Abstract
Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.
This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the “Total Protein Approach.”
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Acknowledgments
This work was supported by the Max-Planck Society for the Advancement of Science.
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Wiśniewski, J.R. (2017). Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and “Total Protein Approach”. In: Mokranjac, D., Perocchi, F. (eds) Mitochondria. Methods in Molecular Biology, vol 1567. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6824-4_6
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DOI: https://doi.org/10.1007/978-1-4939-6824-4_6
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