Prioritization of Biomarker Targets in Human Umbilical Cord Blood: Identification of Proteins in Infant Blood Serving as Validated Biomarkers in Adults

Hansmeier, Nicole, Chao, Tzu-Chiao, Goldman, Lynn R., Witter, Frank R., Halden, Rolf U., Environmental Health Perspectives

BACKGROUND: Early diagnosis represents one of the best lines of defense in the fight against a wide array of human diseases. Umbilical cord blood (UCB) is one of the first easily available diagnostic biofluids and can inform about the health status of newborns. However, compared with adult blood, its diagnostic potential remains largely untapped.

OBJECTIVES: Our goal was to accelerate biomarker research on UCB by exploring its detectable protein content and providing a priority list of potential biomarkers based on known proteins involved in disease pathways.

METHODS: We explored cord blood serum proteins by profiling a UCB pool of 12 neonates with different backgrounds using a combination of isoelectric focusing and liquid chromatography coupled with matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) and by comparing results with information contained in metabolic and disease databases available for adult blood.

RESULTS: A total of 1,210 UCB proteins were identified with a protein-level false discovery rate of -5% as estimated by naive target-decoy and MAYO approaches, signifying a 6-fold increase in the number of UCB proteins described to date. Identified proteins correspond to 138 different metabolic and disease pathways and provide a platform of mechanistically linked biomarker candidates for tracking disruptions in cellular processes. Moreover, among the identified proteins, 38 were found to be approved biomarkers for adult blood.

CONCLUSIONS: The results of this study advance current knowledge of the human cord blood serum proteome. They showcase the potential of UCB as a diagnostic medium for assessing infant health by detection and identification of candidate biomarkers for known disease pathways using a global, non-targeted approach. These biomarkers may inform about mechanisms of exposure-disease relationships. Furthermore, biomarkers approved by the U.S. Food and Drug Administration for screening in adult blood were detected in UCB and represent high-priority targets for immediate validation.

KEY WORDS: body fluid, diagnostics, disease, LC-MALDI-MS, pathways, proteomics. Environ Health Perspect 120:764-769 (2012). http://dx.dolorg/10.1289/ehp.1104190 [Online 27 January 2012]

Many diseases are of early-life origin. Early diagnosis of diseases, toxic exposures, effects, and susceptibilities in the still-developing body of infants will be required to develop successful intervention and treatment strategies to battle diseases. It is well documented that exposures to environmental chemical contaminants, including cigarette smoke constituents, for example, have adverse effects on fetal development and result in unfavorable health trajectories for affected children (Apel berg et al. 2007a; Buczynska and Tarkowski 2005; Mattison 2010; Miranda et al. 2009; Wigle et al. 2007). Long-term outcomes such as diabetes, obesity, and chronic heart and kidney diseases have all been hypothesized or postulated to have their basis in fetal and childhood exposure (Barker et al. 2002) and show an increased prevalence in children and newborns (Bloomgarden 2004; Ferrara 2007). Therefore, the development of early diagnostics as predictors for child health is of paramount importance to enable early intervention.

Umbilical cord blood (UCB) is a very attractive biological specimen, because relatively high volumes (up to tens of milliliters) of this biofluid are available for sampling without posing an added risk and burden to the newborn or its mother in the process. In addition, it has the potential to inform about existing or potential future adverse effects. UCB is already in use for prediagnosis and treatment of immune deficiencies (Notarangelo 2010). However, its primary use at this time is for bone marrow transplantation (Buchheiser et al. 2009).

Mass spectrometry (MS)--based proreomics is a powerful technology, allowing for the identification and quantification of hundreds of proteins in parallel from a single sample without necessitating prior selection. or exclusion of potential analytical targets. It has been employed successfully for a diverse range of organisms, tissues, and biofluids (Aebersold and Mann 2003; Ahrens et al. 2010a, 2010b; Beck et al. 2011; Domon and Aebersold 2010; Nilsson et al. 2010) and offers an ideal platform for fast identification of new protein markers for diseases or adverse exposure (Lemos et al. 2010; Liumbruno et al. 2010). For adult blood, there is a long tradition of analyzing serum proteins using two-dimensional gel electrophoresis (Hughes et al. 1992) or shotgun proteomics (Omeun et al. 2005; Pieper et al. 2003; Richter et al. 1999; Schenk et al. 2008; States et al. 2006), with continuous analytical improvements (Bell et al. 2009; Gaso-Sokac and Josic 2010; Rai et al. 2005). In contrast to adult blood, much less information is available for the cord blood proreome. Recently, two short overviews on cord blood proteomics were published describing 207 and 837 different proteins, respectively (Colquhoun et al. 2009; Song et al. 2009). Unfortunately, the latter report provided information on only 61 of the 837 proteins. The identifiers of the 776 remaining proteins are not published.

More detailed investigations of the UCB proteome are needed to accelerate the pace of discovery and expand the spectrum of infant health diagnostics. In addition, the sampling of UCB for proteome studies has to be successful within the limitations of clinical reality and is therefore much more challenging than the analysis of adult blood. The aim of this study was to provide insights into several important aspects necessary to use the UBC as an effective source for protein-based biomarkers. As a first priority, we aimed to expand the knowledge on the detectable proteome in UCB, which can be obtained from a limited starting volume. This is especially important because large volumes of UCB are required to obtain sufficient stem cells for therapeutic purposes (Buchheiser et al. 2009; Forraz and McGuckin 2011). Hence, reducing the required volume for diagnostic purposes to a minimum is highly desirable. Moreover, we compared the identified proteins with known and proposed biomarkers to provide a short list of potential biomarkers that form a basis for the exploration of molecular mechanisms of exposure-disease relationships.

Materials and Methods

Chemicals. All chemicals were obtained from Sigma-Aldrich (Sigma Aldrich, St. Louis, MO, USA) with the following exceptions: Sequencing-grade modified porcine trypsin was obtained from Promega (Madison, WI, USA), and Bradford reagent was purchased from Bio-Rad (Hercules, CA, USA).

UCB serum samples. The UCB serum samples were acquired from the cord blood cohort collection of the Baltimore Tracking Health-Related Environmental Exposures (THREE) study. This study was approved by the Johns Hopkins Medicine Institutional Review Board (IRB approval 04-04-22-02) and received a waiver from the Health Insurance Portability and Accountability Acr (2002). The study showed U.S.-representative exposure levels (mean and maximum concentrations) of a) perfluorooctanoate (PFOA): 1.6 ng/mL, 7.1 ng/mL; b) perfluorooctane sulfonate (PFOS): 5 …

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Publication information: Article title: Prioritization of Biomarker Targets in Human Umbilical Cord Blood: Identification of Proteins in Infant Blood Serving as Validated Biomarkers in Adults. Contributors: Hansmeier, Nicole - Author, Chao, Tzu-Chiao - Author, Goldman, Lynn R. - Author, Witter, Frank R. - Author, Halden, Rolf U. - Author. Journal title: Environmental Health Perspectives. Volume: 120. Issue: 5 Publication date: May 2012. Page number: 764+. © 2006 National Institute of Environmental Health Sciences. COPYRIGHT 2012 Gale Group.

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