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

Kathryn Stone, Joseph Fernandez, Arie Admon, William Henzel, William Lane, Michael Rohde, Laurey A Steinke

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)26-31
Number of pages6
JournalJournal of Biomolecular Techniques
Volume10
Issue number1
StatePublished - Mar 1 1999

Fingerprint

Cysteine
Amino Acid Sequence
Phenylthiohydantoin
Norleucine
High Pressure Liquid Chromatography
Peptides
Reverse-Phase Chromatography
Tryptophan
Lysine
Sequence Analysis
Mass Spectrometry
propionamide

Keywords

  • Cysteine identification
  • Mass spectrometry
  • Ms/ms
  • Peptide sequencing
  • Protein sequencing
  • Tryptophan identification

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Stone, K., Fernandez, J., Admon, A., Henzel, W., Lane, W., Rohde, M., & Steinke, L. A. (1999). ABRF-97 SEQ: Sequencing results of a low-level sample. Journal of Biomolecular Techniques, 10(1), 26-31.

ABRF-97 SEQ : Sequencing results of a low-level sample. / Stone, Kathryn; Fernandez, Joseph; Admon, Arie; Henzel, William; Lane, William; Rohde, Michael; Steinke, Laurey A.

In: Journal of Biomolecular Techniques, Vol. 10, No. 1, 01.03.1999, p. 26-31.

Research output: Contribution to journalArticle

Stone, K, Fernandez, J, Admon, A, Henzel, W, Lane, W, Rohde, M & Steinke, LA 1999, 'ABRF-97 SEQ: Sequencing results of a low-level sample', Journal of Biomolecular Techniques, vol. 10, no. 1, pp. 26-31.
Stone K, Fernandez J, Admon A, Henzel W, Lane W, Rohde M et al. ABRF-97 SEQ: Sequencing results of a low-level sample. Journal of Biomolecular Techniques. 1999 Mar 1;10(1):26-31.
Stone, Kathryn ; Fernandez, Joseph ; Admon, Arie ; Henzel, William ; Lane, William ; Rohde, Michael ; Steinke, Laurey A. / ABRF-97 SEQ : Sequencing results of a low-level sample. In: Journal of Biomolecular Techniques. 1999 ; Vol. 10, No. 1. pp. 26-31.
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