title: Comparison of detergent-based sample preparation workflows for LTQ-Orbitrap analysis of the Escherichia coli proteome.
abstract: 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.
authors.affiliation: Porto Conte Ricerche, Tramariglio, Alghero, Italy; Dipartimento di Scienze Biomediche, Università di Sassari, Sassari, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Tanca
authors.forename: Grazia
authors.initials: G
authors.lastname: Biosa
authors.forename: Daniela
authors.initials: D
authors.lastname: Pagnozzi
authors.forename: Maria Filippa
authors.initials: MF
authors.lastname: Addis
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D013048:Specimen Handling / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D057188:Workflow
chemical_list: D003902:Detergents; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D020543:Proteome
keywords: FASP; Gram-negative bacteria; Microbiology; RapiGest; SDS; Shotgun proteomics