Dual-Platform Proteomics Study of Plasma Biomarkers in Pediatric Patients Undergoing Cardiopulmonary Bypass

Department of Pediatrics, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
Pediatric Research (Impact Factor: 2.31). 03/2010; 67(6):641-9. DOI: 10.1203/PDR.0b013e3181dceef5
Source: PubMed


Plasma samples from pediatric cardiac patients undergoing cardiopulmonary bypass (CPB) procedures were used to identify and characterize patterns of changes in potential biomarkers related to tissue damage and inflammation. These included proteins associated with systemic inflammatory response syndrome. Potential biomarkers were identified using a dual-platform proteomics approach requiring approximately 150 microL of plasma, which included two-dimensional difference gel electrophoresis (2D-DIGE) and a multiplexed immunoassay. Methods used in the dual approach measured levels of 129 proteins in plasma from pediatric CPB patients. Of these, 70 proteins changed significantly (p<0.05) between time points, and 36 of these retained significance after the highly stringent Bonferroni correction [p<0.001 for 2D-DIGE and p<0.00056 for multianalyte profile (MAP) assays]. Many of the changing proteins were associated with tissue damage, inflammation, and oxidative stress. This study uses a novel approach that combines two discovery proteomics techniques to identify a pattern of potential biomarkers changing after CPB. This approach required only 150 microL of plasma per time point and provided quantitative information on 129 proteins. The changes in levels of expression of these proteins may provide insight into the understanding, treatment, and prevention of systemic inflammation, thereby helping to improve the outcomes of pediatric CPB patients.

Download full-text


Available from: John L Myers, Apr 15, 2014
21 Reads
  • Source
    • "These procedures have been described in detail previously [29,30,123]. Information about the 2D-DIGE study is provided in a form that complies with the most recent version <> of Minimum Information About a Proteomics Experiment – Gel Electrophoresis (MIAPE-GE) standards currently under development by the Human Proteome Organization Proteomics Standards Initiative (see Additional file 6). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Background Male wild type (WT) C57BL/6 mice are less capable of clearing bacteria and surviving from bacterial pneumonia than females. However, if an oxidative stress (acute ozone exposure) occurs before infection, the advantage shifts to males who then survive at higher rates than females. We have previously demonstrated that survival in surfactant protein-A (SP-A) knockout (KO) mice compared to WT was significantly reduced. Because the alveolar macrophage (AM) is pivotal in host defense we hypothesized that SP-A and circulating sex hormones are responsible for these sex differences. We used 2D-DIGE to examine the relationship of sex and SP-A on the AM proteome. The role of SP-A was investigated by treating SP-A KO mice with exogenous SP-A for 6 and 18 hr and studying its effects on the AM proteome. Results We found: 1) less variance between KO males and females than between the WT counterparts by principal component analysis, indicating that SP-A plays a role in sex differences; 2) fewer changes in females when the total numbers of significantly changing protein spots or identified whole proteins in WT or 18 hr SP-A-treated males or females were compared to their respective KO groups; 3) more proteins with functions related to chaperones or protease balance and Nrf2-regulated proteins changed in response to SP-A in females than in males; and 4) the overall pattern of SP-A induced changes in actin-related proteins were similar in both sexes, although males had more significant changes. Conclusions Although there seems to be an interaction between sex and the effect of SP-A, it is unclear what the responsible mechanisms are. However, we found that several of the proteins that were expressed at significantly higher levels in females than in males in WT and/or in KO mice are known to interact with the estrogen receptor and may thus play a role in the SP-A/sex interaction. These include major vault protein, chaperonin subunit 2 (beta) (CCT2), and Rho GDP alpha dissociation inhibitor. We conclude that sex differences exist in the proteome of AM derived from male and female mice and that SP-A contributes to these sex differences.
    Proteome Science 07/2012; 10(1):44. DOI:10.1186/1477-5956-10-44 · 1.73 Impact Factor
  • Source
    • "We have used this procedure in previous studies for other types of protein samples [26,46,143] but a detailed account, including many modifications and refinements appears below. For identification of spots, all 791 protein spots from the experiment were picked from the picking gel using a robot-directed spot picker (Ettan Spot Picker, GE Healthcare). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mice lacking surfactant protein-A (SP-A-/-; knockout; KO) exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL) protein differences between KO and wild-type (WT) are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM) proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse) and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining. We observed: a) significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases). These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis), proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b) SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c) that SP-A treatment altered cell size and F-actin distribution. These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.
    Proteome Science 10/2011; 9(1):67. DOI:10.1186/1477-5956-9-67 · 1.73 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Automotive car bodies are subject to impact by stones either lofted from tires or launched by other passing vehicles. Impact can result either in physical loss of paint and the possibility of failure at the metal/phosphate-polymer interface. A neural network (NN) analysis of electrochemical impedance data is presented. It is shown that electromechanical impedance spectroscopy (EIS) is a very sensitive post impact diagnostic probe to detect delamination at the metal-polymer boundary. Considering the noisy quality of data, the learning of the NN is good. It is shown that the NN is able to make predictions that are in agreement with independent experimental observations. Based on this preliminary work the future use of the NN as a predictive tool will rely on a comprehensive data set obtained under rigorous experimental conditions using stone projectiles, alternate treatments of impedance data, and also taking into account parameters such as stone shape, mass, and density
    Neural Networks, 1993., IEEE International Conference on; 02/1993
Show more