Wiśniewski Jacek R
Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck Institute for Biochemistry, Am Klopferspitz 18, D-82152, Martinsried, Germany.
Methods Mol Biol. 2017;1567:69-77. doi: 10.1007/978-1-4939-6824-4_6.
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."
确定蛋白质组组成和测量蛋白质丰度变化可为线粒体研究提供具有重要价值的信息。本章介绍了一种线粒体蛋白质组定量分析的工作流程，重点是样品制备和数据定量分析。该工作流程涉及多酶消化-滤膜辅助样品制备（MED-FASP）方案，可实现蛋白质的高效提取和高蛋白质-肽转化率。用Lys C和胰蛋白酶连续进行蛋白质消化能够生成重叠最少的肽段，大大增加已鉴定蛋白质的数量，并扩大其序列覆盖范围。可通过“总蛋白质方法”评估由多个肽段鉴定出的蛋白质的丰度。
