Characterisation of isoform-specific tryptic peptides of rat cardiac myosin heavy chains using automated liquid chromatography-matrix assisted laser desorption ionisation (LC-MALDI) mass spectrometry.
Proteomics investigations using 2-dimensional electrophoresis (2-DE) cannot resolve the entire cardiac proteome because some proteins, including myosin heavy chains (MyHC), are insoluble in the buffers required for isoelectric focusing. Here, we report an automated mass spectrometry (MS) method complementary to 2-DE and capable of yielding important additional information. Rat myocardium was homogenised in standard lysis solution and centrifuged to produce a supernatant fraction, suitable for 2-DE. The pelleted fraction, which is normally discarded, was used for the current analysis. Proteins were digested with trypsin and the peptides fractionated by HPLC. Automated spotting of eluent fractions onto 384-well target plates and matrix-assisted laser desorption tandem time of flight (MALDI-ToF/ToF) MS were directed by dedicated software. Peptide ions were fragmented by collision-induced dissociation and the MS/MS spectra searched against the NCBI database using Mascot. This approach confidently identified 13 tryptic peptides specific to cardiac alpha-MyHC and 4 specific to beta-MyHC, which can be used to differentiate these highly homologous protein isoforms in future quantitative MS analyses.
Burniston
Jatin G
JG
Connolly
Joanne B
JB
D000818:Animals; D001331:Automation; D002851:Chromatography, High Pressure Liquid; D008297:Male; D009206:Myocardium / Q000737:chemistry*; D018995:Myosin Heavy Chains / Q000032:analysis*; D020033:Protein Isoforms; D051381:Rats; D017208:Rats, Wistar; D020539:Sequence Analysis, Protein / Q000295:instrumentation*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D024723:Ventricular Myosins / Q000032:analysis*
D020033:Protein Isoforms; D024723:Ventricular Myosins; D018995:Myosin Heavy Chains