ABRF-97 SEQ: Sequencing results of a low-level sample

The ABRF-97SEQ sample was the 10th in a series of studies designed to aid ABRF participant laboratories in determining their abilities to obtain amino acid sequence data.The sample for 1997 was a mixture of two peptides at an approximate picomole ratio of 10:2 and was indicative of a peak that might be obtained by reverse-phase high-performance liquid chromatography (HPLC) of a tryptic digest. Participants were asked to use Edman sequencing or a combination of Edman sequencing and serial mass spectrometry (MS/MS) or postsource decay (PSD) sequence analysis to identify the primary amino acid sequence of the peptides. Cysteine was derivatized to Cys-S-propionamide (Cys-S-PAM) before sending out the sample, and a phenylthiohydantoin (PTH)-Cys-S-PAM standard was included to locate the elution position of this derivative on the participant's HPLC system. An internal standard containing norleucine and succinylated lysine was sent as a control for testing the instrument's performance;it was co-sequenced with the unknown sample. A total of 50 responses were returned, with all participants performing Edman sequencing. The accuracy of the positive correct identifications was 91.5%, with eight participating laboratories correctly identifying the major sequence. Cysteine identification improved from the ABRF-95SEQ sample, which contained an unmodified cysteine, and tryptophan identification was similar to that observed in previous studies.