title: Parallel electrophoretic depletion, fractionation, concentration, and desalting of 96 complex biological samples for mass spectrometry.
abstract: The preparation of complex biological samples for high-throughput mass spectrometric analyses remains a significant bottleneck, limiting advancement of the capabilities of mass spectrometry (MS) and ultimately limiting development of novel clinical assays. The removal of interfering species (e.g., salts, detergents, and buffers), concentration of dilute analytes, and the reduction of sample complexity are required in order to maximize the quality of resultant MS data. This study describes a novel sample preparation method that makes use of electrophoresis to prepare complex biological samples for high-throughput MS analysis. The method provides for integration of key sample preparation steps, including depletion, fractionation, desalting, and concentration. The prepared samples are captured onto a monolithic reversed-phase capture target that can be analyzed directly by a mass spectrometer. Up to 96 individual samples are simultaneously prepared for MS analysis in under 1 h. For standard proteins added to serum, this method provides femtomole level sensitivity and reproducible label-free detection (coefficient of variation <30%). This study demonstrates that this electrophoretic sample preparation system permits high-throughput sample preparation for mass spectrometric analysis of complex biological samples, such as serum, plasma, and tissue extracts.
authors.affiliation: Protein Discovery, Incorporated, 418 South Gay Street, Suite 203, Knoxville, Tennessee 37902, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Harkins
authors.forename: Benjamin B
authors.initials: BB
authors.lastname: Katz
authors.forename: Salvador J
authors.initials: SJ
authors.lastname: Pastor
authors.forename: Peter
authors.initials: P
authors.lastname: Osucha
authors.forename: Dean G
authors.initials: DG
authors.lastname: Hafeman
authors.forename: Charles E
authors.initials: CE
authors.lastname: Witkowski
authors.forename: Jeremy L
authors.initials: JL
authors.lastname: Norris
mesh_terms: D000818:Animals; D001798:Blood Proteins / Q000032:analysis; D053497:Corticotropin-Like Intermediate Lobe Peptide / Q000032:analysis; D004586:Electrophoresis / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D008099:Liver / Q000737:chemistry; D051379:Mice; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014020:Tissue Extracts / Q000032:analysis
chemical_list: D001798:Blood Proteins; D011506:Proteins; D020543:Proteome; D014020:Tissue Extracts; D012710:Serum Albumin, Bovine; D053497:Corticotropin-Like Intermediate Lobe Peptide