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Molecular & Cellular Proteomics 6:305-318, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

A Method for Automatically Interpreting Mass Spectra of ¹⁸O-Labeled Isotopic Clusters^*,S

Christopher J. Mason, Terry M. Therneau, Jeanette E. Eckel-Passow, Kenneth L. Johnson, Ann L. Oberg, Janet E. Olson^¶, K. Sreekumaran Nair^||, David C. Muddiman^** and H. Robert Bergen, III^,

From the Mayo Proteomics Research Center, Divisions of Biostatistics and ^¶ Epidemiology, and ^|| Department of Endocrinology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905 and ^** Department of Chemistry, W. M. Keck FT-ICR Mass Spectrometry Laboratory, North Carolina State University, Raleigh, North Carolina 27695

¹⁶O/¹⁸O labeling is one differential proteomics technology among many that promises diagnostic and prognostic biomarkers of disease. Although the incorporation of ¹⁸O in the C-terminal carboxyl group during endoproteinase digestion in the presence of H₂¹⁸O makes the process of labeling facile, the ease and effectiveness of label incorporation have in some regards been outweighed by the difficulties in interpreting the resulting spectra. Complex isotope patterns result from the composition of unlabeled (¹⁸O₀), singly labeled (¹⁸O₁), and doubly labeled species (¹⁸O₂) as well as contributions from the naturally occurring isotopes (e.g. ¹³C and ¹⁵N). Moreover because labeling is enzymatic, the number of ¹⁸O atoms incorporated can vary from peptide to peptide. Finally it is difficult to distinguish highly up-regulated from highly down-regulated or C-terminal peptides. We have developed an algorithm entitled regression analysis applied to mass spectrometry (RAAMS) that automatically, rapidly, and confidently interprets spectra of ¹⁸O-labeled peptides without requiring chemical composition information derived from product ion spectra. The algorithm is able to measure the effective ¹⁸O incorporation rate due to variable enzyme substrate specificity of the pseudosubstrate during the isotope exchange reaction and corrects for the ¹⁸O₀ abundance that remains in the labeled sample when using a two-step digestion/labeling procedure. We have also incorporated a method for distinguishing pure ¹⁸O₀ from pure ¹⁸O₂ peptides utilizing impure H₂¹⁸O. The algorithm operates on centroided peak lists and is therefore very fast: nine chromatograms of, on average, 1,168 spectra and containing, on average, 6,761 isotopic clusters were interpreted in, on average, 45 s per chromatogram. RAAMS is fast enough (average, 38 ms/spectrum) to allow the possibility of performing information-dependent MS/MS on a chromatographic time scale on species exceeding predetermined ratio thresholds. We describe in detail the operation of the algorithm and demonstrate its use on datasets with known and unknown ratios.

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