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.