Jeremiah Tipton - A Robust 2-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low Mass Proteome

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  Publication Details (including relevant citation   information):

  Lee, J.E.; Kellie, J.F.; Tran, J.C.; Tipton,   J.D.; Catherman, A.C.; Thomas, H.M.; Ahlf, D.R.; Durbin,   K.R.; Vellaichamy, A.; Ntai, I.; Marshall, A.G.; and Kelleher,   N.L. Journal of the American Society for Mass   Spectrometry, 20 (12), 2183-2191 (2009)


  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.

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