, Accuracy and Precision". These conclusions were:
1. A dedicated instrument is required and use of automated hydrolysis is increasing.
2. An average error of less than + or - 10%, is common. There is little difference between performance of pre and post column systems except that post column analysis requires more sample .
3. The average error is less if the quantity of sample is sufficient to permit multiple analyses .
4. Samples containing salt or on PVDF result in larger average error.
5. Cysteine/cystine and tryptophan can be determined with less than 10% error by several different techniques.
In his presentation, ' Characterization Of Synthetic Peptides By A Variety Of Methods Including Mass Spectrometry and Capillary Electrophoresis", Alan Smith compared and summarized the results from the two annual peptide synthesis/mass spectrometry studies that have been completed.
1. The number of participants increased from 32 in 1991 to 58 in 1992.
2. High quality peptides can be synthesized by either tBOC or Fmoc chemistry with the correct peptide being assembled in more than 95% of syntheses. However, due largely to problems with the cleavage and deprotection chemistry, less than hall of the 94 crude samples analyzed in the 1991 and 1992 studies contained >75% desired product.
3. Fmoc chemistry is being used more frequently in 1992 than in 1991.
4. Analysis of product and, when multiple products are obtained, identification of the desired product is d major cause for concern in some facilities.
5. All analytical techniques have limitations; therefore a combined approach using complementary techniques is preferred. The recommended minimum requirements include amino acid analysis, HPLC and mass spectrometry.
6. Only 12% of participants have a mass spectrometer in the facility although 86% occasionally use mass spectrometry as an analytical tool. Although the latter figure indicates a strong appreciation of the value of mass spectrometry for confirming the authenticity of synthetic peptides, the lack of an on-site mass spectrometer was the reason most often cited for not using this analytical tool more routinely. We speculate that in many instances where the wrong peak was purified (i.e., where the % desired product was 1ess in the "purified" as compared to the crude sample), that the submitting facility lacked ready access to the mass spectrometric instrumentation required to identify the peak containing the correct product.
Ron Niece presented the talk, "Protein Sequence Analysis: Current Capabilities And Reasonable Expectations", drawing the following conclusions from the five unknown protein sequencing samples analyzed by ABRF members.
1. One of the main factors in determining the number of amino acid residues that can be sequenced and the overall accuracy of that sequencing is the total amount of sequenceable sample delivered to the sequencer.
2. An equally important factor in determining sequencing accuracy is the interpretation of the data. There is no substitute for an experienced, well trained operator.
3. PVDF is an excellent medium for capturing and transferring small samples of protein (especially from heterogeneous mixtures) to the sequencer. However, it appears to offer no significant advantages in sensitivity, accuracy or distance over samples delivered in solution.
4. The average results of these studies should not be regarded as acceptable performance on a particular sample. One should strive to achieve the performance of the best results presented. Greater than 90% accuracy should be a minimal requirement.
Return to the The ABRF Home Page