Analysis of protein fragmentation inhibition by an MMP-inhibitor in an in vivo model of heart failure using automated chromatography.
Proteomic strategies have continued to demonstrate value in studying disease by exploiting new technologies that can develop significant numbers of measurements from single samples. However, using complex samples such as tissues or blood has continued to be problematic due to the presence of major interfering substances. In this study, a process is described that uses denaturing peptide extraction from whole tissue and automated chromatography in order to allow subsequent analysis of more than 1000 tissue-derived peptides per sample. The process was employed to identify cardiac proteins that were spared degradation by administration of a heart-protecting matrix metalloproteinase (MMP) inhibitor (compound SC-621) following experimental myocardial infarction (MI). HPLC peptide fingerprints were developed from rat heart left ventricles and the resultant integrated peak data was compared across experimental animals. Surprisingly, although protein fragmentation was generally increased in MI hearts, the effect of the MMP inhibitor was only observed on a few species. The results from this study demonstrated that whole-tissue sample enrichment and peptide analysis using HPLC could be linked in order to study the effects of new compounds on a disease state. The system is flexible and amenable to improvements such as incorporating detection by mass spectrometry.
Slemmon
J Randall
JR
Genomics and Biotechnology, Pfizer Global Research and Development, Skokie, IL 60077, USA. Randy.J.Slemmon@Pfizer.com
Painter
Cory L
CL
Nadanaciva
Sashi
S
Catana
Florentina
F
Kaup
Karen
K
Scherrer
Rachel
R
Casadas
Virginia
V
Zhao
Youyang
Y
Heron
Marcia I
MI
D000595:Amino Acid Sequence; D000818:Animals; D002316:Cardiotonic Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D008297:Male; D013058:Mass Spectrometry; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000737:chemistry; D023421:Models, Animal; D008969:Molecular Sequence Data; D009203:Myocardial Infarction / Q000201:enzymology / Q000378:metabolism*; D010449:Peptide Mapping; D011480:Protease Inhibitors / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley
D002316:Cardiotonic Agents; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D020782:Matrix Metalloproteinases