title: A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome.
abstract: For fractionation of intact proteins by molecular weight (MW), a sharply improved two-dimensional (2D) separation is presented to drive reproducible and robust fractionation before top-down mass spectrometry of complex mixtures. The "GELFrEE" (i.e., gel-eluted liquid fraction entrapment electrophoresis) approach is implemented by use of Tris-glycine and Tris-tricine gel systems applied to human cytosolic and nuclear extracts from HeLa S3 cells, to achieve a MW-based fractionation of proteins from 5 to >100 kDa in 1 h. For top-down tandem mass spectroscopy (MS/MS) of the low-mass proteome (5-25 kDa), between 5 and 8 gel-elution (GE) fractions are sampled by nanocapillary-LC-MS/MS with 12 or 14.5 tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometers. Single injections give about 40 detectable proteins, about half of which yield automated ProSight identifications. Reproducibility metrics of the system are presented, along with comparative analysis of protein targets in mitotic versus asynchronous cells. We forward this basic 2D approach to facilitate wider implementation of top-down mass spectrometry and a variety of other protein separation and/or characterization approaches.
authors.affiliation: University of Illinois Urbana-Champaign, Department of Chemistry and The Institute for Genomic Biology, Urbana, Illinois 61801, USA.
authors.forename: Ji Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: John F
authors.initials: JF
authors.lastname: Kellie
authors.forename: John C
authors.initials: JC
authors.lastname: Tran
authors.forename: Jeremiah D
authors.initials: JD
authors.lastname: Tipton
authors.forename: Adam D
authors.initials: AD
authors.lastname: Catherman
authors.forename: Haylee M
authors.initials: HM
authors.lastname: Thomas
authors.forename: Dorothy R
authors.initials: DR
authors.lastname: Ahlf
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Durbin
authors.forename: Adaikkalam
authors.initials: A
authors.lastname: Vellaichamy
authors.forename: Ioanna
authors.initials: I
authors.lastname: Ntai
authors.forename: Alan G
authors.initials: AG
authors.lastname: Marshall
authors.forename: Neil L
authors.initials: NL
authors.lastname: Kelleher
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D005591:Chemical Fractionation / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D009363:Neoplasm Proteins / Q000032:analysis*; D020543:Proteome / Q000032:analysis*; D013048:Specimen Handling / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D020543:Proteome