Preparing to read the ubiquitin code: top‐down analysis of unanchored ubiquitin tetramers

The characterization of polyubiquitin chains has been an analytical challenge for several decades. It has been shown that anchored and unanchored polyubiquitin chains with different isopeptide linkages and lengths exhibit a wide range of profoundly different cellular functions. However, structure function studies have been hindered by the difficulty of characterizing these complex chain structures. This report presents a broadly applicable workflow to characterize ubiquitin tetramers without the need for genetic mutations or reiterative immunoprecipitations. We use a top‐down proteomic strategy that exploits ETciD activation on an orbitrap Fusion Lumos and manual interpretation aided by graphical interpretation of mass shifts to facilitate characterization of chain topography and lysine linkage sites. Our workflow differentiates all topological features of the numerous isomers of tetraubiquitin, which have molecular masses in excess of 34 000 Da and identifies linkage sites in these branched proteins. Copyright © 2016 John Wiley & Sons, Ltd.     

Preparing to read the ubiquitin code: a middle‐out strategy for characterization of all lysine‐linked diubiquitins

Multiple studies demonstrate that ubiquitination of proteins codes for regulation of cell differentiation, apoptosis, endocytosis and many other cellular functions. There is great interest in and considerable effort being given to defining the relationships between the structures of polyubiquitin modifications and the fates of the modified proteins. Does each ubiquitin modification achieve a specific effect, much like phosphorylation, or is ubiquitin like glycosylation, where there is heterogeneity and redundancy in the signal? The sensitive analytical tools needed to address such questions readily are not yet mature. To lay the foundation for mass spectrometry (MS)‐based studies of the ubiquitin code, we have assembled seven isomeric diubiquitins with all‐native sequences and isopeptide linkages. Using these compounds as standards enables the development and testing of a new MS‐based strategy tailored specifically to characterize the number and sites of isopeptide linkages in polyubiquitin chains. Here, we report the use of Asp‐selective acid cleavage, separation by reverse phase high‐performance liquid chromatography and characterization by tandem MS to distinguish and characterize all seven isomeric lysine‐linked ubiquitin dimers. Copyright © 2014 John Wiley & Sons, Ltd.     

A quantitative analysis of light‐driven charge transfer processes using voronoi partitioning of time dependent DFT‐derived electron densities

An analytical method is presented that provides quantitative insight into light‐driven electron density rearrangement using the output of standard time‐dependent density functional theory (TD‐DFT) computations on molecular compounds. Using final and initial electron densities for photochemical processes, the subtraction of summed electron density in each atom‐centered Voronoi polyhedron yields the electronic charge difference, Q ^VECD. This subtractive method can also be used with Bader, Mulliken and Hirshfeld charges. A validation study shows Q ^VECD to have the most consistent performance across basis sets and good conservation of charge between electronic states. Besides vertical transitions, relaxation processes can be investigated as well. Significant electron transfer is computed for isomerization on the excited state energy surface of azobenzene. A number of linear anilinepyridinium donor‐bridge‐acceptor chromophores was examined using Q ^VECD to unravel the influence of its pi‐conjugated bridge on charge separation. Finally, the usefulness of the presented method as a tool in optimizing charge transfer is shown for a homologous series of organometallic pigments. The presented work allows facile calculation of a novel, relevant quantity describing charge transfer processes at the atomic level. © 2017 The Authors Journal of Computational Chemistry Published by Wiley Periodicals, Inc.     

Preparing to read the ubiquitin code: characterization of ubiquitin trimers by top‐down mass spectrometry

The profound effects of ubiquitination on the movement and processing of cellular proteins depend exquisitely on the structures of monoubiquitin and polyubiquitin modifications. Unconjugated polyubiquitins also have a variety of intracellular functions. Structures and functions are not well correlated yet, because the structures of polyubiquitins and polyubiquitin modifications of proteins are difficult to decipher. We are moving towards a robust strategy to provide that structural information. In this report electron transfer dissociation mass spectra of six synthetic ubiquitin trimers (multiply branched proteins with molecular masses exceeding 25 600 Da) are examined using an Orbitrap Fusion Lumos instrument to determine how top‐down mass spectrometry can characterize the chain topology and linkage sites in a single, facile workflow. The efficacy of this method relies on the formation, detection, and interpretation of extensive fragmentation. Copyright © 2016 John Wiley & Sons, Ltd.     

Boosting partial least‐squares discriminant analysis with application to near infrared spectroscopic tea variety discrimination

In the present study, boosting has been combined with partial least‐squares discriminant analysis (PLS‐DA) to develop a new pattern recognition method called boosting partial least‐squares discriminant analysis (BPLS‐DA). BPLS‐DA is implemented by firstly constructing a series of PLS‐DA models on the various weighted versions of the original calibration set and then combining the predictions from the constructed PLS‐DA models to obtain the integrative results by weighted majority vote. Coupled with near infrared (NIR) spectroscopy, BPLS‐DA has been applied to discriminate different kinds of tea varieties. As comparisons to BPLS‐DA, the conventional principal component analysis, linear discriminant analysis (LDA), and PLS‐DA have also been investigated. Experimental results have shown that the inter‐variety difference can be accurately and rapidly distinguished via NIR spectroscopy coupled with BPLS‐DA. Moreover, the introduction of boosting drastically enhances the performance of an individual PLS‐DA, and BPLS‐DA is a well‐performed pattern recognition technique superior to LDA. Copyright © 2012 John Wiley & Sons, Ltd.