Peptide Code-on-a-Microplate for Protease Activity
Analysis via MALDI-TOF Mass Spectrometric Quantitation
- Publication date
- Publisher
Abstract
A peptide-encoded
microplate was proposed for MALDI-TOF mass spectrometric
(MS) analysis of protease activity. The peptide codes were designed
to contain a coding region and the substrate of protease for enzymatic
cleavage, respectively, and an internal standard method was proposed
for the MS quantitation of the cleavage products of these peptide
codes. Upon the cleavage reaction in the presence of target proteases,
the coding regions were released from the microplate, which were directly
quantitated by using corresponding peptides with one-amino acid difference
as the internal standards. The coding region could be used as the
unique “Protease ID” for the identification of corresponding
protease, and the amount of the cleavage product was used for protease
activity analysis. Using trypsin and chymotrypsin as the model proteases
to verify the multiplex protease assay, the designed “Trypsin
ID” and “Chymotrypsin ID” occurred at <i>m</i>/<i>z</i> 761.6 and 711.6. The logarithm value
of the intensity ratio of “Protease ID” to internal
standard was proportional to trypsin and chymotrypsin concentration
in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection
limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively.
The peptide-encoded microplate showed good selectivity. This proposed
method provided a powerful tool for convenient identification and
activity analysis of multiplex proteases