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A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue

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Abstract

A label-free absolute quantitation method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been developed. This methodology was applied to mouse heart in order to quantify cardiac troponin T (cTnT), which is considered to be a sensitive marker of heart damage. The cTnT was extracted, isolated by reversed-phase high-performance liquid chromatography, digested, and analyzed by MALDI-TOF MS. The MS-based quantitation was performed using matrix-matched calibration curves (due to a matrix effect) of two synthetic peptides, one cTnT-specific peptide and one internal standard peptide, respectively. Recoveries at three spiking levels ranged from 87–96%, with relative standard deviations of below 10%. The method detection limit and the method quantitation limit, expressed as the amount of cTnT for the amount of total sarcomeric protein extract, were 0.03 mg g−1 and 0.15 mg g−1, respectively. This method appears to be accurate and generally suitable for improving absolute protein quantitation.

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Acknowledgements

The authors thank the Italian Department of University and Research for funding this work in the framework of the “Improvements in proteomics methodologies programme (PRIN 2006 project), and the Italian Space Agency (ASI) for supporting this research in the “Advanced genomics, metabolomics and proteomic studies in space” (AGEMPROS) project.

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Authors and Affiliations

  1. Department of Experimental Pathology, “La Sapienza” University, via Scarpa 16, 00161, Roma, Italy

    Mariano Bizzarri

  2. Department of Chemistry, “La Sapienza” University, Piazzale Aldo Moro 5, 00185, Roma, Italy

    Chiara Cavaliere, Patrizia Foglia, Chiara Guarino, Roberto Samperi & Aldo Laganà

  3. Dipartimento di Chimica, Università “La Sapienza”, Box n° 34 - Roma 62, Piazzale Aldo Moro 5, 00185, Roma, Italy

    Aldo Laganà

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  1. Mariano Bizzarri
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  2. Chiara Cavaliere
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  3. Patrizia Foglia
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  4. Chiara Guarino
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  5. Roberto Samperi
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  6. Aldo Laganà
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Correspondence to Aldo Laganà.

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Bizzarri, M., Cavaliere, C., Foglia, P. et al. A label-free method based on MALDI-TOF mass spectrometry for the absolute quantitation of troponin T in mouse cardiac tissue. Anal Bioanal Chem 391, 1969–1976 (2008). https://doi.org/10.1007/s00216-008-2113-x

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  • DOI: https://doi.org/10.1007/s00216-008-2113-x

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