bio sphere
Weighing ribosomes with MS Researchers develop a method to accurately measure the masses of large, noncovalent complexes.
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Intensity
the observed mass spectrum. By examthem to correct for adduct formation ecause they are predicted to tip the ining the relative intensities of the 4 solely on the basis of the average peak scales at molecular masses in the components, the researchers estimated width, without any prior knowledge of megadalton range, intact ribosomes have that ~50% of the 30S ribosomal subthe complex’s composition or structure. been difficult to analyze by MS. Howevunits contained the S1 protein. er, Carol Robinson and colleagues (a) (b) Tandem MS of the 30S subunit at the University of Cambridge confirmed the presence of 2 (U.K.) report a new method for major populations, likely correobtaining accurate mass measuresponding to the intact 30S subments of large, noncovalent comunit and the 30S subunit withplexes, such as ribosomes, with out S1. Finally, the researchers MS ( J. Am. Chem. Soc. 2006, applied these techniques to the 128, 11,433–11,442). analysis of the massive 70S riGentle ionization techniques bosome, a ~2.3-MDa complex such as ESI enable MS analyses m /z composed of a 30S and a 50S of noncovalent complexes by subunit. They observed two preserving native interactions, (a) Simulated spectra for heterogeneous ribosomal populations major populations of 70S ribobut spectra from very large com- were summed (bottom) and shifted (middle) to correct for ion somes, reflecting incomplete plexes are difficult to interpret. adducts. Intensities of simulated spectra were adjusted to fit binding of S1, as seen for the According to Robinson, “We the experimental data (top). (b) X-ray crystal structure of the 30S subunit. knew that we could maintain in- T. thermophilus 70S ribosome. Joseph Loo of the University tact ribosomes in the mass specof California, Los Angeles, comments, Next, Robinson and colleagues adtrometer, but because the peaks in the “The simulation and modeling strategy dressed the issue of protein-complex spectra were always very broad, it was presented in this manuscript is really imheterogeneity with an MS analysis of quite difficult to extract information.” The major causes of peak broadening the Thermus thermophilus 30S ribosomal pressive work, and it opens the door to analyze other large protein machines by subunit (~800 kDa). After correcting are small-molecule adducts and the inMS, such as viruses.” David Muddiman for adducts, the researchers found that complete binding of proteins in a comof North Carolina State University says, the mass of the 30S subunit predicted plex. During ESI, inadequate removal “In the absence of suitable molecularfrom protein and RNA sequences was of solvent or buffer ions can cause a weight standards to validate the method, ~28.5 kDa larger than the measured substantial mass increase and broadenone is forced to take this elegant yet emvalue. X-ray crystal structures had reing of MS peaks, because large protein vealed that T. thermophilus 30S subunits pirical approach to elucidate the masses complexes can form adducts with salt of these challenging systems.” sometimes lack the S1 protein compoions. In addition, substoichiometric Robinson notes that the method alnent. In addition, Robinson’s lab had binding of some proteins in a complex lows researchers to probe stoichiomeshown previously that excitation of the complicates the analysis, because the 30S subunit during MS could cause dis- tries of proteins in a large complex. She spectrum is actually a composite of says, “Typically, if you were examining sociation of the S6 protein. The respectra from two or more heterogethe ribosome by MS, you’d use a prosearchers hypothesized, therefore, that neous populations. The researchers deteomics approach, separating all the the observed mass spectrum could be a vised a way to correct for these factors. proteins out. But then you don’t know The researchers examined adduct for- composite of 4 charge-state series, inthe stoichiometry of the proteins in the cluding contributions from the full promation by using a panel of noncovalent complex.” The method is also indepentein complement and the 30S subunit complexes with known molecular comdent of the structure of the complex without S1, S6, or both components. positions. MS analysis revealed masses and of instrumental parameters. These To test this hypothesis, Robinson that were shifted to higher-than-expectand colleagues generated computer sim- features, along with relative ease of imed values. After studying the peaks, the plementation, enable it to be applied ulations of these charge-state distriburesearchers discovered that each mass to a variety of large, noncovalent tions, then summed the spectra and adshift was linearly correlated with the avcomplexes. a justed the respective intensities. The erage width of the peaks in the mass —Laura Tomky Cassiday resulting simulation coincided well with spectrum. This relationship allowed 7926
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