title: Combined cup loading, bis(2-hydroxyethyl) disulfide, and protein precipitation protocols to improve the alkaline proteome of Lactobacillus hilgardii.
abstract: Despite the large number of papers dealing with bacterial proteomes, very few include information about proteins with alkaline pI's, because of the limits inherent in 2-DE technology. Nonetheless, analyses of in silico proteomes of many prokaryotes show a bimodal distribution of their proteins based on their pI's; the most crowded areas lying between pI 4-7 and 9-11. The aim of the present research was to set up a general, simple, and standardizable 2-DE protocol suitable for studying the alkaline proteome of Lactobacillus hilgardii, a Gram-positive bacillus isolated from wine. The method has also been tested on a Gram-negative bacterium able to degrade aromatic pollutants, Acinetobacter radioresistens S13. Optimization of the method was mainly focused on improving protein extraction and IEF (pI 6-11) separation protocols. Concerning IEF, different methods for sample loading (in-gel rehydration and cup loading), and different reducing agents (DTT and bis(2-hydroxyethyl) disulfide (HED)) were tested and compared. The proposed protocol was found to resolve efficiently alkaline proteins from both of our Lactobacillus and Acinetobacter strains, in spite of their different external layers, thus, enabling a more comprehensive study of their proteomes.
authors.affiliation: Dipartimento di Biologia Animale e dell'Uomo, Università di Torino, Torino, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Lamberti
authors.forename: Enrica
authors.initials: E
authors.lastname: Pessione
authors.forename: Maria G
authors.initials: MG
authors.lastname: Giuffrida
authors.forename: Roberto
authors.initials: R
authors.lastname: Mazzoli
authors.forename: Cristina
authors.initials: C
authors.lastname: Barello
authors.forename: Amedeo
authors.initials: A
authors.lastname: Conti
authors.forename: Carlo
authors.initials: C
authors.lastname: Giunta
mesh_terms: D000150:Acinetobacter / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D002457:Cell Extracts / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D000431:Ethanol / Q000031:analogs & derivatives* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D007526:Isoelectric Point; D007778:Lactobacillus / Q000737:chemistry* / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin / Q000378:metabolism; D014920:Wine / Q000382:microbiology
chemical_list: D001426:Bacterial Proteins; D002457:Cell Extracts; D004220:Disulfides; D000431:Ethanol; C031319:2-hydroxyethyl disulfide; D014357:Trypsin