Download Limited Proteolysis

Limited Proteolysis of MDH with Immobilized TPCK-Trypsin
Limited proteolysis of MDH (2 mg/mL) was
performed using varying volumes of immobilized
trypsin (0.1-0.5 mLs) in order to determine the
enzyme’s structural flexibility. The samples were
analyzed using a Brucker OmniFlex MALDI-TOF
mass spectrometer with a matrix of sinapinic acid.
Cleavage sites were computationally predicted
using NickPred (Figure 1).
2a
2b
2c
Figure 2. The MALDI-TOF spectra of (a) undigested, (b) semi-digested for 15 minutes with
0.5 mL immobilized trypsin, and (c) fully-digested for 24hours with 0.5 mL immobilized
trypsin MDH.
A spectrum of the undigested protein is seen in Figure 2a and shows only the
standards used (myoglobin ~17 kDa, and BSA ~ 66 kDa) and native MDH. The
small peak at 50 kDa is most likely due to impurities in the sample. Figure 2b shows
the semi-digested protein 15 minutes after addition of 0.5 mLs of immobilized
trypsin. The fully digested MDH 24 hours after addition of 0.5 mLs of immobilized
trypsin is shown in Figure 2c. The data could be a result of a vast number of different
cleavage combinations and therefore could not be precisely characterized. Also,
small fragments could have been lost due to the poor resolution of the mass
spectrometer.
Figure 1. Predicted Cleavage Sites
(http://wolf.bms.umist.ac.uk/npred/nickpred.html)
QTOF (quadrupole time of flight) analysis would be a more reliable method of mass
spectrometry rather than MALDI-TOF as its ion trap would allow for further
fragmentation of the cleaved peptide, thus enabling more accurate identification of
the fragments.