Quantitative Assessment of In-solution Digestion Efficiency Identifies Optimal Protocols for Unbiased Protein Analysis
Autor: | Ole N. Jensen, Ileana R. León, Richard R. Sprenger, Veit Schwämmle |
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Rok vydání: | 2013 |
Předmět: |
Proteomics
Protein digestion Proteolysis Quantitative proteomics Mitochondria Liver Tandem mass spectrometry Biochemistry Analytical Chemistry Surface-Active Agents Protein sequencing Tandem Mass Spectrometry medicine Animals Urea Trypsin Denaturation (biochemistry) Sample preparation Molecular Biology Chromatography medicine.diagnostic_test Chemistry Technological Innovation and Resources Proteins Sodium Dodecyl Sulfate Rats Solutions Chromatography Liquid Deoxycholic Acid medicine.drug |
Zdroj: | Molecular & Cellular Proteomics. 12:2992-3005 |
ISSN: | 1535-9476 |
Popis: | The majority of mass spectrometry-based protein quantification studies uses peptide-centric analytical methods and thus strongly relies on efficient and unbiased protein digestion protocols for sample preparation. We present a novel objective approach to assess protein digestion efficiency using a combination of qualitative and quantitative liquid chromatography-tandem MS methods and statistical data analysis. In contrast to previous studies we employed both standard qualitative as well as data-independent quantitative workflows to systematically assess trypsin digestion efficiency and bias using mitochondrial protein fractions. We evaluated nine trypsin-based digestion protocols, based on standard in-solution or on spin filter-aided digestion, including new optimized protocols. We investigated various reagents for protein solubilization and denaturation (dodecyl sulfate, deoxycholate, urea), several trypsin digestion conditions (buffer, RapiGest, deoxycholate, urea), and two methods for removal of detergents before analysis of peptides (acid precipitation or phase separation with ethyl acetate). Our data-independent quantitative liquid chromatography-tandem MS workflow quantified over 3700 distinct peptides with 96% completeness between all protocols and replicates, with an average 40% protein sequence coverage and an average of 11 peptides identified per protein. Systematic quantitative and statistical analysis of physicochemical parameters demonstrated that deoxycholate-assisted in-solution digestion combined with phase transfer allows for efficient, unbiased generation and recovery of peptides from all protein classes, including membrane proteins. This deoxycholate-assisted protocol was also optimal for spin filter-aided digestions as compared with existing methods. |
Databáze: | OpenAIRE |
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