Targeted Quantification and Glycosylation Profiling of Protein Biomarkers by Nanoprobe-based Affinity Mass Spectrometry
Date Issued
2016
Date
2016
Author(s)
Rosa, Mira Anne dela Cruz dela
Abstract
Disease biomarker development is plagued by lack of acceptable analytical methods, difficulty and cost for method development, overwhelming need for validation on a large population and the poor performance of biomarkers under development. In this dissertation, we introduce alternative methods for protein biomarker discovery and validation that encompasses quantification and post-translational modification (PTM) profiling in non-invasive specimens. We first designed surfactant-coated monodisperse magnetic nanoprobes to improve detection sensitivity (MNP@IGEPAL). Following oriented antibody immobilization for increased specificity and immuno-activity, the MNP@IGEPAL were found to be superior in solvent dispersibility and enrichment efficiency compared to nanoprobes obtained by conventional co-precipitation method (MNPCP). We then coupled the MNP@IGEPAL-based enrichment to multiple reaction monitoring mass spectrometry (MRM-MS) for multiplexed quantification of alpha-fetoprotein (AFP) and golgi membrane protein 1 (GOLM1), which are low-abundant biomarkers in human serum. The method was found to be sensitive, have good analytical merits (average precision and accuracy of 15%) and wide dynamic range. This method was applied to qualify the biomarkers in serum of liver disease patients, where we found that AFP and GOLM1 had similar diagnostic accuracy for hepatocellular carcinoma (HCC), although AFP has a higher false negative rate (sensitivity = 22%). More importantly, we found complementarity between AFP and GOLM1, where GOLM1 was found to be elevated in 69% of patients with low AFP concentration (<20 ng/mL). To supplement disease diagnoses based on protein concentration, we designed a one-pot dual nanoprobe-based mass spectrometry method to simultaneously quantify the protein and profile its post-translational modification (PTM). Using AFP and another clinically-relevant protein, haptoglobin (Hp), we were able to quantify the protein and profile its glycoforms with superior speed and sensitivity and minimal amount of sample. In addition to obtaining individual biosignatures of AFP in HCC patients, we were able to identify a total of 59 glycoforms, 12 of which were identified on AFP for the first time. Ultimately, we were able to develop methods that can improve disease diagnosis, which can be applied to other cancers and diseases for large-scale biomarker triaging, qualification and PTM profiling.
Subjects
magnetic nanoparticles
protein quantification
glycosylation profiling
hepatocellular carcinoma
mass spectrometry
SDGs
Type
thesis
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