Top-down MS, a powerful complement to the high capabilities of proteolysis proteomics. Academic Article uri icon

abstract

  • For the characterization of protein sequences and post-translational modifications by MS, the 'top-down' proteomics approach utilizes molecular and fragment ion mass data obtained by ionizing and dissociating a protein in the mass spectrometer. This requires more complex instrumentation and methodology than the far more widely used 'bottom-up' approach, which instead uses such data of peptides from the protein's digestion, but the top-down data are far more specific. The ESI MS spectrum of a 14 protein mixture provides full separation of its molecular ions for MS/MS dissociation of the individual components. False-positive rates for the identification of proteins are far lower with the top-down approach, and quantitation of multiply modified isomers is more efficient. Bottom-up proteolysis destroys the information on the size of the protein and the connectivities of the peptide fragments, but it has no size limit for protein digestion. In contrast, the top-down approach has a approximately 500 residue, approximately 50 kDa limitation for the extensive molecular ion dissociation required. Basic studies indicate that this molecular ion intractability arises from greatly strengthened electrostatic interactions, such as hydrogen bonding, in the gas-phase molecular ions. This limit is now greatly extended by variable thermal and collisional activation just after electrospray ('prefolding dissociation'). This process can cleave 287 inter-residue bonds in the termini of a 1314 residue (144 kDa) protein, specify previously unidentified disulfide bonds between eight of 27 cysteines in a 1714 residue (200 kDa) protein, and correct sequence predictions in two proteins, one of 2153 residues (229 kDa).

published proceedings

  • FEBS J

author list (cited authors)

  • McLafferty, F. W., Breuker, K., Jin, M. i., Han, X., Infusini, G., Jiang, H., Kong, X., & Begley, T. P.

citation count

  • 149

complete list of authors

  • McLafferty, Fred W||Breuker, Kathrin||Jin, Mi||Han, Xuemei||Infusini, Giuseppe||Jiang, Honghai||Kong, Xianglei||Begley, Tadhg P

publication date

  • December 2007

publisher

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