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.