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Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal Standard Mass Spectrometry

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Journal of The American Society for Mass Spectrometry

Abstract

Quantitation of cytochrome c (Cyt c) in cell lysates through surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using gold nanoparticles (Au NPs) as the matrix and GR-10 peptide as an internal standard has been demonstrated. To shorten digestion time, temperature sensitive microgels containing trypsin (TR) and Au NPs have been employed. As-prepared functional microgels (TR/Au NPs/MGs) allow digestion of Cyt c within 15 s under microwave irradiation. The internal standard SALDI-MS approach provides linearity (R2 = 0.98) of MS signal ratio (I 1168.6/I 1067.6) of the tryptic digested peptide (m/z 1168.6) to GR-10 peptide (m/z 1067.6) against the concentration of Cyt c ranging from 25 to 200 nM, with a limit of detection (at a signal-to-noise ratio of 3) of 10 nM. This approach has been validated by the analysis of the lysates of HeLa cells, with an average concentration of 13.7 ± 3.5 μM for cytoplasmic Cyt c. Increased concentrations of Cyt c in the HeLa cells treated with etoposide (a commercial drug) or carbon dots (potential drug) have been revealed through this simple, sensitive, and rapid SALDI-MS approach, supporting the drugs induced Cyt c-mediated apoptosis of the cells. This study has shown that this internal standard SALDI-MS approach holds great potential for cell study.

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Acknowledgments

This study was supported by the National Science Council of Taiwan under contract NSC 101-2113M-002-002-MY3. The authors appreciate the assistance of Ya-Yu Yang from the Instrument Center of National Taiwan University (NTU) for TEM measurement.

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Authors and Affiliations

  1. Department of Chemistry, National Taiwan University, Taipei, 10617, Taiwan

    Li-Yi Chen & Huan-Tsung Chang

  2. Department of Engineering and System Science, National Tsing Hua University, Hsinchu, 30013, Taiwan

    Wei-Cheng Wu

  3. Nano Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei, 11529, Taiwan

    Wei-Cheng Wu

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  1. Li-Yi Chen
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  2. Wei-Cheng Wu
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Correspondence to Huan-Tsung Chang.

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Chen, LY., Wu, WC. & Chang, HT. Functional Microgels Assisted Tryptic Digestion and Quantification of Cytochrome c Through Internal Standard Mass Spectrometry. J. Am. Soc. Mass Spectrom. 25, 1944–1952 (2014). https://doi.org/10.1007/s13361-014-0983-z

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  • DOI: https://doi.org/10.1007/s13361-014-0983-z

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