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.
Original language | English (US) |
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Pages (from-to) | 2183-2191 |
Number of pages | 9 |
Journal | Journal of the American Society for Mass Spectrometry |
Volume | 20 |
Issue number | 12 |
DOIs | |
State | Published - Dec 2009 |
Funding
The authors thank Christopher L. Hendrickson, Mark R. Emmett, John P. Quinn, and Greg T. Blakney of the National High Magnetic Field Laboratory for helpful advice and discussion, and the National Science Foundation (DMR-06-54118) for supporting this national facility. The authors also thank Mingxi Li for helping with sample preparations. The laboratory of NLK was supported by the Packard Foundation, the Sloan Foundation, and the National Institutes of Health (GM-067193-07) and JL was supported by the UIUC Neuroproteomics Center on Cell-Cell Signaling supported by the National Institute on Drug Abuse (P30 DA-018310-06).
ASJC Scopus subject areas
- Structural Biology
- Spectroscopy