A mass graph-based approach for the identification of modified proteoforms using top-down tandem mass spectra

Motivation: Although proteomics has rapidly developed in the past decade, researchers are still in the early stage of exploring the world of complex proteoforms, which are protein products with various primary structure alterations resulting from gene mutations, alternative splicing, post-translational modifications, and other biological processes. Proteoform identification is essential to mapping proteoforms to their biological functions as well as discovering novel proteoforms and new protein functions. Top-down mass spectrometry is the method of choice for identifying complex proteoforms because it provides a 'bird's eye view' of intact proteoforms. The combinatorial explosion of various alterations on a protein may result in billions of possible proteoforms, making proteoform identification a challenging computational problem. Results: We propose a new data structure, called the mass graph, for efficient representation of proteoforms and design mass graph alignment algorithms. We developed TopMG, a mass graph-based software tool for proteoform identification by top-down mass spectrometry. Experiments on top-down mass spectrometry datasets showed that TopMG outperformed existing methods in identifying complex proteoforms

Gas Phase Properties of ONOO-anion and ONOO-radical

We report calculated ab initio energies and barriers to internal rotation for ONOO-anion and ONOO-radical species at the 6-31G** level. The anion is 48 kJ/mol more stable than the radical in its lowest 2A" (trans) state. The difference between the more stable trans and less stable cis conformations of the ONOO-anion and ONOO-radical is small, amounting to 4.6 and 3.9 kJ/mol, respectively. However, the energy of the 90° skew form is 68 kJ/mol above the trans form in the ONOO-anion, but only 40 kJ/mol in ONOO radical. Several ways to produce the ONOO-anion for FTMS analysis and determination of its thermodynamic properties are presented

Characterization of proteoforms with unknown post-translational modi cations using the MIScore

Various proteoforms may be generated from a single gene due to primary structure alterations (PSAs) such as genetic variations, alternative splicing, and post-translational modifications (PTMs). Top-down mass spectrometry is capable of analyzing intact proteins and identifying patterns of multiple PSAs, making it the method of choice for studying complex proteoforms. In top-down proteomics, proteoform identification is often performed by searching tandem mass spectra against a protein sequence database that contains only one reference protein sequence for each gene or transcript variant in a proteome. Because of the incompleteness of the protein database, an identified proteoform may contain unknown PSAs compared with the reference sequence. Proteoform characterization is to identify and localize PSAs in a proteoform. Although many software tools have been proposed for proteoform identification by top-down mass spectrometry, the characterization of proteoforms in identified proteoform–spectrum matches still relies mainly on manual annotation. We propose to use the Modification Identification Score (MIScore), which is based on Bayesian models, to automatically identify and localize PTMs in proteoforms. Experiments showed that the MIScore is accurate in identifying and localizing one or two modifications

FRET Imaging of Diatoms Expressing a Biosilica-Localized Ribose Sensor

Future materials are envisioned to include bio-assembled, hybrid, three-dimensional nanosystems that incorporate functional proteins. Diatoms are amenable to genetic modification for localization of recombinant proteins in the biosilica cell wall. However, the full range of protein functionalities that can be accommodated by the modified porous biosilica has yet to be described. Our objective was to functionalize diatom biosilica with a reagent-less sensor dependent on ligand-binding and conformational change to drive FRET-based signaling capabilities. A fusion protein designed to confer such properties included a bacterial periplasmic ribose binding protein (R) flanked by CyPet (C) and YPet (Y), cyan and yellow fluorescent proteins that act as a FRET pair. The structure and function of the CRY recombinant chimeric protein was confirmed by expression in E. coli prior to transformation of the diatom Thalassiosira pseudonana. Mass spectrometry of the recombinant CRY showed 97% identity with the deduced amino acid sequence. CRY with and without an N-terminal Sil3 tag for biosilica localization exhibited characteristic ribose-dependent changes in FRET, with similar dissociation constants of 123.3 µM and 142.8 µM, respectively. The addition of the Sil3 tag did not alter the affinity of CRY for the ribose substrate. Subsequent transformation of T. pseudonana with a vector encoding Sil3-CRY resulted in fluorescence localization in the biosilica and changes in FRET in both living cells and isolated frustules in response to ribose. This work demonstrated that the nano-architecture of the genetically modified biosilica cell wall was able to support the functionality of the relatively complex Sil3-CyPet-RBP-YPet fusion protein with its requirement for ligand-binding and conformational change for FRET-signal generation

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry

AbstractA primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis

De Novo Sequencing of Peptides from High-Resolution Bottom-Up Tandem Mass Spectra using Top-Down Intended Methods

Despite high-resolution mass spectrometers are becoming accessible for more and more laboratories, tandem (MS/MS) mass spectra are still often collected at a low resolution. And even if acquired at a high resolution, software tools used for their processing do not tend to benefit from that in full, and an ability to specify a relative mass tolerance in this case often remains the only feature the respective algorithms take advantage of. We argue that a more efficient way to analyze high-resolution MS/MS spectra should be with methods more explicitly accounting for the precision level, and sustain this claim through demonstrating that a de novo sequencing framework originally developed for (high-resolution) top-down MS/MS data is perfectly suitable for processing high-resolution bottom-up datasets, even though a top-down like deconvolution performed as the first step will leave in many spectra at most a few peaks

Top-down analysis of protein samples by de novo sequencing techniques

Motivation: Recent technological advances have made high-resolution mass spectrometers affordable to many laboratories, thus boosting rapid development of top-down mass spectrometry, and implying a need in efficient methods for analyzing this kind of data. Results: We describe a method for analysis of protein samples from top-down tandem mass spectrometry data, which capitalizes on de novo sequencing of fragments of the proteins present in the sample. Our algorithm takes as input a set of de novo amino acid strings derived from the given mass spectra using the recently proposed Twister approach, and combines them into aggregated strings endowed with offsets. The former typically constitute accurate sequence fragments of sufficiently well-represented proteins from the sample being analyzed, while the latter indicate their location in the protein sequence, and also bear information on post-translational modifications and fragmentation patterns. Availability and Implementation: Freely available on the web at http://bioinf.spbau.ru/en/twister

We Are What We Eat: A Stoichiometric and Ecometabolomic Study of Caterpillars Feeding on Two Pine Subspecies of Pinus sylvestris

Many studies have addressed several plant-insect interaction topics at nutritional, molecular, physiological, and evolutionary levels. However, it is still unknown how flexible the metabolism and the nutritional content of specialist insect herbivores feeding on different closely related plants can be. We performed elemental, stoichiometric, and metabolomics analyses on leaves of two coexisting Pinus sylvestris subspecies and on their main insect herbivore; the caterpillar of the processionary moth (Thaumetopoea pityocampa). Caterpillars feeding on different pine subspecies had distinct overall metabolome structure, accounting for over 10% of the total variability. Although plants and insects have very divergent metabolomes, caterpillars showed certain resemblance to their plant-host metabolome. In addition, few plant-related secondary metabolites were found accumulated in caterpillar tissues which could potentially be used for self-defense. Caterpillars feeding on N and P richer needles had lower N and P tissue concentration and higher C:N and C:P ratios, suggesting that nutrient transfer is not necessarily linear through trophic levels and other plant-metabolic factors could be interfering. This exploratory study showed that little chemical differences between plant food sources can impact the overall metabolome of specialist insect herbivores. Significant nutritional shifts in herbivore tissues could lead to larger changes of the trophic web structure.This research was funded by the research fellowship (JAE) from the CSIC (A.R.-U), the European Research Council Synergy grant SyG-2013-610028 IMBALANCE-P (J.P., J.S.), the Spanish Government projects CGL2016-48074-P and OAPN 022/2008 (PROPINOL) (J.P., J.S.), the Catalan Government project SGR 2014-274 (J.P., J.S.), DOE Office of Science User Facility sponsored by the Office of Biological and Environmental Research at the Pacific Northwest National Laboratory (A.R.-U), and by the Ministry of Education, Youth and Sports of the Czech Republic (grant No. CZ.02.1.01/0.0/0.0/16_013/0001609, and No. LO1415) (O.U., M.O)

Trapped-Ion Cell with Improved DC Potential Harmonicity for FT-ICR MS

The trapped-ion cell is a key component critical for optimal performance in Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS). To extend the performance of FT-ICR MS, we have developed a new cell design that is capable of generating a DC trapping potential which closely approaches that of an ideal Penning trap, i.e., a 3D axial quadrupolar potential distribution. The new cell design was built upon an open cylindrical geometry, supplemented with two pairs of cylindrical compensation segments. Electric potential calculations for trial cell geometries were aimed at minimizing spatial variations of the radial electric field divided by radius. The resulting cell proportions and compensation voltages delivered practically constant effective ion cyclotron frequency that was independent of ion radial and axial positions. Our customized 12 tesla FT-ICR instrument was upgraded with the new cell, and the performance was characterized for a range of ion excitation power and ion populations. Operating the compensated cell at increased postexcitation radii, ∼0.7 of the cell inner radius, resulted in improved mass measurement accuracy together with increased signal intensity. Under these same operating conditions the noncompensated open cell configuration exhibited peak splitting and reduced signal life time. Mass accuracy tests using 11 calibrants covering a wide m/z range reproducibly produced under 0.05 ppm RMS precision of the internal calibration for reduced ion populations and the optimal excitation radius. Conditions of increased ion population resulted in a twofold improvement in mass accuracy compared with the noncompensated cell, due to the larger achievable excitation radii and correspondingly lower space charge related perturbations of the calibration law