Protein identification can be done either by enzymatic digestion of an in-gel sample followed by LC/MSMS analysis or direct n-terminal protein sequencing (Edman chemical sequencing). If the protein is n-terminally blocked (80% of eukaryotic proteins are naturally blocked) then digestion is the next logical step. Once digested the peptides can be separated and collected by off-line HPLC with UV detection (leading to Edman chemical sequencing of individual peptides) or the mixture analyzed on the mass spectrometers by LC/MSMS.
See the Protein Quantity Estimation section to start. Edman chemical sequencing needs 10pmol for high quality data, more for extended sequence runs. Keep in mind the initial yields for this technique is ~50%, with a repetitive yield around 92%. Typically samples can run 30 residues with ~100pmol sample loaded, depending on the protein and sample preparation. Mass spectrometry is several orders of magnitude more sensitive that Edman sequencing, thus it is possible to say we can get high quality data from as little as 10fmol protein. This amount, however, is not what was loaded on the gel, as each step in the sample handling process (reduction, alkylation, digestion, peptide extraction and concentration) incurs protein and peptide loss to surfaces. Maximizing quantity is always the first thing to do for any sample, and in some projects it is simply not possible to get more sample, so trace level analysis must be done with a subsequent loss of protein coverage and spectra quality to some degree. The degree of this impact is difficult to quantify, as each protein will behave differently and thus unpredictably at very low quantities. Also see the FAQ regarding silver stained gel spot or bands.
www.asms.org - American Society for Mass Spectrometry
www.abrf.org - Association of Biomolecular Resource Facilities
expasy.org - ExPASy Proteomics Server
www.hupo.org - Human Proteome Organisation's (HUPO) International Website
www.ushupo.org - Human Proteome Organisation's (HUPO) US Website
www.ionsource.com - Mass Spectrometry and Biotechnology Resource
www.ncbi.nlm.nih.gov - National Center for Biotechnology Information
www.ncbi.nlm.nih.gov/blast - NCBI BLAST search
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