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.