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Investigating Protease Activity Using Mass Spectrometry
David N. Potier1, John R. Griffiths1, Ralf Hoffman2, Anthony D. Whetton1
1: University of Manchester, Manchester, UK; 2: Philips, Eindhoven, Netherlands
Introduction
Proteases have been linked to a range of different diseases, including cancer and arthritis. Many groups have monitored the amount of a protease in the
hopes that this may be a disease biomarker1-3, but very little work has been done on monitoring the activity of a protease of interest. By designing peptides
which are cleaved in specific places by target proteases, these can be added to plasma and their degradation can be monitored by Mass Spectrometry.
This study describes a method of monitoring the activity of a target protease in a plasma sample.
Method
Plasma was obtained from healthy volunteers and prepared as follows.
First, each aliquot was centrifuged at 13,000 rpm for 5 minutes at 4oC. The
supernatant was removed, diluted four-fold with PBS and spin filtered at
4,000 rpm for 30 seconds at 4oC. These were pooled and divided into
25 µl aliquots. Each then had the synthetic peptide eteaaylGGKVdtav (2
µg) added to it along with trypsin in varying amounts. The following
workflow was followed, with samples being taken over a 24 hour period:
Results
Figure 1(a) shows the amount of uncleaved peptide in the sample over a
24 hour period. Figure 1(b) shows the amount of cleaved peptide
generated by the target protease using different Enzyme : Substrate ratios:
Cleaved Peptide Generated
Uncleaved Peptide Removed
35000000.00
30000000.00
30000000.00
25000000.00
Incubate for x hours at 37oC
1:10
20000000.00
1:20
1:50
15000000.00
1:100
20000000.00
Peak Area
Quench Sample with Acid & Freeze
Peak Area
25000000.00
Acknowledgements
We thank BBSRC & Philips for funding
this work. We also thank Dr. Duncan
Smith, Dr. Michael Walker, Dr. Richard
Unwin & Yvonne Connolly for their input
in many helpful discussions.
1:100
0.00
0
NOTE: Amino acids in lower case correspond to the non-naturally
occurring isoform (i.e. the D-isoform) of the amino acid.
1:50
5000000.00
5000000.00
0.00
Monitor Target Peptides by Mass Spectrometry
1:20
15000000.00
10000000.00
10000000.00
Enrich for Peptides (Acetonitrile Precipitation)
1:10
5
10
15
20
25
30
Time (hours)
0
5
10
15
20
25
FIGURE 1: Showing the amount of (a) Cleaved peptide, and
Uncleaved peptide present in a plasma sample over a 24 hour period.
Conclusion
•The amount of uncleaved target peptide is shown to decrease over time,
•The amount of cleaved target peptide produced increases over time,
•Both these changes are dependent on the amount of enzyme present in the sample,
•D-amino acids inhibit enzyme activity, so peptide cleavage occurs in one place,
•Protease activity can be monitored quantitatively by Mass Spectrometry.
30
Time (hours)
(b)
References
1: S Horstmann et. al.,
Int Psychogeriatr. 2010 Sep;22(6):966-72.
2: K Sasajima et. al.,
Hepatogastroenterology. 2009 SepOct;56(94-95):1377-81.
3:B Mroczko et. al., Clin Chem Lab Med.
2009;47(9):1133-9.