title: A cost-benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples.
abstract: Affinity purification coupled to mass spectrometry (AP-MS) is gaining widespread use for the identification of protein-protein interactions. It is unclear, however, whether typical AP sample complexity is limiting for the identification of all protein components using standard one-dimensional LC-MS/MS. Multidimensional sample separation is useful for reducing sample complexity prior to MS analysis and increases peptide and protein coverage of complex samples. Here, we monitored the effects of upstream protein or peptide separation techniques on typical mammalian AP-MS samples, generated by FLAG affinity purification of four baits with different biological functions and/or subcellular distribution. As a first separation step, we employed SDS-PAGE, strong cation exchange LC, or reversed-phase LC at basic pH. We also analyzed the benefits of using an instrument with a faster scan rate, the new TripleTOF 5600 mass spectrometer. While all multidimensional approaches yielded a clear increase in spectral counts, the increase in unique peptides and additional protein identification was modest and came at the cost of increased instrument and handling time. The use of a high duty-cycle instrument achieved similar benefits without these drawbacks. An increase in spectral counts is beneficial when data analysis methods relying on spectral counts, including Significance Analysis of INTeractome (SAINT), are used.
authors.affiliation: Centre for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, Ontario, Canada.
authors.forename: Wade H
authors.initials: WH
authors.lastname: Dunham
authors.forename: Brett
authors.initials: B
authors.lastname: Larsen
authors.forename: Stephen
authors.initials: S
authors.lastname: Tate
authors.forename: Beatriz Gonzalez
authors.initials: BG
authors.lastname: Badillo
authors.forename: Marilyn
authors.initials: M
authors.lastname: Goudreault
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Tehami
authors.forename: Thomas
authors.initials: T
authors.lastname: Kislinger
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gingras
mesh_terms: D002460:Cell Line; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000191:economics / Q000379:methods; D003362:Cost-Benefit Analysis; D006801:Humans; D013058:Mass Spectrometry / Q000191:economics / Q000379:methods; D011506:Proteins / Q000032:analysis; D040901:Proteomics / Q000191:economics / Q000379:methods
chemical_list: D011506:Proteins