Complex Protein Mixture Analysis
Analysis of complex protein mixtures, such as cell extracts, uses the same principles of protein identification as single proteins, but with some differences.
- longer chromatography runs
- possible two dimensional separation of peptides
- much longer data analysis
A longer chromatography gradient can give more separation of peptides. More important, it gives the mass spectrometer time to measure more peptides. In a gradient of 1% acetonitrile/minute, in the 6 seconds to change from 30% to 30.1% acetonitrile, the instrument can analyze 18 peptides. Using a gradient of 0.25% acetonitrile/min, 72 peptides can be analyzed, allowing more proteins to be found.
In some cases, the peptide mixture is fractionated by ion exchange or other method before the reverse phase separation mentioned above. This is 2D-LC, referred to as MUDPIT by some. This increases the number of analyses.
Not every peptide is analyzed. This is always the case. Some peptides are always on a black list not be be analyzed-peptides from trypsin, peptides from keratin-an almost universal contaminant. In an actual sample, after a peptide is analyzed once, it goes on a list of peptides not to be analyzed for a while. Nevertheless, analysis of the data takes much longer than for a single protein.