Bibliographies: 'Tandem Mass Tag Proteomics' – Grafiati (2024)

Abstract:

Bambusa pervariabilis McClure × Dendrocalamopsis grandis (Q.H.Dai & X.l.Tao ex Keng f.) Ohrnb. blight is a widespread and dangerous forest fungus disease, and has been listed as a supplementary object of forest phytosanitary measures. In order to study the control of B. pervariabilis × D. grandis blight, this experiment was carried out. In this work, a toxin purified from the pathogen Arthrinium phaeospermum (Corda) Elli, which causes blight in B. pervariabilis × D. grandis, with hom*ologous heterogeneity, was used as an inducer to increase resistance to B. pervariabilis × D. grandis. A functional analysis of the differentially expressed proteins after induction using a tandem mass tag labeling technique was combined with mass spectrometry and liquid chromatography mass spectrometry in order to effectively screen for the proteins related to the resistance of B. pervariabilis × D. grandis to blight. After peptide labeling, a total of 3320 unique peptides and 1791 quantitative proteins were obtained by liquid chromatography mass spectrometry analysis. Annotation and enrichment analysis of these peptides and proteins using the Gene ontology and Kyoto Encyclopedia of Genes and Genomes databases with bioinformatics software show that the differentially expressed protein functional annotation items are mainly concentrated on biological processes and cell components. Several pathways that are prominent in the Kyoto Encyclopedia of Genes and Genomes annotation and enrichment include metabolic pathways, the citrate cycle, and phenylpropanoid biosynthesis. In the Protein-protein interaction networks four differentially expressed proteins-sucrose synthase, adenosine triphosphate-citrate synthase beta chain protein 1, peroxidase, and phenylalanine ammonia-lyase significantly interact with multiple proteins and significantly enrich metabolic pathways. To verify the results of tandem mass tag, the candidate proteins were further verified by parallel reaction monitoring, and the results were consistent with the tandem mass tag data analysis results. It is confirmed that the data obtained by tandem mass tag technology are reliable. Therefore, the differentially expressed proteins and signaling pathways discovered here is the primary concern for subsequent disease resistance studies.

Abstract:

Most cellular processes are carried out by a multitude of proteins that assemble into multimeric complexes. Thus a precise understanding of the biological pathways that control cellular events relies on the identification and on the biochemical characterization of the proteins involved in such multimeric assemblies. Advances in MS have made possible the identification of multisubunit protein complexes isolated from cell lysates with high sensitivity and accuracy, whereas the TAP (tandem affinity purification) methodology efficiently isolates native protein complexes from cells for proteomics analysis. TAP is a generic method based on the sequential utilization of two affinity tags to purify protein assemblies. During the first purification step, the Protein A moiety of the TAP tag is bound to IgG beads, and protein components associated with the TAP-tagged protein are retrieved by TEV (tobacco etch virus) protease cleavage. This enzyme is a sequence-specific protease cleaving a seven-amino-acid recognition site located between the first and second tags. In the second affinity step, the protein complex is immobilized to calmodulin-coated beads via the CBP (calmodulin-binding peptide) of the TAP tag. The CBP–calmodulin interaction is calcium-dependent and calcium-chelating agents are used in the second elution step to release the final protein complex preparation used for protein identification by MS. The TAP–MS approach has proven to efficiently permit the characterization of protein complexes from bacteria, yeast and mammalian cells, as well as from multicellular organisms such as Caenorhabditis elegans, Drosophila and mice.

Abstract:

Modern mass spectrometry is one of the most frequently used methods of quantitative proteomics, enabling determination of the amount of peptides in a sample. Although mass spectrometry is not inherently a quantitative method due to differences in the ionization efficiency of various analytes, the application of isotope-coded labeling allows relative quantification of proteins and proteins. Over the past decade, a new method for derivatization of tryptic peptides using isobaric labels has been proposed. The labels consist of reporter and balanced groups. They have the same molecular weights and chemical properties, but differ in the distribution of stable heavy isotopes. These tags are designed in such a way that during high energy collision induced dissociation (CID) by tandem mass spectrometry, the isobaric tag is fragmented in the specific linker region, yielding reporter ions with different masses. The mass shifts among the reporter groups are compensated by the balancing groups so that the overall mass is the same for all forms of the reagent. Samples of peptides are labeled with the isobaric mass tags in parallel and combined for analysis. Quantification of individual peptides is achieved by comparing the intensity of reporter ions in the tandem mass (MS/MS) spectra. Isobaric markers have found a wide range of potential applications in proteomics. However, the currently available isobaric labeling reagents have some drawbacks, such as high cost of production, insufficient selectivity of the derivatization, and relatively limited enhancement of sensitivity of the analysis. Therefore, efforts have been devoted to the development of new isobaric markers with increased usability. The search for new isobaric markers is focused on developing a more selective method of introducing a tag into a peptide molecule, increasing the multiplexicity of markers, lowering the cost of synthesis, and increasing the sensitivity of measurement by using ionization tags containing quaternary ammonium salts. Here, the trends in the design of new isobaric labeling reagents for quantitative proteomics isobaric derivatization strategies in proteomics are reviewed, with a particular emphasis on isobaric ionization tags. The presented review focused on different types of isobaric reagents used in quantitative proteomics, their chemistry, and advantages offer by their application.

Abstract:

Ivermectin (IVM), an antiparasitic drug, has a positive effect against Anisakis simplex s.s. infection and has been used for the treatment and prevention of anisakiasis in humans. However, the molecular mechanism of action of IVM on A. simplex s.s. remains unknown. Herein, tandem mass tag (TMT) labeling and extensive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis were used to identify the effect of IVM on the proteome of A. simplex s.s. in vitro. During the study, 3433 proteins, of which 1247 had at least two protein unique peptides, were identified. Comparative proteomics analysis revealed that 59 proteins were differentially regulated (DRPs) in IVM-treated larvae, of which 14 proteins were upregulated and 38 were downregulated after 12 h of culture, but after 24 h, 12 proteins were upregulated and 22 were downregulated. The transcription level of five randomly selected DRPs was determined by real-time PCR as a supplement to the proteomic data. The functional enrichment analysis showed that most of the DRPs were involved in oxidoreductase activity, immunogenicity, protein degradation, and other biological processes. This study has, for the first time, provided comprehensive proteomics data on A. simplex s.s. response to IVM and might deliver new insight into the molecular mechanism by which IVM acts on invasive larvae of A. simplex s.s.

Abstract:

Plasma biomarkers that identify abdominal aortic aneurysm (AAA) rupture risk would greatly assist in stratifying patients with small aneurysms. Identification of such biomarkers has hitherto been unsuccessful over a range of studies using different methods. The present study used an alternative proteomic approach to find new, potential plasma AAA biomarker candidates. Pre-fractionated plasma samples from twelve patients with AAA and eight matched controls without aneurysm were analyzed by mass spectrometry applying a tandem mass tag (TMT) technique. Eight proteins were differentially regulated in patients compared to controls, including decreased levels of the enzyme bleomycin hydrolase. The down-regulation of this enzyme was confirmed in an extended validation study using an enzyme-linked immunosorbent assay (ELISA). The TMT-based proteomic approach thus identified novel potential plasma biomarkers for AAA.

Abstract:

Senecavirus A (SVA), also known as Seneca Valley virus, belongs to the genus Senecavirus in the family Picornaviridae. SVA can cause vesicular disease and epidemic transient neonatal losses in pigs. This virus efficiently propagates in some non-pig-derived cells, like the baby hamster kidney (BHK) cell line and its derivate (BSR-T7/5). Conventionally, a few proteins or only one protein is selected for exploiting a given mechanism concerning cellular regulation after SVA infection in vitro. Proteomics plays a vital role in the analysis of protein profiling, protein-protein interactions, and protein-directed metabolisms, among others. Tandem mass tag-labeled liquid chromatography-tandem mass spectrometry combined with the parallel reaction monitoring technique is increasingly used for proteomic research. In this study, this combined method was used to uncover separately proteomic profiles of SVA- and non-infected BSR-T7/5 cells. Furthermore, both proteomic profiles were compared with each other. The proteomic profiling showed that a total of 361 differentially expressed proteins were identified, out of which, 305 and 56 were upregulated and downregulated in SVA-infected cells at 12 h post-inoculation, respectively. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analyses showed that cellular metabolisms were affected mainly in SVA-inoculated cells at an early stage of infection. Therefore, an integrated metabolic atlas remains to be explored via metabolomic methods.

Abstract:

ABSTRACT IMPACT: Implementation of proteomics as a diagnostic tool for Nontuberculous mycobacteria (NTM) infection can provide a more accurate, efficient and cost-effective means for effectively diagnosing disease and enacting timely management decisions which can revolutionize patient care. OBJECTIVES/GOALS: Proteomic analysis is a proven diagnostic modality enabling rapid identification of microorganisms. We sought to apply proteomics to detect proteins unique to the most clinically relevant NTM. We then determined whether these unique proteomes could be used to successfully identify NTM species from in vitro co*cktail preparations. METHODS/STUDY POPULATION: NTM reference strains for M. avium, m. intracellulare, m.chimaera, m. abscessus abscessus, m. abscessus massiliense and m. abscessus boletti were cultured in vitro and subjected to proteomic analysis using Liquid Chromatography tandem-Mass Spectrometry (LCMS). Tandem Mass Tag (TMT) data acquisition utilized an MS3 approach for data collection using Proteome Discoverer 2.4.A comparative analysis of the proteome of each of these six species was performed quantitatively using LCMS. The process was repeated for three technical replicates and analyzed using the SEQUEST algorithm. Only high scoring peptides were considered utilizing a false discovery rate (FDR) of 1%. Once species-specific proteins were identified, we validated detection in individual and mixed samples of the six reference strains. RESULTS/ANTICIPATED RESULTS: The proteomic profiling of the six NTM reference strains successfully demonstrated proteins unique to each of the MAC species and MABC subspecies. Proteomic MAC species analysis produced between 327 to 2,540 unique peptides for each of the 3 species. MABC proteomic analysis identified between 17-74 unique peptides for each of the 3 subspecies. Fifteen different mixed preparations of MAC and MABC were then subjected to LCMS analysis and compared against the proteome profiles already curated for the six strains. We accurately identified at least one NTM in the majority of the samples (10/15). In three samples (3/15), the NTM was not correctly identified; in two of the samples (2/15) we were unable to determine the identity of NTM within the preparation. Further database curation will be performed to hone these results. DISCUSSION/SIGNIFICANCE OF FINDINGS: Proteomic analysis of in vitro reference strains successfully demonstrated protein fingerprints specific to six common disease-causing strains of NTM. Such findings can be used to evaluate clinical samples enabling more efficient diagnostic specificity. Further research will focus on identification of NTM in sputum samples of infected patients.

Abstract:

Pigeonpea (Cajanus cajan) is a sub-tropical and tropical pulse rich in plant-based protein, carbohydrates, minerals, and vitamins. Helicoverpa armigera is the most devastating insect pest in pigeonpea. This study focussed on deciphering the molecular host plant resistance (HPR) mechanisms applied by Cajanus scarabaeoides a wild pigeonpea against insect using transcriptomic and proteomic studies. These HPR mechanisms were transferred to the cultivated pigeonpea via interspecific hybridisation, and they are stable at F2 generation. The study outcome provides a unique insight into the insect resistance mechanisms employed by C. scarabaeoides and lays the foundation for further studies and applications.

Abstract:

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed August 13, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 187-205).

Abstract:

LC-QTOF tandem mass spectrometers behave according to user controlled switching parameters, duty-cycle and repetition rate, which guide the selection of peptides and the timing of their fragmentation. Using a novel algorithm which analyses all spectra simultaneously, it has been found that the majority of available peptides are not fragmented with the current switching scheme. Unfortunately, it is not practical to experiment with the mass spectrometer to determine optimal switching parameters. In this study, simulation coupled with intensity surface analysis was used as a method of evaluating mass spectrometer performance. Algorithms that mimic the mass spectrometer were created in order to simulate its response to various data sets. The simulations resulted in operating curves displaying the trade-off between quality and quantity of fragment spectra. The optimal operating curve demonstrated that the current switching scheme is sub-optimal, and that new switching parameters with fewer duty cycles and fewer repetitions should be selected.

Abstract:

High-throughput techniques are currently some of the most promising methods to study molecular biology, with the potential to improve medicine and enable new biological applications. In proteomics, the large scale study of proteins, the leading method is mass spectrometry. At present researchers can routinely identify and quantify thousands of proteins in a single experiment with the technique called shotgun proteomics. A challenge of these experiments is the computational analysis and the interpretation of the mass spectra. A shotgun proteomics experiment easily generates tens of thousands of spectra, each thought to represent a peptide from a protein. Due to the immense biological and technical complexity, however, our computational tools often misinterpret these spectra and derive incorrect peptides. As a consequence, the biological interpretation of the experiment relies heavily on the statistical confidence that we estimate for the identifications. In this thesis, I have included four articles from my research on the accuracy of the statistical confidence estimates in shotgun proteomics, how to accomplish and evaluate it. In the first two papers a new method to use pre-characterized protein samples to evaluate this accuracy is presented. The third paper deals with how to avoid statistical inaccuracies when using machine learning techniques to analyze the data. In the fourth paper, we present a new tool for analyzing shotgun proteomics results, and evaluate the accuracy of its statistical estimates using the method from the first papers. The work I have included here can facilitate the development of new and accurate computational tools in mass spectrometry-based proteomics. Such tools will help making the interpretation of the spectra and the downstream biological conclusions more reliable.

Abstract:

Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.
Title from electronic title page (viewed Oct. 10, 2007). " ... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry. Chapel Hill 2006." Discipline: Chemistry; Department/School: Chemistry.

Abstract:

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 16, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Dissertation advisor: Dr. Dong Xu. Vita. Includes bibliographical references.

Abstract:

 Proteomics is the systematic study of the proteome: the total protein expression of a cell or tissue under specified conditions. The multiplicity and complexity of proteins in cells requires sensitive, selective, and comprehensive methodologies for their distinction and characterization. Multidimensional liquid chromatography (MDLC) coupled with biological tandem mass spectrometry (MS/MS) is uniquely suited to fulfill those requirements and has become an indispensable tool in MS-based proteomics. Our laboratory has developed an online high-/low-pH reversed-phase/reversed-phase (RP–RP) LC system exhibiting fully automatable and reproducible performance. It is a promising alternative to the strong cation exchange/reversed-phase (SCX–RP) system commonly used in high-throughput comprehensive proteomics analyses. The first part of this Thesis (Chapter 2) describes the development of a variant of the high-/low-pH RP–RP platform—RP–SCX–RP—that integrates an additional SCX trap column between the two RP columns to enhance sample recovery. This new system allows the detection of larger numbers of hydrophilic peptides. Indeed, in the analyses of a lysate of Arabidopsis chloroplast proteins, it identified approximately 25% more non-redundant proteins than those identified using the previous version of the RP–RP system. The modified platform has been extended for the online removal of sodium dodecyl sulfate and other excess interference chemicals used in Isobaric Tags for Relative and Absolute Quantification (iTRAQ) reactions, thereby avoiding the need for time-consuming offline SCX clean-up prior to RP–RP separation in the quantitative proteomics analyses of crude biological samples at low-microgram levels. A novel online three-dimensional liquid chromatography (3DLC) system was derived from the RP–SCX–RP design, exhibiting remarkably enhanced orthogonality, resolution, and peak capacity. Peptides were separated in the first-dimension high-pH RP column based on their hydrophobicity, followed by sub-fractionation in the second-dimension SCX column, primarily based on charge; the third dimension was a typical low-pH RP separation, prior to MS analysis. The overall performance of the system was evaluated through analysis of a cell lysate of mouse embryonic fibroblasts. Relative to the two-dimensional high-/low-pH RP–RP system, the new 3D system yielded significant increases in the number of unique peptides and proteins identified, making it a good alternative to SCX–RP and high-/low-pH RP–RP as an efficient automated MDLC platform for high-throughput shotgun proteomics. An optimized and miniaturized variant of the three-dimensional LC platform was also developed. Its simplified setup and operation, by decreasing the number of six-port switching valves (from three to two) and the number of SCX fractionation steps, minimized both the potential sample loss and the total analysis time (by ca. 30%). Thus, a variety of novel, automatable, and robust RP–SCX–RP-based MDLC platforms have been developed for high-throughput qualitative and quantitative analysis. The performance of these systems complements conventional MDLC systems, with enhanced quality, quantity, reproducibility, and throughput of protein identification and quantification.
published_or_final_version
Chemistry
Doctoral
Doctor of Philosophy

Abstract:

Although molybdenum blue solutions have been known for more than twocenturies, an understanding of their chemical nature is only beginning to emerge.This dissertation aimed at elucidating the structural nature of the polydisperse,nanoscopic components in the solution phases and the solid states of partiallyreduced polyoxomolybdate (Mo-POM). The study offered at least fourcontributions to the area: (1) a rational protocol for the molecular recognition ofMo-POM with de novo organic hosts. (2) demonstration of kinetic precipitation ofa dynamic mixture of polyoxomolybdates and application of the technique to thestudy of the dynamic mixture by TEM (3) characterization of the Mo-POMnanostructures by an unusual combination of complementary analyticaltechniques. (4) a general approach for the synthesis of crown-ethers-containingtripodal molecules.The molecular recognition of Mo-POM with designer tripodal hexaminetris-crown ethers opened a window to the solution phase structures of Mo-POMnanoscopic components. Studies with a series of structurally analogous hostsprobed the relationship between the structure of the molecular host and theformation of nanostructures.An unusual combination of complementary analytical protocols: flow fieldflowfractionation, electron microscopy (transmission and scanning), andinductively coupled plasma – emission spectroscopy, was used to monitor thesolution-phase evolution of Mo-POM nanostructures. The crystallization – drivenformation of keplerate Mo-POM and solution-phase evolution of structurallyrelated nanoscopic species were apparent in the self-assembling process ofpartially reduced Mo-POM.

Abstract:

Tandem mass spectrometry is the most widely used technology to identify proteins in a complex biological sample, which produces a large number of spectra representative of protein subsequences named peptide. In this paper, we propose a hierarchical multi-stage framework, referred as DeepTag, to identify the peptide sequence for each given spectrum. Compared with the traditional one-stage generation, our sequencing model starts the inference with a selected high-confidence guiding tag and provides the complete sequence based on this guiding tag. Besides, we introduce a cross-modality refining module to asist the decoder focus on effective peaks and fine-tune with a reinforcement learning technique. Experiments on different public datasets demonstrate that our method achieves a new state-of-the-art performance in peptide identification task, leading to a marked improvement in terms of both precision and recall.

Abstract:

Maximizing food production with minimal negative effects on the environment remains a long-term challenge for sustainable food production. Microbial pathogens cause devastating diseases, minimizing crop losses by controlling plant diseases can contribute significantly to this goal. All plants possess an innate immune system that is activated after recognition of microbial-derived molecules. The fungal protein Eix induces defense responses in tomato and tobacco. Plants recognize Eix through a leucine-rich-repeat receptor- like-protein (LRR-RLP) termed LeEix. Despite the knowledge obtained from studies on tomato, relatively little is known about signaling initiated by RLP-type immune receptors. The focus of this grant proposal is to generate a foundational understanding of how the tomato xylanase receptor LeEix2 signals to confer defense responses. LeEix2 recognition results in pattern triggered immunity (PTI). The grant has two main aims: (1) Isolate the LeEix2 protein complex in an active and resting state; (2) Examine the biological function of the identified proteins in relation to LeEix2 signaling upon perception of the xylanase elicitor Eix. We used two separate approaches to isolate receptor interacting proteins. Transgenic tomato plants expressing LeEix2 fused to the GFP tag were used to identify complex components at a resting and activated state. LeEix2 complexes were purified by mass spectrometry and associated proteins identified by mass spectrometry. We identified novel proteins that interact with LeEix receptor by proteomics analysis. We identified two dynamin related proteins (DRPs), a coiled coil – nucleotide binding site leucine rich repeat (SlNRC4a) protein. In the second approach we used the split ubiquitin yeast two hybrid (Y2H) screen system to identified receptor-like protein kinase At5g24010-like (SlRLK-like) (Solyc01g094920.2.1) as an interactor of LeEIX2. We examined the role of SlNRC4a in plant immunity. Co-immunoprecipitation demonstrates that SlNRC4a is able to associate with different PRRs. Physiological assays with specific elicitors revealed that SlNRC4a generally alters PRR-mediated responses. SlNRC4a overexpression enhances defense responses while silencing SlNRC4 reduces plant immunity. We propose that SlNRC4a acts as a non-canonical positive regulator of immunity mediated by diverse PRRs. Thus, SlNRC4a could link both intracellular and extracellular immune perception. SlDRP2A localizes at the plasma membrane. Overexpression of SlDRP2A increases the sub-population of LeEIX2 inVHAa1 endosomes, and enhances LeEIX2- and FLS2-mediated defense. The effect of SlDRP2A on induction of plant immunity highlights the importance of endomembrane components and endocytosis in signal propagation during plant immune . The interaction of LeEIX2 with SlRLK-like was verified using co- immunoprecipitation and a bimolecular fluorescence complementation assay. The defence responses induced by EIX were markedly reduced when SlRLK-like was over-expressed, and mutation of slrlk-likeusing CRISPR/Cas9 increased EIX- induced ethylene production and SlACSgene expression in tomato. Co-expression of SlRLK-like with different RLPs and RLKs led to their degradation, apparently through an endoplasmic reticulum-associated degradation process. We provided new knowledge and expertise relevant to expression of specific be exploited to enhance immunity in crops enabling the development of novel environmentally friendly disease control strategies.