Detection and Selective Dissociation of Intact Ribosomes in a Mass Spectrometer

Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2000-05, Vol.97 (10), p.5185-5190
Main Authors: Rostom, Adam A., Fucini, Paola, Benjamin, Dennis R., Juenemann, Ralf, Nierhaus, Knud H., Hartl, F. Ulrich, Dobson, Christopher M., Robinson, Carol V.
Format: Article
Language:English
Subjects:
Average linear density
Bacteria
Biochemistry
Biological Sciences
Cross-Linking Reagents
Escherichia coli
Escherichia coli - ultrastructure
Ions
Magnesium - pharmacology
Mass spectrometers
Mass Spectrometry - methods
Mass spectroscopy
Molecular Weight
Molecules
Particle collisions
Ribosomal proteins
Ribosomal Proteins - chemistry
ribosomal subunit 30S
ribosomal subunit 50S
Ribosomes
Ribosomes - chemistry
Ribosomes - drug effects
Ribosomes - ultrastructure
RNA
RNA, Ribosomal - chemistry
rRNA
Vapor phases
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Summary:Intact Escherichia coli ribosomes have been projected into the gas phase of a mass spectrometer by means of nanoflow electrospray techniques. Species with mass/charge ratios in excess of 20,000 were detected at the level of individual ions by using time-of-flight analysis. Once in the gas phase the stability of intact ribosomes was investigated and found to increase as a result of cross-linking ribosomal proteins to the rRNA. By lowering the Mg2+concentration in solutions containing ribosomes the particles were found to dissociate into 30S and 50S subunits. The resolution of the charge states in the spectrum of the 30S subunit enabled its mass to be determined as 852,187 ± 3,918 Da, a value within 0.6% of that calculated from the individual proteins and the 16S RNA. Further dissociation into smaller macromolecular complexes and then individual proteins could be induced by subjecting the particles to increasingly energetic gas phase collisions. The ease with which proteins dissociated from the intact species was found to be related to their known interactions in the ribosome particle. The results show that emerging mass spectrometric techniques can be used to characterize a fully functional biological assembly as well as its isolated components.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.97.10.5185