Top-down identification and characterization of biomolecules by mass spectrometry.
The most widely used modern mass spectrometers face severe performance limitations with molecules larger than a few kDa. For far larger biomolecules, a common practice has been to break these up chemically or enzymatically into fragments that are sufficiently small for the instrumentation available. With its many sophisticated recent enhancements, this "bottom-up" approach has proved highly valuable, such as for the rapid, routine identification and quantitation of DNA-predicted proteins in complex mixtures. Characterization of smaller molecules, however, has always measured the mass of the molecule and then that of its fragments. This "top-down" approach has been made possible for direct analysis of large biomolecules by the uniquely high (>10(5)) mass resolving power and accuracy ( approximately 1 ppm) of the Fourier-transform mass spectrometer. For complex mixtures, isolation of a single component's molecular ions for MS/MS not only gives biomolecule identifications of far higher reliability, but directly characterizes sequence errors and post-translational modifications. Protein sizes amenable for current MS/MS instrumentation are increased by a "middle-down" approach in which limited proteolysis forms large (e.g., 10 kDa) polypeptides that are then subjected to the top-down approach, or by "prefolding dissociation." The latter, which extends characterization to proteins >200 kDa, was made possible by greater understanding of how molecular ion tertiary structure evolves in the gas phase.
Institute of Organic Chemistry and Center for Molecular Biosciences Innsbruck, University of Innsbruck, Innsbruck, Austria.
Kathrin
K
Breuker
Mi
M
Jin
Xuemei
X
Han
Honghai
H
Jiang
Fred W
FW
McLafferty
D000818:Animals; D004583:Electrons; D005740:Gases / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000639:trends*; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*
D005740:Gases; D011506:Proteins