A robust two-dimensional separation for top-down tandem mass spectrometry of the low-mass proteome.
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.
Lee
Ji Eun
JE
University of Illinois Urbana-Champaign, Department of Chemistry and The Institute for Genomic Biology, Urbana, Illinois 61801, USA.
Kellie
John F
JF
Tran
John C
JC
Tipton
Jeremiah D
JD
Catherman
Adam D
AD
Thomas
Haylee M
HM
Ahlf
Dorothy R
DR
Durbin
Kenneth R
KR
Vellaichamy
Adaikkalam
A
Ntai
Ioanna
I
Marshall
Alan G
AG
Kelleher
Neil L
NL
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*
D014408:Biomarkers, Tumor; D009363:Neoplasm Proteins; D020543:Proteome