Mass graphs and their applications in top-down proteomics

Although proteomics has made rapid progress 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 view" of intact proteoforms. The combinatorial explosion of possible proteoforms, which may result in billions of possible proteoforms for one protein, makes proteoform identification a challenging computational problem. Here we propose a new data structure, called the mass graph, for efficiently representing proteoforms. In addition, we design mass graph alignment algorithms for proteoform identification by top-down mass spectrometry. Experiments on a histone H4 mass spectrometry data set showed that the proposed methods outperformed MS-Align-E in identifying complex proteoforms

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

Identification of ultramodified proteins using top-down tandem mass spectra

Post-translational modifications (PTMs) play an important role in various biological processes through changing protein structure and function. Some ultramodified proteins (like histones) have multiple PTMs forming PTM patterns that define the functionality of a protein. While bottom-up mass spectrometry (MS) has been successful in identifying individual PTMs within short peptides, it is unable to identify PTM patterns spreading along entire proteins in a coordinated fashion. In contrast, top-down MS analyzes intact proteins and reveals PTM patterns along the entire proteins. However, while recent advances in instrumentation have made top-down MS accessible to many laboratories, most computational tools for top-down MS focus on proteins with few PTMs and are unable to identify complex PTM patterns. We propose a new algorithm, MS-Align-E, that identifies both expected and unexpected PTMs in ultramodified proteins. We demonstrate that MS-Align-E identifies many proteoforms of histone H4 and benchmark it against the currently accepted software tools

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

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

Vegetative and dendrological characteristics of Hober forest park in Korčula

Sustavnih istraživanja biološko-ekoloških obilježja cjelokupnog područja i same park-šume Hober nije bilo do sada. Strukturna obilježja sastojine nalazimo tek u Planu gospodarenja za park-šumu Hober (2006-2015). Šumarsku problematiku nalazimo u radu Vojinovića (1997). Dendrološka istraživanja imamo u radovima Denich i Draganović (1985), Vojinović (1997),Fabris (2001), Onofri (2002). Izostala je njegova cjelovita inventarizacija.Današnje stanje park-šume Hober karakterizira njegova zapuštenost i neuređenost kako šumske vegetacije i dendrološko-hortikulturnih, tako i vrtno-arhitektonskih sadržaja. Prisutna je koncepcijska nedefiniranost cjelokupnog područja Hobera.Park-šuma Hober u vegetacijskom smislu pripada šumskoj zajednici šuma alepskog bora i hrasta crnike (Querco ilicis – Pinetum halepensis Loisel 1971). Upravo iz razloga vegetacijske i strukturne neujednačenosti, postavljeno je 7 pokusnih ploha u samoj park-šumi, te još 4 izvan nje. Cilj je detaljnom vegetacijskom i strukturnom analizom izdvojiti pojedina područja, dati njihov opis i prikazati njihovo stanje, što se kasnije može upotrijebiti u svrhu zoniranja prostora.Analizirana istraživanja pokazuju da u sloju drveća alepski bor prevladava na plohama 1,2,3 i 9; crnika na plohama 4 i 5 te obični čempres na plohama 6 i 7. U sloju grmlja najzastupljenija je lemprika koja dolazi na svim plohama. Zatim su tu širokolisna zelenika i tršlja. Iz tablice 2 vidimo da je na svim plohama pokrovnost vegetacijom potpuna osim na plohi 6, gdje je rijetka makija. Sloj drveća je prevladavajući osim na plohama 8, 10 i 11, gdje je prisutna makija. Zamjetno je da je kod razvijenih sastojina podjednako gust sloj drveća i grmlja. Tu odstupaju plohe 6 i dijelom ploha 9 te plohe 10 gdje je makija te ploha 11 koja je prirodna sukcesija šumske vegetacije na zapuštenim poljoprivrednim površinama. Osim kod makije imamo vrlo visok drvni volumen (drvna zaliha) koji se ovisno o razvoju sastojine kreće od 100 do čak 270 m3/ha. Broj stabala je isto vrlo velik i kreće se od 1000 do 2700 po ha. Svi ti podaci ukazuju na neuređenost i izostanak bilo kakvih uzgojnih radova. Cilj ovog rada je istražiti vegetacijska, dendrološka, šumsko-uzgojna i strukturna obilježja cjelokupnog područja Hober. Ona su primarni pokazatelj stanje i dat će smjernice za njegovu buduću biološku i prostornu valorizaciju.A systematic research of biological and ecological characteristics of the overall Hober area and Hober forest park itself has not been performed thus far. Structural characteristics of the stand were included only in The Management Plan for Hober Forest Park (2006-2015). Forestry issues were addressed in the paper by Vojinović (1997). Dendrological research was included in the papers by Denich and Draganović (1985), Vojinović (1997), Fabris (2001) and Onofri (2002). Its overall inventory records are missing.The current condition of Hober forest park is characterised by its neglect and no landscape design in terms of both forest vegetation, dendrological, horticultural and garden and architectural content. Moreover, there is conceptual indefiniteness of the overall Hober area.Hober forest park in terms of vegetation belongs to the forest community of the Aleppo pine and Holm oak (Querco ilicis – Pinetum halepensis Loisel 1971). It is primarily due to vegetative and structural unevenness that 7 experimental surfaces were placed in the forest park itself and 4 more outside of it. A comprehensive vegetation and structural analysis was intended for the selection of individual areas, in order to provide their description and present their condition, which can subsequently be used for the purpose of zoning the area.According to the analysed research the Aleppo pine prevails in the layer of trees on the surfaces 1, 2, 3 and 9; Holm oak on the surfaces 4 and 5 and Common cypress on the surfaces 6 and 7. Laurestine is the most common species in the shrub layer, which appears on all the surfaces. Then there are green olive trees with broad leaves and mastic trees. The tree layer prevails with the exception of the surfaces 8, 10 and 11 where there is maquis. It is evident that in developed stands the tree layer and the shrub layer are almost equally dense. The latter is not the case on the surface 6, partially on the surface 9 and the surface 10 where maquis prevails and the surface 11, which is a natural succession of forest vegetation on abandoned agricultural surfaces. With the exception of maquis, there is a very high wood volume which, depending on stand development, ranges from 100 to even 270 m3/ha. The number of trees is also very large and it ranges from 1,000 to 2,700 per hectare. All this data indicates no landscape design and absence of any silvicultural work. The objective of this paper is to explore vegetative, dendrological, silvicultural and structural characteristics of the overall Hober area. They are the primary indicators of the condition and will provide the guidelines for its future biological and spatial valorisation

Landscape and ecological valorisation in the context of protection and revitalisation of Hober forest park in Korčula, the Republic of Croatia

O nastanku gradskog parka Hober ima malo novijih radova koji se uglavnom temelje na prijepisima onih prijašnjih. Od vremena nastanka do danas prostor gradskog parka, danas i park-šume Hober, doživljava niz promjena uvjetovanih različitim društveno-ekonomskim i prirodnim okolnostima koje su se događale kroz povijest, a koje se događaju i danas. Prostor Hobera prolazi kroz različite razvojne faze, od prirodne šume iz koje nastaje preko gradskog parka koji se održava i njeguje, sve do razine zakonske zaštite u park-šumu. Godine 1969. je sukladno Odluci o proglašenju gradskog parka u Korčuli rezervatom prirodnog predjela, Hober zaštićen prema Zakonu o zaštiti prirode u kategoriji park-šuma. Nakon zakonske zaštite slijedi najveće njegovo zapuštanje. Gubitak identiteta gradskog parka i park-šume Hober događa se dugi niz godina kao posljedica nedostatka neophodnih mjera održavanja, uslijed širenja naselja i izostanka potreba i kulture građana, što uvjetuje njegovu degradaciju i gubitak kompozicijske osnove. Nedostatak istraživanja i naklonosti za obnovom i uređenjem ovog vrijednog prostora dovelo je zone ugroženosti sve do samih granica parka ali i unutar njega. Grad Korčula prepoznaje vrijednosti gradskog parka Hober koji traže adekvatnu valorizaciju i obnovu. Unatoč tomu, područje Hobera i gradskog parka ima značajnu biološko-ekološku, prostornu, estetsku, rekreacijsku i turističku važnost u kontekstu grada Korčule. Cilj je rada inventarizirati, analizirati i valorizirati postojeće stanje park-šume Hober i cjelokupnog prostora Hobera u odnosu na stanje kroz povijest i danas, te obrazložiti njegovu vrijednost i mogućnosti uređenja i obnove. Kroz istraživanja je obuhvaćeno prikupljanje postojeće arhivske građe, dokumentacije, kartografskih prikaza i katastarskih podloga vezanih za park. Kompleksno terensko istraživanje je obuhvaćalo i inventarizaciju te analizu postojećeg stanja parka, vrtno-arhitektonskih i biološko-ekoloških elemenata. Na osnovi provedenih istraživanja određena su područja prirodnog i kulturnog krajobraza na istraživanom području. Odabrani su kriteriji koji najcjelovitije prezentiraju istraživano područje. Definiranjem zajedničkih mjerila za određivanje krajobraznih vrijednosti za oba tipa krajobraza, njihova usporedba daje potpuni pregled vrijednosti i mogućeg vrednovanja svakog posebno i njihovu biološko-ekološku i socio-gospodarsku cjelovitost. Ocjene mjerila krajobraznih vrijednosti ujedno predstavljaju na određeni način njihovu osjetljivost na promjene i negativne utjecaje. Svrha dane klasifikacije i vrednovanja istraživanih krajobraza trebaju biti preporuke pomoću kojih se donose dugoročne strategije razvoja i korištenja. Uređenjem i obnovom parka treba naglasiti sve njegove vrijednosti koje se trebaju valorizirati u širem kontekstu Hobera. Dobiveni rezultati daju doprinos postizanja ravnoteže između zaštite i razvoja.The number of recent papers about the establishment of Hober City Park in Korčula is small and they are primarily based on copies of the previous papers. Since its inception, both the area of the City Park and currently also Hober Forest Park have seen a wide range of changes caused by different social and economic, as well as natural circumstances that occurred both throughout history and to date. The Hober area has passed through different development stages, from a natural forest from which it developed, through the city park that was maintained and cared for until it reached the level of legal protection in the Forest Park. In 1969, in accordance with the Decision on the Declaration of the City Park in Korčula a Natural Landscape Reserve, Hober was protected in accordance with Nature Protection Act in the Forest Park Category. After, legal protection was followed by its neglect. The loss of identity of the City Park and Hober Forest Park has been occurring for a large number of years as a consequence of a lack of the necessary maintenance measures, amid the expansion of the settlements and a lack both of the needs and culture of the citizens, which caused its degradation and the loss of its compositional basis. A lack of research and propensity towards renovation and landscaping of this valuable area resulted in an endangered zone both inside the park and to its very boundaries. The City of Korčula recognises the value of Hober City Park. Nevertheless, the areas of Hober and the City Park have a vital importance from the aspect of biology and ecology, as well as from spatial, aesthetic, recreational and tourism aspect in the context of the City of Korčula. The aim of this paper is to inventory, analyze and valorize the current state of the Hober park-forest and the entire Hober area in relation to its condition throughout history and nowadays and explain its value and the possibilities for landscaping and renovation. The research included the collection of the existing archival materials, documentation, cartographic images and cadastral databases linked with the Park. A complex field research also included making of an inventory and analysis of the pre-existing condition of the Park, as well as both of its garden and architectural components and its biological and ecological components. On the basis of the conducted research, there were determined areas of natural and cultural landscape in the researched area. The criteria that most comprehensively represent the research area was selected. By means of defining common criteria for determining the landscape values ​​for both types of landscapes, with their comparison we have a complete overview of the values ​​and possible evaluation of each one separately, and also of their biological-ecological and socio-economic integrity. In a certain way, estimates of landscape values ​​also represent their sensitivity to changes and negative impacts. The purpose of the given classification and evaluation of the researched landscapes should be the recommendations by means of which long-term strategies of development and usage are to be adopted. By the arrangement and renovation of the park it should be emphasized all of its values ​​that need to be valorised in the broader context of Hober. The obtained results on such a way, contribute to achievement of a balance between protection and development