title: Critical comparison of multidimensional separation methods for increasing protein expression coverage.
abstract: We present a comparison of two-dimensional separation methods and how they affect the degree of coverage of protein expression in complex mixtures. We investigated the relative merits of various protein and peptide separations prior to acidic reversed-phase chromatography directly coupled to an ion trap mass spectrometer. The first dimensions investigated were density gradient organelle fractionation of cell extracts, 1D SDS-PAGE protein separation followed by digestion by trypsin or GluC proteases, strong cation exchange chromatography, and off-gel isoelectric focusing of tryptic peptides. The number of fractions from each first dimension and the total data accumulation RP-HPLC-MS/MS time was kept constant and the experiments were run in triplicate. We find that the most critical parameters are the data accumulation time, which defines the level of under-sampling and the avoidance of peptides from high expression level proteins eluting over the entire gradient.
authors.affiliation: Protein Technology, Department of Immunotechnology, CREATE Health, Lund University, Sweden.
authors.forename: Linn
authors.initials: L
authors.lastname: Antberg
authors.forename: Paolo
authors.initials: P
authors.lastname: Cifani
authors.forename: Marianne
authors.initials: M
authors.lastname: Sandin
authors.forename: Fredrik
authors.initials: F
authors.lastname: Levander
authors.forename: Peter
authors.initials: P
authors.lastname: James
mesh_terms: D000143:Acids / Q000737:chemistry; D002458:Cell Fractionation / Q000379:methods*; D045744:Cell Line, Tumor; D002499:Centrifugation, Density Gradient; D002852:Chromatography, Ion Exchange; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D013058:Mass Spectrometry; D015388:Organelles / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*; D059748:Proteolysis; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
chemical_list: D000143:Acids; D010455:Peptides; D020543:Proteome; D014357:Trypsin