title: Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".
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."
authors.affiliation: Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany. jwisniew@biochem.mpg.de.
authors.forename: Jacek R
authors.initials: JR
authors.lastname: Wiśniewski
mesh_terms: D002853:Chromatography, Liquid; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D057188:Workflow
chemical_list: D024101:Mitochondrial Proteins; D010455:Peptides; D020543:Proteome
keywords: Absolute protein quantitation; Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP); Proteomic sample preparation; WF-assay; “Total Protein Approach”