Use of cumulative distribution functions to characterize mass spectra of intact proteins |
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Authors: | Paul S. Blank Christin M. Sjomeling Peter S. Backlund Alfred L. Yergey |
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Affiliation: | Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA. |
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Abstract: | The MH+ ions of matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) spectra for a series of closely related but otherwise indistinguishable proteins were analyzed for singularity using a distribution free statistic, the Kolmogorov-Smirnov non-parametric statistic, K-S. The approach allows spectra which might otherwise be taken as identical, to be distinguished. Such analysis of the spectra may lead to a greater understanding of the chemistry of the proteins under investigation. The analysis requires only standard instrumentation. A standard data analysis protocol was developed and applied to generate a normalized cumulative distribution function (NCDF) for each spectrum. Differences in the NCDF for two different spectra were calculated and the maximum difference, deltamax compared to critical values of K-S. Values of deltamax exceeding the critical value of K-S are taken as the basis for rejecting the statistical null-hypothesis and assigning statistical significance to the differences in the two spectra. We have shown that this approach allows spectra of 1:1 mixtures of closely related recombinant proteins to be distinguished from either protein alone, and that mixtures of a 45 kDa protein and a labeled version of that protein can be distinguished from the pure material and from one another at the level of about 25%. In addition, we are able to use this approach to characterize the extent to which a synthetic glyococonjugation reaction has proceeded under circumstances of differing reaction times. |
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