This work presents a comparative evaluation of several detergent-based sample 
preparation workflows for the MS-based analysis of bacterial proteomes, 
performed using the model organism Escherichia coli. Initially, RapiGest- and 
SDS-based buffers were compared for their protein extraction efficiency and 
quality of the MS data generated. As a result, SDS performed best in terms of 
total protein yields and overall number of MS identifications, mainly due to a 
higher efficiency in extracting high molecular weight (MW) and membrane 
proteins, while RapiGest led to an enrichment in periplasmic and fimbrial 
proteins. Then, SDS extracts underwent five different MS sample preparation 
workflows, including: detergent removal by spin columns followed by in-solution 
digestion (SC), protein precipitation followed by in-solution digestion in 
ammonium bicarbonate or urea buffer, filter-aided sample preparation (FASP), and 
1DE separation followed by in-gel digestion. On the whole, about 1000 proteins 
were identified upon LC-MS/MS analysis of all preparations (>1100 with the SC 
workflow), with FASP producing more identified peptides and a higher mean 
sequence coverage. Each protocol exhibited specific behaviors in terms of MW, 
hydrophobicity, and subcellular localization distribution of the identified 
proteins; a comparative assessment of the different outputs is presented.