Proteomics of neurodegenerative diseases using novel online isoelectric point separation

The work presented in this thesis describes an instrument, developed for separation of proteins and peptides based on corresponding isoelectric point (pI) values, to empower mass spectrometry-based proteomics analysis. The main objectives are the instrument development and optimization, as well as clinical applications in biomarker discovery, particularly for neurodegenerative disorders. The thesis is based on five scientific papers that focus on three main stages; (i) development and optimization of the device for separation of proteins and peptides by pI that are well integrated with tandem mass spectrometry (ii) optimization of the device for intact blood plasma protein fractionation for application in biomarker discovery, and (iii) biomarker discovery in blood plasma for early diagnosis of Alzheimer disease. Within the first part of the work, a novel multiple-junction capillary isoelectric focusing fractionator (MJ-CIEF) is developed (Paper I). Subsequently, the resolving power and reproducibility of fractionation are improved, and in addition, a novel algorithm is developed to calculate the identified peptides’ pI (Paper II). Moreover, to achieve the aim of deep proteomics, a multi-parameter optimization of the LC-MS/MS pipeline is performed (Paper III). In the second part, an online desalinator is developed and coupled to the device, for direct buffer-exchange and isoelectric separation of intact human blood plasma/serum proteins. The developed pipeline achieves the increased depth of the proteome analysis and provides additional information on the pI of identified proteins, as another dimension of information in biomarker discovery by proteomics (Paper IV). The last part of the thesis is focused on the application of the developed method in biomarker discovery for early diagnosis of Alzheimer disease. A panel of new potential biomarkers is introduced based on the abundance changes, as well as shifts in the pI values. By means of the pI information, the protein concentration in a narrow pH range around 7.4 reveals increased levels in patients with progressive Alzheimer disease compared to stable ones. Proteome analysis of this particular pI region also suggests several potential proteins as biomarkers for early diagnosis of the disease (Paper V). Taken together, this thesis demonstrates emerging applications of peptides and proteins fractionation by pI in deep proteomics. The development of MJ-CIEF facilitates online separation of peptides and proteins from small amounts of samples in a fast format, automatable, cost-effective and compatible with mass spectrometry analysis. Further biomarker discovery in the narrow range of pH around 7.4 of blood proteome is suggested for early diagnosis of neurodegenerative diseases

SURFACE ENABLED LAB-ON-A-CHIP (LOC) DEVICE FOR PROTEIN DETECTION AND SEPARATION

Sensitive and selective chemical/biological detection/analysis for proteins is essential for applications such as disease diagnosis, species phenotype identification, product quality control, and sample examination. Lab-on-a-chip (LOC) device provides advantages of fast analysis, reduced amount of sample requirements, and low cost, to magnificently facilitate protein detection research. Isoelectric focusing (IEF) is a strong and reliable electrophoretic technique capable of discerning proteins from complex mixtures based on the isoelectric point (pI) differences. It has experienced plenty of fruitful developments during previous decades which has given it the capability of performing with highly robust and reproducible analysis. This progress has made IEF devices an excellent tool for chemical/biological detection/analysis purposes. In recent years, the trends of simple instrument setting, rapid analysis, small sample requirement, and light labor intensity have inspired the LOC concept to be combined with IEF to evolve it into an “easily-handled chip with hours of analysis” from the earlier method of “working with big and heavy machines in a few days.” Although IEF is already a mature technique being applied, further LOC-IEF developments are still experiencing challenges related to its limitations such as miniaturizing the device scale without harming the resolving/discerning ability. With the facilitation of newly technologically advanced/improved fabrication tools, it is completely possible to address challenges and approach new limits of LOC-IEF. In this dissertation, a surface enabled printing technique, which can transfer liquid to a surface with prescribed patterns, was firstly introduced to IEF device fabrication. By employing surface enabled printing, a surface enabled IEF (sIEF) device running at a scale of 100 times smaller than those previously reported was designed and fabricated. Commercial carrier ampholytes (PharmalyteTM) with different pH range were engaged to generate a continuous pH gradient on sIEF device. Device design and optimized fabrication conditions were practically investigated; establishment of pH gradient was verified by fluorescent dyes; dependencies of electric field strength and carrier ampholytes concentration were systematically examined. To further optimize the sIEF system, dependencies of surface treatment and additive chemicals were explored. Fluorescent proteins and peptides were tested for the separation capability of sIEF. Finally, the well optimized sIEF system was used as a tool for real protein (hemoglobin variants and monoclonal antibody isoforms) separations. Hemoglobin variants test results revealed that sIEF is capable of separating amphoteric species with pI difference as small as 0.2. Monoclonal protein tests demonstrated the capability of sIEF to be a ready-to-use tool for protein structural change monitoring. In conclusion, this new sIEF approach has lower applied voltages, smaller sample requirements, a relatively quick fabrication process, and reusability, making it more attractive as a portable, user-friendly platform for qualitative protein detection and separation

A comprehensive guide for performing sample preparation and top-down protein analysis

© 2017 by the authors. Methodologies for the global analysis of proteins in a sample, or proteome analysis, have been available since 1975 when Patrick O'Farrell published the first paper describing two-dimensional gel electrophoresis (2D-PAGE). This technique allowed the resolution of single protein isoforms, or proteoforms, into single 'spots' in a polyacrylamide gel, allowing the quantitation of changes in a proteoform0s abundance to ascertain changes in an organism's phenotype when conditions change. In pursuit of the comprehensive profiling of the proteome, significant advances in technology have made the identification and quantitation of intact proteoforms from complex mixtures of proteins more routine, allowing analysis of the proteome from the 'Top-Down'. However, the number of proteoforms detected by Top-Down methodologies such as 2D-PAGE or mass spectrometry has not significantly increased since O'Farrell's paper when compared to Bottom-Up, peptide-centric techniques. This article explores and explains the numerous methodologies and technologies available to analyse the proteome from the Top-Down with a strong emphasis on the necessity to analyse intact proteoforms as a better indicator of changes in biology and phenotype. We arrive at the conclusion that the complete and comprehensive profiling of an organism0s proteome is still, at present, beyond our reach but the continuing evolution of protein fractionation techniques and mass spectrometry brings comprehensive Top-Down proteome profiling closer

Proteoforms

A proteoform is the basic unit in a proteome, defined as its amino acid sequence + post-translational modifications + spatial conformation + localization + cofactors + binding partners + a function, which is the final functional performer of a gene. Studies on proteoforms offer in-depth insights and can lead to the discovery of reliable biomarkers and therapeutic targets for effective prediction, diagnosis, prognostic assessment, and therapy of disease. This book focuses on the concept, study, and applications of proteoforms. Chapters cover such topics as methodologies for identifying and preparing proteoforms, proteoform pattern alteration in pituitary adenomas, and proteoforms in leukemia

Platforms and Protocols for the Multidimensional Microchip Electrophoretic Analysis of Complex Proteomes

The need for rapid, portable and high-throughput systems in proteomics is now prevalent because of demands for generating new protein-based disease biomarkers. However, 2-D protein profile patterns are lending themselves as potential diagnostic tools for biomarker discovery. It is difficult to identify protein biomarkers which are low abundant in the presence of highly abundant proteins, especially in complex biological samples like serum. Protein profiles from 2-D separation of the protein content of cells or body fluids, which are unique to certain physiological or pathological states, are currently available on internet databases. In this work, we demonstrate the ability to separate a complex biological sample using low cost, disposable, polymer-based microchips suitable for a multidimensional techniques that employed sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) in the 1st dimension and micellar electrokinetic capillary chromatography (MEKC) or microemulsion electrokinetic capillary chromatography (MEEKC) in the 2nd dimension. The peak capacity generated by this microchip technique was about 3-fold greater compared to conventional 2-D separation methods and the complete separation time was 60X faster. To minimize electroosmotic flow effects, we dynamically coated the channels with methylhydroxyethyl cellulose. Proteins were detected by laser-induced fluorescence following their labeling with dyes. To mitigate challenges posed by labeling the proteins, we investigated the use of a label-free technique that relied upon conductivity measurements. Preliminary data are presented on the fabrication of on-chip electrodes using a conductive SU-8 polymer via lithography

Integrating Micro-Scale Separations to Matrix Assisted Laser Desorption and Ioniation Time of Flight Mass Spectrometry (MALDI-TOF-MS) for Protein Analysis

This dissertation describes the integration of micro-scale separations to matrix assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI TOF MS) for protein analysis. MALDI MS provides unsurpassed accurate mass measurements of intact bio-molecules, for example peptides and proteins, which in turn generate high molecular specificity enabling the identity, function and structure of these molecules to be characterized. However, in order to realize the full potential of MS in proteomic studies, integrated sample processing on automated and high throughput platforms is required to address the complexity, diversity and the dynamic range of proteomic analysis. The work described here contributes towards the development of automated and high throughput micro-total analysis systems (µ-TAS) for proteomics. An overview of mass spectrometry instrumentation and techniques used in protein analysis is presented to highlight the significance of the work described. Microfluidics devices can serve as automated and high throughput platforms for integrating proteomics sample processing steps such as whole cell lyses, enrichment, solubilization, denaturation, protein separations, proteolytic digestion and chromatographic separations of peptides prior to MALDI TOF MS analysis. Therefore, coupling microfluidics devices to biological mass spectrometry is the first logical step towards developing fully integrated and automated systems for protein analysis. On-line and off-line approaches for analysis from microfluidic devices are discussed. The development of a specially tailored rotating ball inlet for automated on-line MALDI MS sample introduction from an electrophoresis-based separation platform is described. Electrophoresis-based micro-scale separations of peptides on fused silica capillary and polymer-based microfluidic devices were coupled to on-line MALDI TOF MS using a rotating ball inlet. The rotating ball inlet allowed for individual technique optimization and automation thereby eliminating the need for fractionation and routine MALDI sample preparation. High throughput solid phase micro-reactors for efficient enzymatic cleavages and improved protein identification with MALDI MS in a microfluidic device were also developed for incorporation in an integrated protein analysis microfluidic system. Future work that outlines the framework and focus geared towards integrating the modules discussed in this dissertation into a functional micro-total analysis system for protein sample processing is discussed

New analytical tools combining gel electrophoresis and mass spectrometry

Proteomics has been one of the main projects challenging biological and analytical chemists for many years. The separation, identification and quantification of all the proteins expressed within biological systems remain the main objectives of proteomics. Due to sample complexity, the development of fractionation, separation, purification and detection techniques that possess appropriate resolution to separate a large number of proteins, as well as being sensitive and fast enough for high throughput protein analysis are required. The objective of this thesis was to develop new separation strategies for protein/peptide fractionation using gel electrophoresis and its further detection by mass spectrometry analysis. A multi-electrode set up based on OFFGEL electrophoresis was developed. The objective was to provide a more efficient application of the electric field for fast and improved protein separation. The results showed that using a multi-electrode setup provides not only a higher protein separation but also better protein collection efficiency. Electrophoretic separation using an ultra narrow pH gradient (UNPG) gel was adapted for a three-well OFFGEL device for fast sample purification and desalting. Purification of an E.coli extract was applied to demonstrate that electrophoretic separation with UNPG gels provides an efficient strategy for fast purification of complex biological samples and can be utilized as a preparative technique in proteomics. UNPG gels were also used to separate charge molecules taking place in a new electro-elution device. The molecules were washed from the gel surface by an aqueous buffer and collected for further analysis by mass spectrometry. The electroelution device provides a fast approach that avoids time-consuming steps of extraction from the polyacrylamide gel. An electrostatic spray ionization (ESTASI) mass spectrometry technique was developed in our group to ionize sample solutions on different substrates. ESTASI has been here coupled to isoelectric focusing and demonstrated to be a powerful tool, which can improve the detection sensitivity compared to standard visualizing protocols. Paper-ESTASI-MS was developed and applied for rapid perfume analysis of six authentic fragrances as a high throughput and sample preparation-free method. ESTASI was also applied for quantitative analysis of caffeine in different beverages using a standard addition strip. The results were compared to classical standard addition methods for MS or LC. It was shown that the strip strategy could be utilized for fast and accurate food analysis and quality control. Desalting and direct analysis of samples from ZipTip by ESTASI-MS has been demonstrated

Proteomes Are of Proteoforms: Embracing the Complexity

Proteomes are complex—much more so than genomes or transcriptomes. Thus, simplifying their analysis does not simplify the issue. Proteomes are of proteoforms, not canonical proteins. While having a catalogue of amino acid sequences provides invaluable information, this is the Proteome-lite. To dissect biological mechanisms and identify critical biomarkers/drug targets, we must assess the myriad of proteoforms that arise at any point before, after, and between translation and transcription (e.g., isoforms, splice variants, and post-translational modifications [PTM]), as well as newly defined species. There are numerous analytical methods currently used to address proteome depth and here we critically evaluate these in terms of the current ‘state-of-the-field’. We thus discuss both pros and cons of available approaches and where improvements or refinements are needed to quantitatively characterize proteomes. To enable a next-generation approach, we suggest that advances lie in transdisciplinarity via integration of current proteomic methods to yield a unified discipline that capitalizes on the strongest qualities of each. Such a necessary (if not revolutionary) shift cannot be accomplished by a continued primary focus on proteo-genomics/-transcriptomics. We must embrace the complexity. Yes, these are the hard questions, and this will not be easy…but where is the fun in easy?Natural Sciences and Engineering Research Council of Canad