title: Identification of cardiac myofilament protein isoforms using multiple mass spectrometry based approaches.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  Three different workflows were used to isolate and fractionate 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 postsearch analysis was performed by MASPECTRAS. All MS data have been deposited in the ProteomeXchange with identifier PXD000874 (http://proteomecentral.proteomexchange.org/dataset/PXD000874).
  
  
  RESULTS
  The combination of multiple workflows and search engines resulted in a larger number of nonredundant proteins identified than with individual methods. A total of 102 myofilament annotated 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: ten cardiac myofilament and 17 cardiac myofilament-associated proteins were identified with 39 isoforms and subisoforms.
  
  
  CONCLUSIONS
  We have identified multiple isoforms of myofilament proteins that are present in cardiac tissue using unique tryptic peptides. Changes in distribution of these protein isoforms under pathological conditions could ultimately allow for clinical diagnostics or as therapeutic targets.
authors: Department of medicine, Division of Cardiology, The Johns Hopkins University, Baltimore, USA.
authors: Viola
authors: V
authors: Kooij
authors: Department of medicine, Division of Cardiology, The Johns Hopkins University, Baltimore, USA.|Advanced Clinical Biosystems Research Institute, Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, USA.
authors: Vidya
authors: V
authors: Venkatraman
authors: Department of medicine, Division of Cardiology, The Johns Hopkins University, Baltimore, USA.
authors: Jonathan A
authors: JA
authors: Kirk
authors: Department of Molecular and Comparative Pathobiology, The Johns Hopkins University, Baltimore, MD, USA.
authors: Ceereena
authors: C
authors: Ubaida-Mohien
authors: Department of medicine, Division of Cardiology, The Johns Hopkins University, Baltimore, USA.|Department of Molecular and Comparative Pathobiology, The Johns Hopkins University, Baltimore, MD, USA.
authors: David R
authors: DR
authors: Graham
authors: Erasmus MC-Sophia, Department of Pediatrics, Division of Pediatric Cardiology, Rotterdam, The Netherlands.
authors: Matthijs J
authors: MJ
authors: Faber
authors: Department of medicine, Division of Cardiology, The Johns Hopkins University, Baltimore, USA.|Advanced Clinical Biosystems Research Institute, Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, USA.
authors: Jennifer E
authors: JE
authors: Van Eyk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009210:Myofibrils / Q000378:metabolism*; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000378:metabolism
chemical_list: D020033:Protein Isoforms; D014357:Trypsin
keywords: Cardiac; Myofilament proteins; Protein isoforms; Protein subisoforms