Separate & analyze: Improved mass spectrometry-based clinical proteomics by fractionation

Research output: Types of ThesisDoctoral ThesisInternal

Abstract

The proteome of clinical specimens is complex, and its entirety cannot be fully captured by current methods. By separating the proteome into fractions, it becomes possible to analyze it in greater depth, i.e. to identify and quantify proteins that are present in relatively low amounts. The separation of the proteome and the analytic disassembly into its components can be performed in various ways at different stages of an analysis. This thesis describes the realization of clinical proteomic studies using analytical methods for separation and fractionation of cells, proteins, and peptides.

In Chapter 2, we sampled isolated populations of epithelial and stromal cells in non-dysplastic and dysplastic/carcinogenic tissue samples taken from the esophagus of patients with different stages of the dysplastic progression from Barrett’s esophagus to esophageal adenocarcinoma. This approach allowed us to determine proteomic alterations in dysplastic/cancerous cell compartments, which quantitatively represent only a small proportion of the entire tissue specimen. Chapters 3 and 4 address biomarker discovery and validation in cerebrospinal fluid (CSF) using mass spectrometry-based proteomics. In preceding stages, we investigated the applicability of two different separation techniques. In Chapter 3, we removed albumin and immunoglobulins (Ig) from CSF through immunoaffinity depletion and conducted a label-free quantitative proteomic discovery study on depleted CSF fractions. In Chapter 4, we conducted a preceding pilot study to explore the use of two-dimensional (2D) chromatography to increase proteome coverage and depth in CSF samples. In Chapter 5, we applied 2D chromatography and ion mobility gas-phase fractionation to improve the detection of antibody variable region peptides. Chapter 6 involved the assessment of the phosphoproteome in fresh-frozen and FFPE brain tissue samples using IMAC phosphopeptide enrichment. The work of Chapter 6 was adapted and extended in Chapter 7, using the fraction of phosphopeptides as an epitope substrate for serum antibodies targeting tumor-specific phosphosites. The antibody-peptide binding assay presented in this chapter indicated the presence of a serum antibody against a glioblastoma multiforme-associated phosphosite. In Chapter 8, we introduced a data processing method for the analysis of quantitative LC-MS data. Parallel reaction monitoring (PRM) is a common and versatile quantitative MS technique, which was applied in Chapters 3, 4, 6, and 7. The tool presented in Chapter 8 enhances reproducibility, confidence, and speed during data analysis.

Original languageEnglish
Awarding Institution
  • Erasmus University Rotterdam
Supervisors/Advisors
  • Sillevis Smitt, Peter, Supervisor
  • Bergman, Jacques J. G. H. M., Supervisor, External person
  • Luider, Theo, Co-supervisor
Award date18 Jun 2024
Place of PublicationRotterdam
Print ISBNs978-94-6496-127-0
Publication statusPublished - 18 Jun 2024

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