Dahabiyeh, Lina
(2016)
Targeted mass spectrometry for plasma glycoprotein profiling in pre-eclampsia.
PhD thesis, University of Nottingham.
Abstract
Pre-eclampsia is a common hypertensive disorder of pregnancy that substantially affects maternal and neonatal morbidity and mortality worldwide. Despite decades of research, the aetiology of the disease remains poorly understood, and the clinical management of it is hampered by the lack of reliable diagnostic tests and effective therapy. Several screening tests have been suggested for the prediction of preeclampsia; however, none possess the sufficient specificity and sensitivity. Moreover, multiple pathways are known to be involved in the pathogenesis of pre-eclampsia, so it is very unlikely that a single or a small group of biomarkers will accurately predict the disease. In this thesis, two separate targeted LC-MS/MS methods were developed and validated to quantify plasma glycoproteins in pre- eclampsia to increase the pool of preeclampsia biomarker candidates and explore new pathways associated with the disease. The first method was hypothesis-driven and aimed to quantify the oxidation level of the plasma glycoprotein angiotensinogen (AGT), which has been proposed to be involved in the increased blood pressure characteristic of pre-eclampsia. The second method was hypothesis-generating and aimed to detect glycoprotein fold changes between different disease conditions using a simple and cost-effective conventional LC-MS/MS workflow.
For both methods, a reproducible workflow for efficient glycoprotein/AGT extraction from human plasma was developed by coupling ConA lectin affinity chromatography with reversed-phase solid phase extraction fractionation (RP-SPE). Analysis of the enzymatically digested proteins was conducted using targeted LC-MS/MS working under the multiple reaction monitoring mode. For the quantification of the two distinct forms of AGT in the plasma (the sulphydryl-bridged oxidised form and the free thiol reduced form), a differential alkylation strategy was coupled with targeted LC-MS/MS to recognise and quantify the cysteine (Cys) peptides involved in the redox switch of AGT. The developed method enabled the reproducible detection of the two distinct forms of AGT in the plasma with CV% <15%, and confirmation of the identity of the differentially alkylated Cys peptides was supported by LC-MS/MS. Analysis of clinical plasma samples using the developed method showed a significantly higher level of the oxidised AGT in pre-eclamptic women compared to gestational age-matched normotensive controls (P=0.008), whilst maintaining a similar total AGT level in the plasma. The research findings indicate that the elevated level of oxidised AGT rather than its total level might be a contributing factor to the hypertension characteristic of pre-eclampsia, and provide an extra line of evidence linking the oxidative state and the generation of reactive oxygen species with hypertension in pre-eclampsia.
In the second part of the research, 54 clinically relevant glycoproteins were selected to be profiled by label-free targeted LC-MS/MS. Measurement of the analytical precision of the method revealed acceptable CV values for the majority of the assays (median CV 11.8%). Analysis of plasma samples collected from early- and late-onset preeclamptic women using the developed glycoprotein profiling methodology successfully identified significant changes in the level of several proteins in pre-eclampsia. Two of them, apolipoprotein D and kallikrein, are reported for the first time to be altered in the plasma of pre-eclamptic women suggesting that they could be further evaluated as novel biomarkers. Some pre-eclampsia-relevant pathways and biological processes, including iron transport and metabolism, coagulation, and lipid metabolism and oxidative stress were found to be altered in the disease. Moreover, different glycoproteins were changed in early-onset compared to late-onset pre-eclampsia which might reflect different pathophysiological mechanisms. Additionally, the method was applied to identify any altered glycoproteins in plasma samples from women with polycystic ovary syndrome (PCOS). These were subsequently compared with those found to be altered in pre-eclampsia, resulting in the proposal of possible underlying pathophysiological mechanisms that may explain the reported association between the two conditions, such as hypofibrinolysis and thrombophilia and iron overload.
Moreover, the study detected, for the first time, significant changes in the plasma levels of vitronectin and insulin growth factor acid labile subunit, suggesting that these may also be further appraised as potential biomarkers for the diagnosis of PCOS.
Taken together, the two targeted LC-MS/MS methods developed in this thesis provided relevant information regarding pre-eclampsia by identifying potential pre-eclampsia protein biomarkers. This sheds light on the different biochemical processes altered in the disease and points to possible pathophysiological mechanisms that might assist in explaining the link between PCOS and pre eclampsia, all of which should improve the understanding of the molecular mechanisms of the disease. The present work offers information that may play a key role in improving the health care of women with preeclampsia and serves as a foundational cornerstone for future work.
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