This year's study sample (ABRF-95SEQ) was a mixture of two variants of a recombinant protein that contained standard amino acids including cysteine and tryptophan as well as sequence heterogeneity at cycles six through nine. One objective of this year's study was to determine how far this sample, which contained 30 pmol of variant 1 and 15 pmol of variant 2, could be sequenced.
The longest continuous stretch of positively correct sequence was 49 residues followed closely by responses with 47, 42, and 41 positively correct assignments. The heterogeneity in cycles 6 through 9 caused an average decrease of 20% in assignment accuracy compared to the surrounding residues. A few facilities made 0 to 1 positive assignment errors, but for most facilities about 20% of positive assignments were erroneous. The number of correct assignments per response ranged from 0 to 62.
Results for cysteine assignment were similar to those of the previous study, ABRF-94SEQ. Perhaps because this year's study did not stress the identification of cysteine and tryptophan, as in the 1994 study, less than half (25 of 71) of the respondents performed reduction and alkylation. However, several facilities that did not derivatize cysteine residues made positive assignments even when no cysteine degradation peaks were visible. Accuracy of tryptophan assignment was lower than reported previously, indicating that identification of this residue remains problematic.
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