The low-molecular weight proteome is of great interest due to the vast number of smaller proteins and peptides present within it, some of which are likely to be disease-related and may provide insights into disease mechanisms or even prediction and diagnosis. Serum is often the most preferred specimen for disease prediction or diagnosis, as it can be obtained less-invasively and its collection is fairly routine in a clinical setting. Although serum samples are one of the most preferred specimens for disease studies, additional disease information may also be obtained using other specimens, such as tissue. The study of tissue specimens can be extremely valuable in disease studies as it likely contains the highest concentration of potential markers for tissue related diseases. Therefore, despite the difficulty of sample collection, as compared to serum specimens, tissue specimens can be invaluable in studying tissue-related diseases. Tissue proteomics approaches for studying the low-molecular weight proteome of tissue have been infrequently described in the literature, which suggests the need to develop new methods or improve upon current proteomics approaches to better study this subset of the proteome in tissue. In this dissertation, I first report on a low-molecular weight tissue proteomics approach that we have developed based on a previously reported serum approach. As reported here, we conclude that this tissue proteomics approach cannot only distinguish the low-molecular weight proteome of different tissue types, but it also appears to be within the reproducibility range of other currently used proteomic approaches. Additionally, we also report on the use of a serum proteomics approach for biomarker discovery and identification in preeclampsia and endometriosis. These studies reveal many candidate biomarkers for preeclampsia prediction and endometriosis diagnosis. In the preeclampsia study we discovered several candidate biomarkers early in pregnancies (12-14 weeks gestation) that were able to predict preeclampsia later in pregnancy with statistical significance. In the endometriosis study, several candidate biomarkers were discovered that were statistically significant in diagnosing endometriosis. Further, for both preeclampsia and endometriosis, combinations of 3 or 4 biomarkers yielded an improved sensitivity and specificity in disease prediction and diagnosis with combined area under the curves of greater than 0.8. Thus, the low-molecular weight proteome of both tissue and serum could potentially provide important information regarding disease physiology, prediction, and diagnosis that may supplement our current understanding of these processes.

Preeclampsia (PE) is a potentially fatal complication of pregnancy characterized by an increase in blood pressure (>140/90 mmHg) and proteinuria (>300 mg/24 hrs), often accompanied by edema. Symptoms of PE start after 20 weeks of gestation. If PE remains untreated, it can lead to eclampsia, grand-mal seizures responsible for most fatalities. PE is believed to affect 2-10% of pregnancies worldwide, and claims the lives of over 75,000 mothers and 500,000 newborns yearly. No therapeutic agents have been developed to prevent or cure PE. Part of the reason for this is the absence of a complete understanding of the pathogenesis of this disease. PE has long been regarded as a “disease of theories”, and the pathophysiology of PE continues to be the subject of debate. Nonetheless, several abnormalities have been observed to precede established, clinical PE and have in turn been proposed to be involved in the causation of this disease, all with involvement of the mother's placenta as a central feature. Removal of placenta is the only cure for PE and results in a rapid resolution of the symptoms. Thus, the placenta remains an organ of substantial interest and many research groups have attempted to identify abnormal placental features occurring in PE. None of these studies have focused on less abundant, low molecular weight (LMW) biomolecules, which play important roles in the pathophysiology of many diseases. There are a number of alterations that are believed to affect the placenta and contribute to the pathogenesis of PE. The most widely accepted ones include hypoxia, oxidative stress, and an increase of pro-inflammatory mediators in the mother's placenta. The goal of my initial study was to identify which of these hypothesized causative pathways has a significance in the etiology of this syndrome as well as to investigate which less abundant, low molecular weight biomolecules change in response to these abnormalities. For this purpose, we first adapted and optimized a previously developed methodology that studied LMW biomolecules in tissue specimens to study placental biomolecules. This approach involved a tissue homogenization step followed by protein depletion using acetonitrile. We compared two regions of human placenta: the chorionic plate and the basal plate to find differences in the LMW fraction. We discovered 16 species with statistically significant differences between the two sides, and identified 12 of them using tandem mass spectrometry. In the second study we collected normal human term placentas from elective C-section deliveries and exposed explants to each of the above-mentioned provocative agents or stress conditions for 48 hrs. Other explants without any stressors were cultured in parallel for the same amount of time. The processing of explants was divided into five steps: 1) explant culture; 2) tissue homogenization; 3) acetonitrile precipitation to remove high abundance, high molecular weight proteins; 4) injection of the protein-depleted specimen into a capillary liquid chromatography–mass spectrometer; 5) analysis of MS data to identify quantitative differences between cases (stressed explants) and controls (normal explants). In total, we observed 146 molecules changed in abundance between the treated explants and the controls with 75 of these molecules changed in response to hypoxic treatment, 23 changed due to hypoxia-reoxygenation, a process generating reactive oxygen species, and 48 changed due to tumor necrosis factor–alpha (TNFα), a pro-inflammatory cytokine. We were successful in identifying 45% of all these molecules by tandem MS. Statistical modeling that applied LASSO analysis allowed for the development of a model that used 16 of the 146 differentially expressed biomolecules to accurately classify and differentiate each of the 4 stressed conditions. In my third study, I then submitted actual preeclamptic and non-diseased placental tissue to our established homogenization and acetonitrile precipitation protocol to see if any of the differences in LMW biomolecules produced under stress conditions in normal placenta were recapitulated in actual diseased placenta. In a preliminary statistical analysis, 8 of the original 146 differentially expressed species, displayed significant or near significant changes in the actual disease placenta. After applying two stringent statistical tests that eliminated any potential influence of gestational age, four out of the 146 biomarkers previously studied, continued to be differentially expressed in both stringent analyses. Of the four, 1 biomarker (m/z 649.49 (+1)) showed an increased abundance in hypoxic placental explants as well as in PE placenta; 2 (461.06 (+1), 476.24 (+1)) were increased in response to TNFα-exposed placental explants and in these PE placentas and 1 (426.35 (+1)) increased in response to hypoxia-reoxygenation-treated placental explants was also increased in PE placenta. We have chemically characterized 2 of the 4 biomarkers. One was a phospholipid (m/z 476.24) while the other was an acyl-carnitine (m/z 426.35). This suggests that features of PE appear to arise from the predicted early abnormalities that affect the placenta. In conclusion, I was successful in developing an ‘omics’ approach to study less abundant, low molecular weight biomolecules in human placenta as well as investigate which biomarkers show differential expression in human placenta when exposed to proposed abnormalities of PE and have data to suggest that these same responses are present in PE placenta.