Adaptation of proteomic techniques for the identification and characterization of protein species from murine heart.
Disturbed energy metabolism with impaired fatty acid oxidation, ATP synthesis and changing levels of contractile proteins has been observed during the development and manifestation of cardiovascular diseases, with the latter showing sexual differences in terms of onset, manifestation and progress. Estrogenic compounds, such as estrogens and phytoestrogens, are known to exert beneficial effects on several cardiovascular parameters. However, global studies implying the normal, non-failing myocardium are rare. Thus, identifying and characterizing protein patterns involved in the maintenance of normal heart physiology at the protein species level will help understanding disease conditions. In this study, we performed an adapted 2-DE/MS approach in order to identify and characterize post-translational modified and truncated protein species from murine heart. Female and male animals of different age were receiving the phytoestrogen genistein and comparative analyses were performed to identify sex and genistein treatment-related effects. Selected 2-DE spots that exposed varying abundance between animal groups and identified as identical proteins were subject to multi-protease cleavage to generate an elevated sequence coverage enabling characterization of post-translational modifications and truncation loci via high-resolution MS. Several truncated, phosphorylated and acetylated species were identified for mitochondrial ATP synthase, malate dehydrogenase and trifunctional enzyme subunit alpha. However, confirmation of several of these modifications by manual spectra interpretation failed. Thus, our results warrant caution for the blind trust in software output. For the regulatory light chain of myosin, we identified an N-terminal processed species, which so far has been related to ischemic conditions only. We tried to unravel the information content of protein species separated by high-resolution 2-DE as an alternative to high-throughput proteomics, which mainly is interested in lists of protein names, ignoring the protein species identity.
Schwab
Karima
K
Center for Cardiovascular Research, Institute of Pharmacology, Charité Universitätsmedizin, Berlin, Germany. karima.schwab@charite.de
Neumann
Boris
B
Scheler
Christian
C
Jungblut
Peter R
PR
Theuring
Franz
F
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D019833:Genistein / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D048789:Phytoestrogens / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D057825:Two-Dimensional Difference Gel Electrophoresis
D024101:Mitochondrial Proteins; D018994:Myosin Light Chains; D010750:Phosphoproteins; D048789:Phytoestrogens; D020543:Proteome; D019833:Genistein