Simultaneous Quantification of Human Cardiac α- and β-Myosin Heavy Chain Proteins by MALDI-TOF Mass Spectrometry
| Autor: | Chia-Yu Yen, Steve M. Helmke, Karin Nunley, Krzysztof J. Cios, Mark W. Duncan, M. Benjamin Perryman, Michael R. Bristow |
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| Rok vydání: | 2004 |
| Předmět: |
Protein isoform
chemistry.chemical_classification Chromatography Myosin Heavy Chains Chemistry Calibration curve Myocardium Analytical chemistry Peptide macromolecular substances musculoskeletal system Mass spectrometry Analytical Chemistry Ventricular Myosins Standard curve Matrix-assisted laser desorption/ionization Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization Humans Protein Isoforms Beta protein Peptides Quantitative analysis (chemistry) |
| Zdroj: | Analytical Chemistry. 76:1683-1689 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | We have developed a novel method for quantifying protein isoforms, in both relative and absolute terms, based on MALDI-TOF mass spectrometry. The utility of the approach is demonstrated by quantifying the alpha and beta protein isoforms of myosin heavy chain (MyHC) in human atrial tissue. Alpha-MyHC (726-741) and beta-MyHC (724-739) were identified as isoform-specific tryptic peptides. A calibration curve was constructed by plotting ion current ratios against molar ratios of the two peptides prepared synthetically. MyHC was digested by trypsin and the ion current ratio determined for the two tryptic peptides. The ion current ratio was converted to the peptide ratio and hence the isoform ratio by reference to the standard curve. The accuracy of the method was confirmed by a comparison between these results and those determined by an established method of MyHC isoform ratio determination. So that the molar ratio could be converted to absolute values, a third peptide, an analogue of the two peptides being measured, was synthesized for use as an internal standard (IS). The measured ion current ratios of synthetic alpha-MyHC (726-741), beta-MyHC (724-739), and IS peptides were used to generate standard curves. A known quantity of the IS was added to the MyHC digests. The measured ion current ratios were converted to the actual quantities of the isoform-specific peptides and hence the actual quantity of each protein isoform by reference to the standard curves. This method is of general applicability, especially when isoform quantification is required. |
| Databáze: | OpenAIRE |
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