Upping the mass limit of top-down proteomics

Top-down protein mass spectrometry permits proteins to be analyzed by fragmenting the intact protein in the mass analyzer rather than first digesting the protein with an enzyme. So far, the top-down approach has been limited to proteins with masses up to about 75 kilodaltons. Fred W. McLafferty of Cornell University and his coworkers there and at the University of Innsbruck, Austria, have now pushed that mass limit beyond 200 kDa (Science 2006, 314, 109). They extended the mass limit by preventing protein folding, which is accomplished by using additives in the electrospray solution, heating the inlet capillary, and adjusting the ion acceleration voltages so that noncovalent and covalent bonds are cleaved immediately after electrospray ion introduction. The researchers used this method, which they call "prefolding dissociation" because it disrupts intramolecular interactions involved in protein folding, to analyze 144-, 200-, and 229-kDa proteins. The spectra allowed them to correct the amino acid sequence predictions for the 144-kDa protein (formylglycinamide ribonucleotide amidotransferase) and identify eight previously unknown disulfide bonds in the 200-kDa protein (human complement C4 glycoprotein).