title: Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes.
abstract: In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.
authors.affiliation: Biomedical Proteomics Research Group, Department of Structural Biology and Bioinformatics, Geneva University, 1 Michel Servet, Geneva, Switzerland.
authors.forename: Ali R
authors.initials: AR
authors.lastname: Vaezzadeh
authors.forename: Jacques M P
authors.initials: JM
authors.lastname: Deshusses
authors.forename: Patrice
authors.initials: P
authors.lastname: Waridel
authors.forename: Patrice
authors.initials: P
authors.lastname: François
authors.forename: Catherine G
authors.initials: CG
authors.lastname: Zimmermann-Ivol
authors.forename: Pierre
authors.initials: P
authors.lastname: Lescuyer
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
authors.forename: Denis F
authors.initials: DF
authors.lastname: Hochstrasser
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D006868:Hydrolysis; D008872:Microwaves; D020543:Proteome / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D013211:Staphylococcus aureus / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D014357:Trypsin