Towards high throughput and spatiotemporal proteomics : analytical workflows and quantitative label-free mass spectrometry

A large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject__s phenotype. Proteomics describes the state of the system from the perspective of expression, structure, localization, interaction and function of the proteins. It is a complex field which requires a combination of various disciplines such as cellular biology and biochemistry for sample preparation, analytical chemistry for sample measurement and bioinformatics for the processing of data. Different chapters of this thesis illustrate various aspects of the proteomics pipeline and emphasize the importance and connection between them. Therefore the information in this dissertation can be divided into three major parts depicting these aspects of a proteomics experiment. First, sample preparation for proteomics has been addressed through two studies aimed at decreasing sample complexity and increasing proteome coverage. The second part is dedicated mostly to the technical step of the mass spectrometry measurement and the analysis of obtained spectra. The final part describes example- and proof-of-principle applications.UBL - phd migration 201

Identifying Proteins in Zebrafish Embryos Using Spectral Libraries Generated from Dissected Adult Organs and Tissues

Proteomic

Towards high throughput and spatiotemporal proteomics : analytical workflows and quantitative label-free mass spectrometry

A large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject__s phenotype. Proteomics describes the state of the system from the perspective of expression, structure, localization, interaction and function of the proteins. It is a complex field which requires a combination of various disciplines such as cellular biology and biochemistry for sample preparation, analytical chemistry for sample measurement and bioinformatics for the processing of data. Different chapters of this thesis illustrate various aspects of the proteomics pipeline and emphasize the importance and connection between them. Therefore the information in this dissertation can be divided into three major parts depicting these aspects of a proteomics experiment. First, sample preparation for proteomics has been addressed through two studies aimed at decreasing sample complexity and increasing proteome coverage. The second part is dedicated mostly to the technical step of the mass spectrometry measurement and the analysis of obtained spectra. The final part describes example- and proof-of-principle applications

Cloud Parallel Processing of Tandem Mass Spectrometry Based Proteomics Data

Proteomic

Особливості функціонального стану печінки у дітей з хворобою Крона

Objective: Investigation of the functional state of the liver in the children with Crohn's disease activity and taking into account the length of bowel disease. Patients and methods. The study has involved 45 children with Crohn's disease in an age of 3 to 18 years old. Clinical study was conducted under the order of Ministry of Health of Ukraine (No.59 from 29.01.2013). We examined the degree of the alanine aminotransferase (ALT), the aspartate aminotransferase, (AST), the g'glutamyl transpeptidase (GGT), the alkaline phosphatase (ALP), the direct bilirubin, the crude protein, the proteynohramma, for the study of liver parenchyma used ultrasound. Results. It was found that an increase in transaminases more than one episode had 42.2% of children with CD. Violation of the functional state of the liver in children with CD is describe by predominantly cytolytic syndrome — a severe form of the disease increased ALT>40 IU/l were observed in 50% (n=6), and at moderate form — in 9% (n=3) children (χ2=9,2; p=0.008); increase in AST>40 IU/l more likely to have children with a severe form of the disease — 58,3% (n=7) than with moderate 12,2% (n=4) (χ2=10,18; p=0.001). Cytolytic option biochemical changes significantly more likely to have children with a severe form of the disease than the disease of moderate severity, — 8 (80%) and 3 (33,3%) (χ2=8,03; p=0.008), respectively. Conclusions. Violation of the functional state of the liver mainly in the form of a periodic hypertransaminasemia occurred in 42.2% of children with CD. The children with CD have version prevails cytolytic violation functional state of the liver, the frequency is higher in children with a severe form of CD (р<0,05). Key words: children, Crohn's disease, liver functional status, activity, duration of illness.Цель: исследовать особенности функционального состояния печени у детей с болезнью Крона (БК) в зависимости от активности и длительности заболевания. Пациенты и методы. Было обследовано 45 детей с БК в возрасте от 3 до 18 лет. Комплекс обследований проводился согласно приказу МЗ Украины №59 от 29.01.2013. У детей с БК были исследованы функциональное состояние печени — уровни аланинаминотрансферазы (АЛТ), аспартатаминотрансферазы (АСТ), гамма-глютамилтранспептидазы (ГГТ), щелочная фосфатаза (ЩФ), общий билирубин, общий белок, протеинограмма; для оценки эхоструктуры печени проводили ультразвуковое исследование. Результаты. Установлено, что повышение трансаминаз больше одного эпизода имели 42,2% детей с БК. Нарушение функционального состояния печени у детей с БК преимущественно характеризуется цитолитическим синдромом — при тяжелой форме заболевания повышение уровня АЛТ >40 Ед/л наблюдалось у 50% (n=6), а при среднетяжелой форме — у 9% (n=3) детей (χ2=9,2; р=0,008); повышение уровня АСТ>40 Ед/л чаще имели дети с тяжелой формой заболевания, чем со среднетяжелой, — 58,3% (n=7) и 12,2% (n=4) соответственно (χ2=10,18; р=0,001). Цитолитический вариант биохимических изменений достоверно чаще имели дети с тяжелой формой заболевания, чем со среднетяжелой, — 8 (80%) и 3 (33,3%) соответственно (χ2=8,03; р=0,008). Выводы. Нарушение функционального состояния печени, преимущественно в виде периодической гипертрансаминаземии, имело место у 42,2% детей с БК. Установлено, что у детей с БК превалирует цитолитический вариант нарушения функционального состояния печени, частота котрого достоверно выше у детей с тяжелой формой БК. Ключевые слова: дети, болезнь Крона, печень, клиника, активность и длительность заболевания.Мета: дослідити особливості функціонального стану печінки у дітей із хворобою Крона (ХК) залежно від активності і тривалості захворювання. Пацієнти і методи. Було обстежено 45 дітей із ХК віком від 3 до 18 років. Комплексне обстеження проводилося відповідно до наказу МОЗ України №59 від 29.01.2013. У дітей із ХК досліджувалися функціональний стан печінки — рівні аланінамінотрансферази (АЛТ), аспартатамінотрансферази (АСТ), гамма"глютамілтранспептидази (ГГТ), лужна фосфатаза (ЛФ), загальний білірубін, загальний білок, протеїнограма; для оцінки ехоструктури печінки проводили ультразвукове дослідження. Результати. Установлено, що підвищення трансаміназ більше одного епізоду мали 42,2% дітей із ХК. Порушення функціонального стану печінки у дітей із ХК переважно характеризується цитолітичним синдромом — за важкої форми захворювання підвищення рівня АЛТ>40 Ед/л спостерігалося у 50% (n=6), а за середньоважкої форми — у 9% (n=3) дітей (χ2=9,2; р=0,008); підвищення рівня АСТ>40 Ед/л частіше мали діти з важкою формою захворювання, ніж із середньоважкою, — 58,3% (n=7) і 12,2% (n=4) відповідно (χ2=10,18; р=0,001). Цитолітичний варіант біохімічних змін достовірно частіше мали діти з важкою формою захворювання, ніж із середньоважкою, — 8 (80%) і 3 (33,3%) відповідно (χ2=8,03; р=0,008). Висновки. Порушення функціонального стану печінки, переважно у вигляді періодичної гіпертрансаміназемії, спостерігається у 42,2% дітей із ХК. Установлено, що у дітей із ХК превалює цитолітичний варіант порушення функціонального стану печінки, частота якого достовірно вища у дітей із важкою формою ХК. Ключові слова: діти, хвороба Крона, функціональний стан печінки, активність та тривалість хвороби

A Novel Mass Spectrometry Cluster for High-Throughput Quantitative Proteomics

We have developed and implemented a novel mass spectrometry (MS) platform combining the advantages of high mass accuracy and resolving power of Fourier transform ion cyclotron resonance (FTICR) with the economy and speed of multiple ion traps for tandem mass spectrometry. The instruments are integrated using novel algorithms and software and work in concert as one system. Using chromatographic time compression, a single expensive FTICR mass spectrometer can match the throughput of multiple relatively inexpensive ion trap instruments. Liquid chromatography (LC)-mass spectrometry data from the two types of spectrometers are aligned and combined to hybrid datasets, from which peptides are identified using accurate mass from the FTICR data and tandem mass spectra from the ion trap data. In addition, the high resolving power and dynamic range of a 12 tesla FTICR also allows precise label-free quantitation. Using two ion traps in parallel with one LC allows simultaneous MS/MS experiments and optimal application of collision induced dissociation and electrontransfer dissociation throughout the chromatographic separation for increased proteome coverage, characterization of post-translational modifications and/or simultaneous measurement in positive and negative ionization mode. An FTICR-ion trap cluster can achieve similar performance and sample throughput as multiple hybrid ion trap-FTICR instruments, but at a lower cost. We here describe the first such FTICR-ion trap cluster, its performance and the idea of chromatographic compression. (J Am Soc Mass Spectrom 2010, 21, 1002-1011) (C) 2010 American Society for Mass SpectrometryProteomic

Protein fractionation for quantitative plasma proteomics by semi-selective precipitation

Blood plasma is a highly complex mixture of proteins, metabolites and lipids, and a rich source of potential biomarkers for a range of diseases and conditions. The wide range in protein abundance poses a tremendous challenge for plasma proteomics. However, as a relatively small number of proteins make up most of the total protein pool, the concentration range can be compressed by depletion of abundant proteins, such as albumin. To reduce sample complexity and increase the protein coverage, we have developed a sample preparation method based on semi-selective precipitation with acetonitrile at different pH and built a data analysis pipeline, combining different search strategies. The method we propose is reproducible and easily parallelised (high throughput), and may be well suited to fractionate plasma for label-free quantitative proteomics in large clinical studies. Up to 90% of albumin and other abundant proteins were removed by adding an equal volume of acetonitrile to the samples adjusted to pH 5

Protein fractionation for quantitative plasma proteomics by semi-selective precipitation

Blood plasma is a highly complex mixture of proteins, metabolites and lipids, and a rich source of potential biomarkers for a range of diseases and conditions. The wide range in protein abundance poses a tremendous challenge for plasma proteomics. However, as a relatively small number of proteins make up most of the total protein pool, the concentration range can be compressed by depletion of abundant proteins, such as albumin. To reduce sample complexity and increase the protein coverage, we have developed a sample preparation method based on semi-selective precipitation with acetonitrile at different pH and built a data analysis pipeline, combining different search strategies. The method we propose is reproducible and easily parallelised (high throughput), and may be well suited to fractionate plasma for label-free quantitative proteomics in large clinical studies. Up to 90% of albumin and other abundant proteins were removed by adding an equal volume of acetonitrile to the samples adjusted to pH 5

Partially sequenced organisms, decoy searches and false discovery rates.

Tandem mass spectrometry is commonly used to identify peptides, typically by comparing their product ion spectra with those predicted from a protein sequence database and scoring these matches. The most reported quality metric for a set of peptide identifications is the false discovery rate (FDR), the fraction of expected false identifications in the set. This metric has so far only been used for completely sequenced organisms or known protein mixtures. We have investigated whether FDR estimations are also applicable in the case of partially sequenced organisms, where many high-quality spectra fail to identify the correct peptides because the latter are not present in the searched sequence database. Using real data from human plasma and simulated partial sequence databases derived from two complete human sequence databases with different levels of redundancy, we could demonstrate that the mixture model approach in PeptideProphet is robust for partial databases, particularly if used in combination with decoy sequences. We therefore recommend using this method when estimating the FDR and reporting peptide identifications from incompletely sequenced organisms