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Cardiac myofilament isoforms - Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches

ABSTRACT: Purpose: The identification of protein isoforms in complex biological samples is challenging. We, therefore, used a mass spectrometry (MS) approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform. Experimental design: Three different workflows were used to isolate and fractionate the rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were identified using various search engines (Mascot, X!Tandem, X!Tandem Kscore and OMSSA) with results combined via PepArML Meta-Search Engine, and a post-search analysis performed by MASPECTRAS. Results: The combination of multiple workflows and search engines resulted in a larger number of non-redundant proteins identified than individual methods. A total of 461 proteins were observed overlapping in two or three of the workflows. Literature search for myofilament presence with manual validation of the MS spectra was carried out for unambiguous identification: 10 cardiac myofilament and 17 cardiac myofilament-associated proteins were confirmed to have multiple isoforms or sub-isoforms. Conclusion and clinical relevance: We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes of distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets.

INSTRUMENT(S): LTQ Orbitrap

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Cardiac Muscle, Cardiac Muscle Cell

DISEASE(S): Cardiovascular System Disease

SUBMITTER: Vidya Venkatraman

LAB HEAD: Dr. Jennifer E Van Eyk

PROVIDER: PXD000874 | Pride | 2014-08-28

REPOSITORIES: Pride

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			Action	DRS
	050802_VanEykJ_ShewngS_MF01.RAW	Raw
	050802_VanEykJ_ShewngS_MF01.mzxml	Mzxml
	050802_VanEykJ_ShewngS_MF02.RAW	Raw
	050802_VanEykJ_ShewngS_MF02.mzxml	Mzxml
	050802_VanEykJ_ShewngS_MF03.RAW	Raw

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Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches.

Kooij Viola V Venkatraman Vidya V Kirk Jonathan A JA Ubaida-Mohien Ceereena C Graham David R DR Faber Matthijs J MJ Van Eyk Jennifer E JE

Proteomics. Clinical applications 20140801 7-8

<h4>Purpose</h4>The identification of protein isoforms in complex biological samples is challenging. We, therefore, used an MS approach to unambiguously identify cardiac myofilament protein isoforms based on the observation of a tryptic peptide consisting of a sequence unique to a particular isoform.<h4>Experimental design</h4>Three different workflows were used to isolate and fractionate rat cardiac myofilament subproteomes. All fractions were analyzed on an LTQ-Orbitrap MS, proteins were ident ...[more]

PMID: 24974818

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Project description:Background The axon guidance cue Slit2 recently has been found to regulate calcium homeostasis and molecular signaling in various stress events in different organs. However, whether Slit2 plays a role in cardiac ischemia-reperfusion (IR) injury has not been reported. Here, we aimed to investigate the role of Slit2 and the underlying mechanisms in cardiac IR injury. Methods Langendorff-perfused isolated hearts from Slit2-overexpressing (Slit2-Tg) mice and their background strain C57BL/6J mice were subjected to 20 min of global ischemia followed by 40 min of reperfusion. Left ventricular function of isolated hearts was monitored. Infarct size of post-IR hearts was determined by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and histological changes of cardiac tissues and cells were determined with hematoxylin-eosin (HE) staining and transmission electron microscopy. Transcriptomic analysis was used to predict the biological processes and signaling pathways affected by Slit2 overexpression in the post-IR myocardium. Pro-Q staining and Western blotting was used to assess the phosphorylation levels of cardiac myofilaments and expression levels of myofilament-associated protein kinase and phosphatases. Results Slit2 overexpression increased post-IR left ventricular developed pressure (LVDP) by 35% and reduced infarct size by 53%, along with decreased myofibrillar disruption, mitochondrial swelling, and mitochondrial cristae dissolution. Slit2 overexpression significantly changed post-IR gene expression profiles. Functional products of these genes include regulation of cation transmembrane transport, cation homeostasis, collagen fibril organization, and regulation of heart rate. And post-IR myocardial KEGG pathways upregulated by Slit2 overexpression include ECM-receptor interaction, PI3K-Akt signaling pathway, and adrenergic signaling. Slit2 overexpression impacted myofilament phosphorylation together with myofilament-associated protein kinase C (PKC) isoforms and protein phosphatases (PPs). IR in C57BL/6J hearts upregulated phosphorylation of cardiac troponin-I (cTnI), which was suppressed by Slit2 overexpression. Myofilament‐associated PKCε, PKCδ, and PP2A were significantly increased post‐IR in C57BL/6J hearts, but in Slit2‐Tg hearts, myofilament‐associated PKCε and PP2A were increased and PKCδ was suppressed. Conclusions Our results demonstrate that Slit2 overexpression protects cardiac function and reduces IR injury, which is associated with Slit2‐induced gene profile shifts. The suppression of MyBP‐C and troponin‐I phosphorylation, and myofilament‐associated PKCδ levels induced by Slit2 overexpression could contribute to the cardioprotection of Slit2 in post-IR myocardium.