A Robust Two-Dimensional Separation for Top-Down Tandem Mass Spectrometry of the Low-Mass Proteome

• Published in: Journal of the American Society for Mass Spectrometry 2009-12, Vol.20 (12), p.2183-2191
• Main Authors: Lee, Ji Eun, Kellie, John F., Tran, John C., Tipton, Jeremiah D., Catherman, Adam D., Thomas, Haylee M., Ahlf, Dorothy R., Durbin, Kenneth R., Vellaichamy, Adaikkalam, Ntai, Ioanna, Marshall, Alan G., Kelleher, Neil L.
• Format: Article
• Language: English
• Subjects: Analytical Chemistry; Analytical, structural and metabolic biochemistry; Bioinformatics; Biological and medical sciences; Biomarkers, Tumor - analysis; Biotechnology; Chemical Fractionation - methods; Chemistry; Chemistry and Materials Science; Cyclotron resonance; Electrophoresis, Gel, Two-Dimensional - methods; Elution; Entrapment; Fourier transforms; Fractionation; Fundamental and applied biological sciences. Psychology; General aspects, investigation methods; Glycine; HeLa Cells; Humans; Ions; Mass spectrometers; Mass spectrometry; Neoplasm Proteins - analysis; Organic Chemistry; Proteins; Proteome - analysis; Proteomics; Reproducibility; Scientific imaging; Separation; Specimen Handling - methods; Spectrometers; Spectroscopy; Spectroscopy, Fourier Transform Infrared - methods
• ISSN: 1044-0305; 1879-1123
• DOI: 10.1016/j.jasms.2009.08.001

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. Molecular weight-based separation of intact proteins coupled to LC-FTMS/MS facilitated low-mass top-down proteomics. A heat map visualization of LC-MS injections is shown.