Protein Sequencing
In the 1980's and early 1990's amino-terminal (Edman) sequencing was the primary means of identifying proteins. Mass spectrometry of peptides from enzymatic digests is now the most common means of protein identification due to the speed (multiple peptides identified over a 2-3 hour HPLC gradient being fed into an electrospray mass spectrometer vs. 1 amino acid per hour by Edman sequencing) and ability to identify N-terminally blocked peptides and proteins in complex mixtures. However, Edman sequencing can provide data unavailable by mass spectrometric methods and is a powerful complimentary technique.
Edman sequencing is carried out on an Applied Biosystems protein sequencer equipped with an on-line HPLC system. The limit of detection on this Procise 494 HT instrument is about 300 fmol of an individual phenylthiohydantoin (Pth) amino acid. In general, 10 pmole is sufficient to sequence from 5 to 25 or more residues, with the quality of data and length of sequence that can be assigned increasing with the amount of sample. The minimum sample load is 1-2 pmole while the maximum is approximately 1 nmole. The best results are generally obtained on samples in the 10-200 pmole range.
