Algorithms for Peptide Identification from Mixture Tandem Mass Spectra

The large amount of data collected in an mass spectrometry experiment requires effective computational approaches for the automated analysis of those data. Though extensive research has been conducted for such purpose by the proteomics community, there are still remaining challenges, among which, one particular challenge is that the identification rate of the MS/MS spectra collected is rather low. One significant reason that contributes to this situation is the frequently observed mixture spectra, which result from the concurrent fragmentation of multiple precursors in a single MS/MS spectrum. However, nearly all the mainstream computational methods still take the assumption that the acquired spectra come from a single precursor, thus they are not suitable for the identification of mixture spectra. In this research, we focused on developing effective algorithms for the purpose of interpreting mixture tandem mass spectra, and our research work is mainly comprised of two components: de novo sequencing of mixture spectra and mixture spectra identification by database search. For the de novo sequencing approach, firstly we formulated the mixture spectra de novo sequencing problem mathematically, and proposed a dynamic programming algorithm for the problem. Additionally, we use both simulated and real mixture spectra datasets to verify the efficiency of the algorithm described in the research. For the database search identification, we proposed an approach for matching mixture tandem mass spectra with a pair of peptide sequences acquired from the protein sequence database by incorporating a special de novo assisted filtration strategy. Besides the filtration strategy, we also introduced in the research a method to give an reasonable estimation of the mixture coefficient which represents the relative abundance level of the co-sequenced precursors. The preliminary experimental results demonstrated the efficiency of the integrated filtration strategy and mixture coefficient estimating method in reducing examination space and also verified the effectiveness of the proposed matching scheme

Algorithms for Glycan Structure Identification with Tandem Mass Spectrometry

Glycosylation is a frequently observed post-translational modification (PTM) of proteins. It has been estimated over half of eukaryotic proteins in nature are glycoproteins. Glycoprotein analysis plays a vital role in drug preparation. Thus, characterization of glycans that are linked to proteins has become necessary in glycoproteomics. Mass spectrometry has become an effective analytical technique for glycoproteomics analysis because of its high throughput and sensitivity. The large amount of spectral data collected in a mass spectrometry experiment makes manual interpretation impossible and requires effective computational approaches for automated analysis. Different algorithmic solutions have been proposed to address the challenges in glycoproteomics analysis based on mass spectrometry. However, new algorithms that can identify intact glycopeptides are still demanded to improve result accuracy. In this research, a glycan is represented as a rooted unordered labelled tree and we focus on developing effective algorithms to determine glycan structures from tandem mass spectra. Interpreting the tandem mass spectra of glycopeptides with a de novo sequencing method is essential to identifying novel glycan structures. Thus, we mathematically formulated the glycan de novo sequencing problem and propose a heuristic algorithm for glycan de novo sequencing from HCD tandem mass spectra of glycopeptides. Characterizing glycans from MS/MS with a de novo sequencing method requires high-quality mass spectra for accurate results. The database search method usually has the ability to obtain more reliable results since it has the assistance of glycan structural information. Thus, we propose a de novo sequencing assisted database search method, GlycoNovoDB, for mass spectra interpretation

Parallel algorithms for real-time peptide-spectrum matching

Tandem mass spectrometry is a powerful experimental tool used in molecular biology to determine the composition of protein mixtures. It has become a standard technique for protein identification. Due to the rapid development of mass spectrometry technology, the instrument can now produce a large number of mass spectra which are used for peptide identification. The increasing data size demands efficient software tools to perform peptide identification. In a tandem mass experiment, peptide ion selection algorithms generally select only the most abundant peptide ions for further fragmentation. Because of this, the low-abundance proteins in a sample rarely get identified. To address this problem, researchers develop the notion of a `dynamic exclusion list', which maintains a list of newly selected peptide ions, and it ensures these peptide ions do not get selected again for a certain time. In this way, other peptide ions will get more opportunity to be selected and identified, allowing for identification of peptides of lower abundance. However, a better method is to also include the identification results into the `dynamic exclusion list' approach. In order to do this, a real-time peptide identification algorithm is required. In this thesis, we introduce methods to improve the speed of peptide identification so that the `dynamic exclusion list' approach can use the peptide identification results without affecting the throughput of the instrument. Our work is based on RT-PSM, a real-time program for peptide-spectrum matching with statistical significance. We profile the speed of RT-PSM and find out that the peptide-spectrum scoring module is the most time consuming portion. Given by the profiling results, we introduce methods to parallelize the peptide-spectrum scoring algorithm. In this thesis, we propose two parallel algorithms using different technologies. We introduce parallel peptide-spectrum matching using SIMD instructions. We implemented and tested the parallel algorithm on Intel SSE architecture. The test results show that a 18-fold speedup on the entire process is obtained. The second parallel algorithm is developed using NVIDIA CUDA technology. We describe two CUDA kernels based on different algorithms and compare the performance of the two kernels. The more efficient algorithm is integrated into RT-PSM. The time measurement results show that a 190-fold speedup on the scoring module is achieved and 26-fold speedup on the entire process is obtained. We perform profiling on the CUDA version again to show that the scoring module has been optimized sufficiently to the point where it is no longer the most time-consuming module in the CUDA version of RT-PSM. In addition, we evaluate the feasibility of creating a metric index to reduce the number of candidate peptides. We describe evaluation methods, and show that general indexing methods are not likely feasible for RT-PSM

Developing a bioinformatics framework for proteogenomics

In the last 15 years, since the human genome was first sequenced, genome sequencing and annotation have continued to improve. However, genome annotation has not kept up with the accelerating rate of genome sequencing and as a result there is now a large backlog of genomic data waiting to be interpreted both quickly and accurately. Through advances in proteomics a new field has emerged to help improve genome annotation, termed proteogenomics, which uses peptide mass spectrometry data, enabling the discovery of novel protein coding genes, as well as the refinement and validation of known and putative protein-coding genes. The annotation of genomes relies heavily on ab initio gene prediction programs and/or mapping of a range of RNA transcripts. Although this method provides insights into the gene content of genomes it is unable to distinguish protein-coding genes from putative non-coding RNA genes. This problem is further confounded by the fact that only 5% of the public protein sequence repository at UniProt/SwissProt has been curated and derived from actual protein evidence. This thesis contends that it is critically important to incorporate proteomics data into genome annotation pipelines to provide experimental protein-coding evidence. Although there have been major improvements in proteogenomics over the last decade there are still numerous challenges to overcome. These key challenges include the loss of sensitivity when using inflated search spaces of putative sequences, how best to interpret novel identifications and how best to control for false discoveries. This thesis addresses the existing gap between the use of genomic and proteomic sources for accurate genome annotation by applying a proteogenomics approach with a customised methodology. This new approach was applied within four case studies: a prokaryote bacterium; a monocotyledonous wheat plant; a dicotyledonous grape plant; and human. The key contributions of this thesis are: a new methodology for proteogenomics analysis; 145 suggested gene refinements in Bradyrhizobium diazoefficiens (nitrogen-fixing bacteria); 55 new gene predictions (57 protein isoforms) in Vitis vinifera (grape); 49 new gene predictions (52 protein isoforms) in Homo sapiens (human); and 67 new gene predictions (70 protein isoforms) in Triticum aestivum (bread wheat). Lastly, a number of possible improvements for the studies conducted in this thesis and proteogenomics as a whole have been identified and discussed

Molecular Formula Identification using High Resolution Mass Spectrometry: Algorithms and Applications in Metabolomics and Proteomics

Wir untersuchen mehrere theoretische und praktische Aspekte der Identifikation der Summenformel von Biomolekülen mit Hilfe von hochauflösender Massenspektrometrie. Durch die letzten Forschritte in der Instrumentation ist die Massenspektrometrie (MS) zur einen der Schlüsseltechnologien für die Analyse von Biomolekülen in der Proteomik und Metabolomik geworden. Sie misst die Massen der Moleküle in der Probe mit hoher Genauigkeit, und ist für die Messdatenerfassung im Hochdurchsatz gut geeignet. Eine der Kernaufgaben in der MS-basierten Proteomik und Metabolomik ist die Identifikation der Moleküle in der Probe. In der Metabolomik unterliegen Metaboliten der Strukturaufklärung, beginnend bei der Summenformel eines Moleküls, d.h. der Anzahl der Atome jedes Elements. Dies ist der entscheidende Schritt in der Identifikation eines unbekannten Metabolits, da die festgelegte Formel die Anzahl der möglichen Molekülstrukturen auf eine viel kleinere Menge reduziert, die mit Methoden der automatischen Strukturaufklärung weiter analysiert werden kann. Nach der Vorverarbeitung ist die Ausgabe eines Massenspektrometers eine Liste von Peaks, die den Molekülmassen und deren Intensitäten, d.h. der Anzahl der Moleküle mit einer bestimmten Masse, entspricht. Im Prinzip können die Summenformel kleiner Moleküle nur mit präzisen Massen identifiziert werden. Allerdings wurde festgestellt, dass aufgrund der hohen Anzahl der chemisch legitimer Formeln in oberen Massenbereich eine exzellente Massengenaugkeit alleine für die Identifikation nicht genügt. Hochauflösende MS erlaubt die Bestimmung der Molekülmassen und Intensitäten mit hervorragender Genauigkeit. In dieser Arbeit entwickeln wir mehrere Algorithmen und Anwendungen, die diese Information zur Identifikation der Summenformel der Biomolekülen anwenden

Improving the Performance and Precision of Bioinformatics Algorithms

Recent advances in biotechnology have enabled scientists to generate and collect huge amounts of biological experimental data. Software tools for analyzing both genomic (DNA) and proteomic (protein) data with high speed and accuracy have thus become very important in modern biological research. This thesis presents several techniques for improving the performance and precision of bioinformatics algorithms used by biologists. Improvements in both the speed and cost of automated DNA sequencers have allowed scientists to sequence the DNA of an increasing number of organisms. One way biologists can take advantage of this genomic DNA data is to use it in conjunction with expressed sequence tag (EST) and cDNA sequences to find genes and their splice sites. This thesis describes ESTmapper, a tool designed to use an eager write-only top-down (WOTD) suffix tree to efficiently align DNA sequences against known genomes. Experimental results show that ESTmapper can be much faster than previous techniques for aligning and clustering DNA sequences, and produces alignments of comparable or better quality. Peptide identification by tandem mass spectrometry (MS/MS) is becoming the dominant high-throughput proteomics workflow for protein characterization in complex samples. Biologists currently rely on protein database search engines to identify peptides producing experimentally observed mass spectra. This thesis describes two approaches for improving peptide identification precision using statistical machine learning. HMMatch (HMM MS/MS Match) is a hidden Markov model approach to spectral matching, in which many examples of a peptide fragmentation spectrum are summarized in a generative probabilistic model that captures the consensus and variation of each peak's intensity. Experimental results show that HMMatch can identify many peptides missed by traditional spectral matching and search engines. PepArML (Peptide Identification Arbiter by Machine Learning) is a machine learning based framework for improving the precision of peptide identification. It uses classification algorithms to effectively utilize spectra features and scores from multiple search engines in a single model-free framework that can be trained in an unsupervised manner. Experimental results show that PepArML can improve the sensitivity of peptide identification for several synthetic protein mixtures compared with individual search engines

De novo sequencing of heparan sulfate saccharides using high-resolution tandem mass spectrometry

Heparan sulfate (HS) is a class of linear, sulfated polysaccharides located on cell surface, secretory granules, and in extracellular matrices found in all animal organ systems. It consists of alternately repeating disaccharide units, expressed in animal species ranging from hydra to higher vertebrates including humans. HS binds and mediates the biological activities of over 300 proteins, including growth factors, enzymes, chemokines, cytokines, adhesion and structural proteins, lipoproteins and amyloid proteins. The binding events largely depend on the fine structure - the arrangement of sulfate groups and other variations - on HS chains. With the activated electron dissociation (ExD) high-resolution tandem mass spectrometry technique, researchers acquire rich structural information about the HS molecule. Using this technique, covalent bonds of the HS oligosaccharide ions are dissociated in the mass spectrometer. However, this information is complex, owing to the large number of product ions, and contains a degree of ambiguity due to the overlapping of product ion masses and lability of sulfate groups; as a result, there is a serious barrier to manual interpretation of the spectra. The interpretation of such data creates a serious bottleneck to the understanding of the biological roles of HS. In order to solve this problem, I designed HS-SEQ - the first HS sequencing algorithm using high-resolution tandem mass spectrometry. HS-SEQ allows rapid and confident sequencing of HS chains from millions of candidate structures and I validated its performance using multiple known pure standards. In many cases, HS oligosaccharides exist as mixtures of sulfation positional isomers. I therefore designed MULTI-HS-SEQ, an extended version of HS-SEQ targeting spectra coming from more than one HS sequence. I also developed several pre-processing and post-processing modules to support the automatic identification of HS structure. These methods and tools demonstrated the capacity for large-scale HS sequencing, which should contribute to clarifying the rich information encoded by HS chains as well as developing tailored HS drugs to target a wide spectrum of diseases