The ABRF94-AAA1 and -AAA2 samples were analyzed by a record 62 participating sites. These samples were similar proteins, pre-hydrolyzed and unhydrolyzed, and their analyses, together with the strong support of our membership which increased the statistical significance of the study, provided a clear conclusion that the hydrolysis process is a major source of error in amino acid analysis.
The results of this study were summarized in an oral presentation and a poster at the 1994 ABRF Meeting and via a poster at the Protein Society meeting in San Diego. A manuscript detailing the study is in press in "Techniques in Protein Chemistry VI". The highlights from the study are: (i) the pre-hydrolysate was analyzed more accurately than the protein, with overall average errors of 6.52% and 8.36%, respectively (statistically significant using the Mann- Whitney ranking test, p=0.002); (ii) the pre-hydrolysate was analyzed significantly better by ninhydrin than by PTC sites (4.7% vs 7.9% error, p=0.0007), while no significant differences were seen for analyses of the protein sample; (iii) cystine analyses show a continuing trend of successful use of disulfide exchange reagents; (iv) histidine analyses of the protein sample indicate positively biased results by ninhydrin sites (+4.2%), negatively biased by PTC sites (- 6.8%), whereas no such bias was seen for the pre-hydrolyzed sample (0.12, -1.4%); (v) glucosamine, spiked into the pre- hydrolysate, was identified by only 8 sites.
A question arose during the ABRF meeting in San Diego regarding the use of the pre-hydrolysate sample (ABRF- 94AAA2) as a standard. We want to emphasize that the specific methodology by which this hydrolysate was prepared is not germane to the conclusions of the study. What is germane is that everyone analyzed the same hydrolysate (i.e., all samples were hydrolyzed simultaneously in one reaction vessel). The error calculations were specifically based on the average composition determined by the participants of the study, and not on an ideal composition.
During the workshop at the ABRF meeting the AAA Committee presented a session on "Practical Amino Acid Analysis", led by Tom Andersen, with Umit Yuksel, Lowell Ericsson, Tom Andersen, Izydor Apostol and Jay Fox discussing various practical aspects of hydrolysis, post- and pre-column methodologies and data handling.
With the retirement of most members of this committee, the renewal process is in high gear, with five new members about to join: Steve Cohen (Waters), Nancy Denslow (U. Florida), Alan Mahrenholz (Purdue U.), Kathleen Schegg (U. Nevada) and our first European member, Karl-Heinz Mann (Max Planck, Martinsried).
The planning for the 1995 study is already under way, and the samples will be distributed earlier than in previous years, to allow time for data reduction before the European meeting at the end of May 1995. This study will address needs expressed by members during the San Diego meeting.
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