Statistical Study of Ions and Peptides Found in LC/MS, LC/MSE Analysis of Human Serum Digest

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Marc V. Gorenstein, Guo-Zhong Li, Scott Geromanos, Jeffrey C. Silva, Craig A. Dorschel, Robert S. Plumb, Chris L. Stumpf, and Timothy Riley Waters
Waters ASMS 2004 poster
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Data-analysis algorithms are critical to the success of proteomic investigations. This paper describes, first, two algorithms and their application to LC/MS data. It then describes the application of these algorithms to a novel, LC/MS-based, proteomics method. The first data-analysis algorithm detects the ions obtained in an LC/MS separation. This convolution-based technique is designed to measure three key properties of each ion: retention time, mass-to-charge ratio, and intensity. The second algorithm simplifies spectra by selecting only those ions whose retention times fall within restricted ranges. As an example, consider a peptide that elutes at retention time. All its ions must also elute at tr. Variations from are due only to measurement error. By selecting ions that have the same retention time (to within measurement error), the algorithm simplifies spectra. Such simplified spectra can more clearly reveal the unique, multi-ion signature of peptides. Both algorithms are applied to a novel, LC/MS-based, exact-mass, proteomics method.1,2,3 This method collects spectra that alternate uniformly in time between a (low-energy) MS-mode and an (elevated-energy) MSE–mode. There is no MS spectral selection applied prior to MSE fragmentation. Thus, the MSE spectra contain fragment ions of all precursor peptides. Rather than relying on MS selection, this alternating-energy method simplifies spectra using retention- time selection. The convolution technique detects all the precursor ions and all the fragment ions seen in both modes. The retention time of a fragment ion must be the same as the retention time of its respective precursor. Given an MS precursor, the spectral-simplification method selects only MSE ions that have the same retention times (to within measurement error) as that precursor peptide. The MSE spectra are greatly simplified, and the remaining, accurately-massmeasured, fragment ions can be searched against a database.

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