title: Coupling a detergent lysis/cleanup methodology with intact protein fractionation for enhanced proteome characterization.
abstract: The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples ( Escherichia coli  K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.
authors.affiliation: UT-ORNL Graduate School of Genome Science and Technology, University of Tennessee, Knoxville-Tennessee 37996, United States.
authors.forename: Ritin
authors.initials: R
authors.lastname: Sharma
authors.forename: Brian D
authors.initials: BD
authors.lastname: Dill
authors.forename: Karuna
authors.initials: K
authors.lastname: Chourey
authors.forename: Manesh
authors.initials: M
authors.lastname: Shah
authors.forename: Nathan C
authors.initials: NC
authors.lastname: VerBerkmoes
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D002725:Chloroform / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000737:chemistry*; D048168:Escherichia coli K12 / Q000737:chemistry; D060587:Groundwater / Q000382:microbiology; D000432:Methanol / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D016958:Pseudomonas putida / Q000737:chemistry; D012680:Sensitivity and Specificity; D020614:Shewanella putrefaciens / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012996:Solutions / Q000737:chemistry; D053719:Tandem Mass Spectrometry / Q000379:methods; D014238:Trichloroacetic Acid / Q000737:chemistry; D014871:Water Microbiology
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D020543:Proteome; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D014238:Trichloroacetic Acid; D002725:Chloroform; D000432:Methanol