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.

List of scientific papers

I. Chingin K., Astorga-Wells J., Pirmoradian M., Lavold T., Zubarev R. A. (2012) Separation of Polypeptides by Isoelectric Point Focusing in Electrospray-Friendly Solution Using a Multiple-Junction Capillary Fractionator. Analytical Chemistry. 84: 6856-6862.
https://doi.org/10.1021/ac3013016

II. Pirmoradian M., Zhang B., Chingin K., Astorga-Wells J., Zubarev R. A. (2014) Membrane-Assisted Isoelectric Focusing Device As a Micropreparative Fractionator for Two-Dimensional Shotgun Proteomics. Analytical Chemistry. 86 (12), 5728-5732.
https://doi.org/10.1021/ac404180e

III. Pirmoradian M., Budamgunta H., Chingin K., Zhang B., Astorga-Wells J., Zubarev R. A. (2013) Rapid and Deep Human Proteome Analysis by Single-Dimension Shotgun Proteomics. Mol Cell Proteomics. 12: 3330-3338.
https://doi.org/10.1074/mcp.O113.028787

IV. Pirmoradian M., Astorga-Wells J., Zubarev R. A. (2015) Multijunction Capillary Isoelectric Focusing Device Combined with Online Membrane-Assisted Buffer Exchanger Enables Isoelectric Point Fractionation of Intact Human Plasma Proteins for Biomarker Discovery. Analytical Chemistry. 87(23):11840-6.
https://doi.org/10.1021/acs.analchem.5b03344

V. Pirmoradian M., Årsland D., Zubarev R. A. Blood Proteins with Isoelectric Point close to 7.4 as Alzheimer Disease Biomarker. [Manuscript]