Thesis

Automated sample preparation for streamlined proteomic profiling of clinical specimens

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Abstract

The genetic information of all life is encoded within DNA molecules that are translated into functional entities, so-called proteins. They are responsible for operating and controlling a vast array of molecular mechanisms in any biological system and ubiquitous in (patho)physiology as a result. Besides, proteins are the primary target of drugs and can have a central role as biomarkers for diagnostic, prognostic, or predictive purposes. Here, many regulatory mechanisms and spatiotemporal influences prevent an accurate prediction of a proteins’ abundance and its associated functionality based on the genome information alone. Nowadays, it has become possible to measure and quantify thousands of proteins simultaneously, however, involving comprehensive sample preparation procedures. Currently, no universally standardized method enables a routine application of proteome profiling in a clinical environment. In this thesis, an automated workflow for the efficient processing of the most common and quantity-limited specimens is described. In order to demonstrate the usefulness of the end-to- end pipeline, which was termed autoSP3, it was applied to the proteome profiling of histologically defined and WHO recognized growth patterns of pulmonary adenocarcinoma (ADC) that currently have a limited clinical implication. Secondly, we investigated the proteome composition of a molecularly well-defined cohort of Ependymoma (EPN) pediatric brain tumors. Despite the availability of substantial NGS data and their ability to differentiate nine distinct subgroups, the majority of tumors remained without a functional insight. Here, the proteome profiling could provide a missing link and emphasize several subgroup-specific protein targets. In summary, this thesis describes the optimization of SP3 and its automation into a robust and cost-efficient pipeline for quantity-limited sample preparation and biological insight into the proteome composition of ADC growth patterns and EPN tumor subgroups.

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Selecting a sample preparation strategy for mass spectrometry-based proteomics is critical to the success of quantitative workflows. Here we present a universal, solid-phase protein preparation (USP³) method which is rapid, robust and scalable, facilitating high-throughput protein sample preparation for bottom-up and top-down mass spectrometry (MS) analysis. This technique builds upon the single-pot solid-phase-enhanced sample preparation (SP3) where we now demonstrate its scalability (low to high μg protein) and the influence of variables such as bead and enzyme amounts on the efficiency of protein digestion. We also incorporate acid hydrolysis of DNA and RNA during complete proteome extraction resulting in a more reliable method that is simple and easy to implement for routine and high throughput analysis of proteins. We benchmarked the performance of this technique against filter-aided sample preparation (FASP) using 30 μg total HeLa protein lysate. We also show that the USP³ method is compatible with top-down MS where we reproducibly detect over 1800 proteoforms from 50 μg HeLa protein lysate. The USP³ protocol allows for efficient and reproducible data to be generated in a cost-effective and robust manner with minimal down time between sample collection and analysis by MS.
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Recent developments in proteomics have enabled signaling studies where >10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited in throughput, reproducibility, and robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, and network inference analysis. To address these challenges, we introduce rapid-robotic-phosphoproteomics (R2-P2), an end-to-end automated method that uses magnetic particles to process protein extracts to deliver mass spectrometry-ready phosphopeptides. R2-P2 is robust, versatile, high-throughput, and achieves higher sensitivity than classical protocols. To showcase the method, we applied it, in combination with data-independent acquisition mass spectrometry, to study signaling dynamics in the mitogen-activated protein kinase (MAPK) pathway in yeast. Our results reveal broad and specific signaling events along the mating, the high-osmolarity glycerol, and the invasive growth branches of the MAPK pathway, with robust phosphorylation of downstream regulatory proteins and transcription factors. Our method facilitates large-scale signaling studies involving hundreds of perturbations opening the door to systems-level studies aiming to capture signaling complexity.
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The understanding and management of Wilson disease (WD) have dramatically improved since the first description of the disease by K. Wilson more than a century ago. However, the persistent long delay between the first symptoms and diagnosis emphasizes challenges in diagnosing earlier this copper overload disorder. As a treatable disease, WD should be detected early in the course of the disease by any health professionals at any care level, but the rare prevalence of the disease explains the lack of awareness of referring physicians. The most important challenge is to train physicians to recognize atypical or rare symptoms of WD that will lead to discuss the diagnosis more systematically. Atypia can come from the age of onset, the liver [non-alcoholic steatohepatitis (NASH) presentation], the central or peripheral nervous system (neuropathy, epilepsy, sleep disorders…) or may be due to lesions of other organs (renal manifestations, osteo-articular disorders or endocrine disturbances). Isolated biological anomalies, rare radiological findings or inadequate interpretation of copper test may also lead to misdiagnosis. The second challenge is to confirm the diagnosis faster and more effectively so as not to delay the initiation of treatment, and expand family screening as the genetic prevalence is higher than previously expected. Generalization of the exchangeable copper assay and the next generation sequencing (NGS) are two promising ways to overcome this ultimate challenge. By drawing attention to the earliest and rare symptoms and to new biomarkers and diagnostic tools, we hope that this article will increase diagnostic awareness and reduce delays so that patients can start their treatment earlier in the course of the illness and thus have a better disease prognosis.
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The polycomb repressive complex 2 (PRC2) is a chromatin-associated methyltransferase catalyzing mono-, di-, and trimethylation of lysine 27 on histone H3 (H3K27). This activity is required for normal organismal development and maintenance of gene expression patterns to uphold cell identity. PRC2 function is often deregulated in disease and is a promising candidate for therapeutic targeting in cancer. In this review, we discuss the molecular mechanisms proposed to take part in modulating PRC2 recruitment and shaping H3K27 methylation patterns across the genome. This includes consideration of factors influencing PRC2 residence time on chromatin and PRC2 catalytic activity with a focus on the mechanisms giving rise to regional preferences and differential deposition of H3K27 methylation. We further discuss existing evidence for functional diversity between distinct subsets of PRC2 complexes with the aim of extracting key concepts and highlighting major open questions toward a more complete understanding of PRC2 function.
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Peptide cleanup is essential for the removal of contaminating substances that may be introduced during sample preparation steps in bottom-up proteomic workflows. Recent studies have described benefits of carboxylate-modified paramagnetic particles over traditional reversed-phase methods for detergent and polymer removal, but challenges with reproducibility have limited the widespread implementation of this approach among laboratories. To overcome these challenges, the current study systematically evaluated key experimental parameters regarding the use of carboxylate-modified paramagnetic particles and determined those that are critical for maximum performance and peptide recovery and those for which the protocol is tolerant to deviation. These results supported the development of a detailed, easy-to-use standard operating protocol, termed SP2, which can be applied to remove detergents and polymers from peptide samples while concentrating the sample in solvent that is directly compatible with typical LC-MS workflows. We demonstrate that SP2 can be applied to phosphopeptides and glycopeptides, and that the approach is compatible with robotic liquid handling for automated sample processing. Altogether, the results of this study and accompanying detailed operating protocols for both manual and automated processing are expected to facilitate reproducible implementation of SP2 for various proteomics applications and will especially benefit core or shared resource facilities where unknown or unexpected contaminants may be particularly problematic.