Before being analyzed by LC-MS/MS, about 10% of each sample is subjected to separation on a monolithic nano-HPLC column. This is in most cases done after the proteolytic digest. The obtained UV-chromatogram is used as a quality measurement of the sample. We can estimate the amount of generated peptides, the digest efficiency and the contamination by detergents, protease inhibitors or other interfering components. The outcome of this test run helps us to decide on both the amount of the sample and the method for the analysis of the peptides by LC-MS/MS.
Example of our reference sample consisting of 500fm tryptically digested BSA
Samples which are successfully digested and are free of detectable intact protein or other contaminations will resemble our reference sample.
Example of a mixture of tryptically digested BSA plus the intact protein BSA
On a monolithic column peptides can be separated from proteins, which allows for the evaluation of the digest efficiency.