Nitric oxide (NO) acts mainly via the cGMP/PKG signaling pathway leading to protein phosphorylation and muscle relaxation. To explore the pathophysiological significance of NO derived from inducible nitric oxide synthase (iNOS) in the heart, we compared the cardiac phosphoproteome of isolated hearts of double transgenic mice with cardiac specific iNOS-overexpression in a myoglobin deficient background (iNOS+/Myo-/-) pefused with and without L-arginine.

Stable isotope dimethyl labeling of primary amino groups of tryptic peptides was applied for mass spectrometry (MS) based gel-free relative quantification. Phosphorylated peptides were enriched on titanium dioxide (TiO₂), further separated by strong cation exchange (SCX) chromatography and measured with a nano-flow reverse phase liquid chromatography coupled tandem mass spectrometer. Peptides identified via MASCOT database search were quantified using MSQuant. Additionally, regulated peptides were manually validated based on the relative ratio of integrated peak area of stable isotope dimethyl labeled peptides.

The method was validated using the standard phosphoprotein casein and showed excellent linearity over a wide range. Spiked casein and remaining not phosphorylated peptide pairs of heart samples served as an internal standard and showed a one to one ratio in each SCX fraction. Hence, differences in phosphopeptide ratios are due to changes in phosphorylation levels induced by in vivo NO formation.

Using LTQ Orbitrap XL we were able to identify 128 phosphorylation sites (45 novel) on 105 cardiac phosphoproteins from as little as 200 mg cytosolic protein fraction. Starting with a higher amount of sample, the number of identified phosphopeptides was 753 (453 proteins, 38% novel phosphoproteins). As expected, most of the identified phosphorylation sites were not influenced by iNOS derived NO, however, 8% and 2.7% were found to be up or downregulated, respectively. These proteins are cardiac contractile proteins (myosin regulatory polypeptide 7, myosin binding protein c), sarcomeric proteins (titin), and calmodulin binding proteins (calnexin). Additionaly, proteins involved in calcium ion homeostasis (phospholamban, ryanodine receptor) were altered, which are known targets of NO-induced phosphorylation. Interestingly, there are also enzymes (pyruvate dehydrogenase,H⁺ transporting mitochondrial ATP synthase F1) as well as transcriptional regulators (histone deacetylase 4), many kinases (mitogen activated protein kinase kinase kinase 3) and some phosphatases that were altered.

We have elaborated a new method to study global differences in the cardiac phosphoproteome. The results suggest that signal transduction of NO in the failing heart is very likely more complex than previously thought. The physiological relevance of some of the newly detected phosphorylation sites remain to be investigated.