This year's study (ABRF-97SEQ) was the tenth in a series of studies designed to aid participating laboratories in determining their abilities to obtain amino acid sequence data. The sample was a mixture of two peptides (with compositions similar to ABRF-96SEQ) at an approximate ratio of 10:2 intended to simulate a peak that might be obtained by RP-HPLC of a tryptic digest. The major peptide was IWTCM EGANS YQCAS WAGLF K and the minor peptide HYAEG DSVAT KPAR. Therefore, this sample was amenable to sequencing using Edman chemistry, post source decay (PSD), or MS/MS. A matrix assisted laser desorption ionization (MALDI) mass spectrum of the sample was included, and participants were encouraged to use the data to help determine the length of the peptide, and verify that the correct sequence had been called.
The Committee modified the cysteines to Cys-S-propionamide using acrylamide . This was done to eliminate the variability of reduction/alkylation procedures and to minimize sample handling. Acrylamide was chosen as the alkylation reagent because proteins separated by SDS PAGE could potentially contain this modification. A PTH-Cys-S-PAM standard was included so that the separation of this derivative from the other PTH amino acid standards could be determined before analyzing the sample. A 17-residue internal sequencing standard (composed of norleucine at residues 1, 6, 11, and 16 and succinylated lysine at the remaining residues) was also included and was to be co-sequenced with the ABRF-97SEQ sample. The purpose of this standard was to allow an independent monitoring of sequencer performance.
A total of 50 responses were returned to the Committee in time for inclusion in this year's study, with all laboratories choosing to perform Edman sequencing. The accuracy of positive correct calls for the major sequence was 91.5%, and for the minor sequence 71.7%. Those accuracy figures were slightly lower than the 95.8% observed in the ABRF-96SEQ study. There were three responses where both the major and minor sequences were 100% correct, including tentative assignments. Problematic residues in the major sequence were the isoleucine in cycle 1, tryptophan in cycle 16, and serine in cycles 10 and 15. In the minor sequence, the histidine in cycle 1 and alanine in cycle 9 were called incorrectly most often.
Cysteine identification was greatly improved over previous years (excluding ABRF-96SEQ) with an accuracy of 88% for cysteine in cycle 4 and 97% for cycle 13. In the ABRF-95SEQ sample, the cysteine accuracy was only 63%. Thus, it appears that difficulties are encountered in cysteine derivatization and not in identification of the derivative itself.
Finally, it appears that MS is not being widely used to assist in Edman sequence analysis. This conclusion is based on the fact that the majority of the laboratories that provided longer than expected sequences indicated they did not use the MALDI-MS spectra provided to them.
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