Deconvolution and Database Search of Complex Tandem Mass Spectra of Intact Proteins

Abstract

Top-down proteomics studies intact proteins, enabling new opportunities for analyzing post-translational modifications. Because tandem mass spectra of intact proteins are very complex, spectral deconvolution (grouping peaks into isotopomer envelopes) is a key initial stage for their interpretation. In such spectra, isotopomer envelopes of different protein fragments span overlapping regions on the m/z axis and even share spectral peaks. This raises both pattern recognition and combinatorial challenges for spectral deconvolution. We present MSDeconv, a combinatorial algorithm for spectral deconvolution. The algorithm first generates a large set of candidate isotopomer envelopes for a spectrum, then represents the spectrum as a graph, and finally selects its highest scoring subset of envelopes as a heaviest path in the graph. In contrast with other approaches, the algorithm scores sets of envelopes rather than individual envelopes. We demonstrate that MS-Deconv improves on Thrash and Xtract in the number of correctly recovered monoisotopic masses and speed. We applied MS-Deconv to a large set of top-down spectra from Yersinia rohdei (with a still unsequenced genome) and further matched them against the protein database of related and sequenced bacterium Yersinia enterocolitica. MS-Deconv is available at http://proteomics.ucsd.edu/Software.html. Molecular & Cellular Proteomics 9:2772-2782, 2010.Top-down proteomics is a mass spectrometry-based approach for identification of proteins and their post-translational modifications (PTMs) 1 (1-14). Unlike the "bottom-up" approach where proteins are first digested into peptides and then a peptide mixture is analyzed by mass spectrometry, the top-down approach analyzes intact proteins. Thus, it has advantages in detecting and localizing PTMs as well as identifying multiple protein species (e.g. proteolytically processed protein species). Despite its advantages, top-down proteomics presents many challenges. These include requirement of high sample quantity, sophisticated instrumentation, protein separation, and robust computational analysis tools. For this reason, top-down proteomics has rarely been used for analyzing complex mixtures (12-18), and it is typically used to study single purified proteins. However, this situation is quickly changing with recent top-down studies of complex protein mixtures (14,19).Because of the existence of natural isotopes, fragment ions of the same chemical formula and charge state are usually represented by a collection of spectral peaks in tandem mass spectra called an isotopomer envelope. The monoisotopic mass of a chemical formula is the sum of the masses of the atoms using the principal (most abundant) isotope for each element. Spectral deconvolution focuses on grouping spectral peaks into isotopomer envelopes. By doing so, the charge state and monoisotopic mass of each envelope are effectively determined. A complex multi-isotopic peak list in the m/z space is translated into a simple monoisotopic mass list that is easier to analyze.Given ...

Three Dimensional Liquid Chromatography Coupling Ion Exchange Chromatography/Hydrophobic Interaction Chromatography/Reverse Phase Chromatography for Effective Protein Separation in Top-Down Proteomics

“…48 These MSAlign files, which contain the monoisotopic mass, intensity, and charge of all the detected ions, were then searched with the NCBI Human database (Uniprot-Swissprot database, released January, 2013, containing 20,232 protein sequences) with alignment-based MS-Align+ algorithm for top-down intact protein identification based on protein-spectrum matches. 49 A fragment mass tolerance of 15 ppm was used for the assignment of b and y ions.…”

De novo sequencing of proteins and peptides: algorithms, applications, perspectives

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Middle-down electron capture dissociation and electron transfer dissociation for histone analysis

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