Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis

Analytical advantages of using multiple enzymes for sample digestion (MED), primarily an increase of sequence coverage, have been reported in several studies. However, this approach is only rarely used, mainly because it requires additional sample and mass spectrometric measurement time. We have previously described Filter Aided Sample Preparation (FASP), a type of proteomic reactor, in which samples dissolved in sodium dodecyl sulfate (SDS) are digested in an ultrafiltration unit. In FASP, such as in any other preparation protocol, a portion of sample remains after digestion and peptide elution. Making use of this fact, we here develop a protocol enabling consecutive digestion of the sample with two or three enzymes. By use of the FASP method, peptides are liberated after each digestion step and remaining material is subsequently cleaved with the next proteinase. We observed excellent performance of the ultrafiltration devices in this mode, allowing efficient separation of orthogonal populations of peptides, resulting in an increase in the numbers of identified peptides and proteins. At the low microgram level, we found that the consecutive use of endoproteinases LysC and trypsin enabled identification of up to 40% more proteins and phosphorylation sites in comparison to the commonly used one-step tryptic digestion. MED-FASP offers efficient exploration of previously unused sample material, increasing depth of proteomic analyses and sequence coverage.