ABRF-98SEQ: Evaluation of peptide sequencing at high sensitivity.

The ABRF-98SEQ sample was the 11th in a series of amino acid sequencing studies performed by the Protein Sequence Research Group of the Association of Biomolecular Resource Facilities. This study was designed to aid participants' laboratories in determining their abilities to analyze the amino acid sequence of a peptide at high sensitivity using Edman degradation, mass spectrometry (MS), or both. ABRF-98SEQ is a 17-amino acid synthetic peptide (IFDDEIEEVQALYPTER) that resembles a typical tryptic peptide. It was distributed at the 2.8-pmol level. The sample was sent dried in a microfuge tube accompanied by instructions on solubilizing the sample and by a survey form. Including tentative calls, the correct sequence was obtained by 16% of the responding participants, compared with only 6% in the 1997 study when the low-level peptide was a minor component of a mixture. This increase probably reflects the purity of ABRF-98SEQ. A secondary factor in the increase in correct calls may be the larger number of respondents this year reporting that they perform sequence analysis at the 1- to 10-pmol level. Most respondents who obtained the correct sequence used a combination of Edman sequencing and molecular weight determination by MS. Overall, the accuracy and sensitivity of peptide sequencing by Edman degradation continue to improve and are clearly aided by the use of MS for molecular weight determination. Although peptide sequencing by MS is not yet routinely practiced by the participating laboratories, results of this study indicate that MS-derived sequence data, when properly interpreted, are valuable for correcting, completing, or corroborating sequence assignments derived by Edman.

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