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

Area of Science:

• Biomolecular analysis
• Proteomics
• Analytical chemistry

Background:

• The Association of Biomolecular Resource Facilities (ABRF) conducts annual studies to assess peptide sequencing capabilities.
• Previous studies indicated challenges in low-level peptide analysis.
• The ABRF-98SEQ sample was designed as a pure, synthetic peptide for high-sensitivity sequencing assessment.

Purpose of the Study:

• To evaluate the performance of laboratories in determining the amino acid sequence of a synthetic peptide.
• To assess the effectiveness of Edman degradation and mass spectrometry (MS) for high-sensitivity peptide sequencing.
• To track improvements in peptide sequencing accuracy and sensitivity over time.

Main Methods:

• Distribution of a 17-amino acid synthetic peptide (ABRF-98SEQ) at the 2.8-pmol level.
• Participants utilized Edman degradation, mass spectrometry (MS), or a combination of both for sequence analysis.
• Data collection via survey forms accompanying the peptide sample.

Main Results:

• 16% of participants successfully determined the correct amino acid sequence of ABRF-98SEQ.
• This represents a significant improvement from the 6% success rate in the 1997 study.
• Laboratories reporting higher sensitivity analysis (1-10 pmol) showed increased success rates.
• A combination of Edman sequencing and MS molecular weight determination yielded the best results.

Conclusions:

• Peptide sequencing accuracy and sensitivity, particularly with Edman degradation, are improving.
• Mass spectrometry (MS) significantly aids in correcting, completing, and corroborating sequence data.
• While not yet routine, MS-derived sequence data is valuable for peptide analysis.
• The purity of the ABRF-98SEQ sample likely contributed to the improved results.