Undergraduate Honors Thesis Projects

Date of Award

Spring 4-15-2015

Document Type

Honors Paper

Degree Name

Biochemistry and Molecular Biology-BS

Department

Biochemistry and Molecular Biology

Advisor

Lisa Marr, M.D.

First Committee Member

John Tansey, Ph.D.

Second Committee Member

Stephanie Patridge, Ph.D.

Keywords

Preeclampsia, Congo Red, Misfolded Proteins, Proteinuria, Trypsin Digest, Pregnancy

Subject Categories

Analytical Chemistry | Biochemistry | Biology | Obstetrics and Gynecology

Abstract

Preeclampsia is a perinatal complication of unknown etiology, resulting in 76,000 maternal deaths and 500,000 infant deaths annually1. The Nationwide Children’s Center for Perinatal Research was the first cohort to propose that preeclampsia is rooted in protein misfolding. We aimed to characterize the misfolded protein components of maternal urine samples and to determine if distinct molecular profiles exist for clinical preeclampsia phenotypes. A total of 86 maternal urine samples from Yale-New Haven Hospital were analyzed for this study, from six clinical preeclampsia subphenotypes- pregnant control (n=10), idiopathic proteinuria (n=15), mild preeclampsia (n=14), superimposed preeclampsia (n=12), typical severe preeclampsia (n=16), and atypical severe preeclampsia (n=19). The maternal urine samples were subjected to Congo Red dye, followed by a trypsin digest. The Congo Red binds to the misfolded protein regions in the sample, and the trypsin digest acts to cleave the proteins into peptide fragments. The samples were then analyzed via liquid chromatography-tandem mass spectrometry (LC-MS/MS). From this experimentation, we found that the Congo Red precipitate was very diverse in terms of protein composition. We were also able to analyze the Congo Red retention time of the six groups, which provided information supporting the hypothesis that the protein profiles between the preeclampsia subphenotypes are indeed quite different. By a linear discriminate analysis (LDA), using 732 unique protein IDs, we were able to partition the 86 urine samples into six LDA axes. These groups clustered in a way that supports the hypothesis that clinical manifestations and diagnoses of preeclampsia can be determined by analyzing the misfolded proteins present in maternal urine. This experimentation also proved that the Congo Red precipitate is more complex in terms of protein composition than that of crude urine (which has no Congo Red precipitate). It has also allowed us to demonstrate that the protein profiles of maternal urine samples can provide insight into the mechanisms of various hypertensive and proteinuria pregnancy complications. With this knowledge, the potential exists to alter clinical practice and to provide therapeutic interventions in the future, with the ultimate goal of lessening maternal and fetal deaths due to preeclampsia.

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