Translational Research Efforts in Biomarkers and Biology of Early Transplant-Related Complications  Sophie Paczesny, Maribel Diaz-Ricart, Enrique Carerras, Kenneth R. Cooke  Biology of Blood and Marrow Transplantation  Volume 17, Issue 1, Pages S101-S108 (January 2011) DOI: 10.1016/j.bbmt.2010.11.025 Copyright © 2011 Terms and Conditions

Figure 1 Overview of 3 proteomics strategies used for discovery of GVHD biomarkers (1) Differential in-gel electrophoresis (DIGE). Pooled plasma samples from GVHD-negative and GVHD-positive individuals are labeled with 2 fluorescent dyes, combined, and separated in a 2-dimensional gel by charge and molecular weight, followed by HPLC to separate proteins based on hydrophobicity. Spots of interest are picked and digested with trypsin for identification by mass spectrometry. (2) Antibody arrays. Plasma samples from GVHD-negative and GVHD-positive individuals are combined to form a reference pool, which is labeled with Biotin-ULS (red). A second aliquot of each individual plasma sample is labeled with Fluorescein-ULS (green). The labeled plasma proteins from 1 individual sample and 1 aliquot of the reference pool are hybridized to each side of the antibody array overnight. After repeated washes, the arrays are probed with the fluorescent reporter molecules streptavidin-DY647 and antifluorescein-DY547, washed, dried, and scanned at 2 wavelengths within 24-48 hours before bioinformatic analysis. (3) Intact Protein Analysis System. Plasma samples from ten GVHD-positive patients and 10 GVHD-negative patients are independently pooled. Pools are individually immunodepleted of albumin, IgG, IgA, transferrin, haptoglobin, and antitrypsin, and intact proteins are labeled at cysteine residues with acrylamide. The GVHD-negative pool receives the light C12-acrylamide isotope and the GVHD-positive pool receives the heavy C13-acrylamide isotope. The pools are mixed together for analysis by anion exchange chromatography, reverse-phase chromatography, and LC-MS/MS. The spectra are automatically processed by the Computational Proteomics Analysis System to identify individual proteins. Biology of Blood and Marrow Transplantation 2011 17, S101-S108DOI: (10.1016/j.bbmt.2010.11.025) Copyright © 2011 Terms and Conditions

Figure 2 Translational research initiatives for complications following allogeneic hematopoietic stem cell transplantation: Translational research initiatives can be conceptualized as translational “arcs of discovery” that can be both launched and completed. Mechanistic insights generated at the bench during the last several years regarding the role of TNFα in the development of GVHD and IPS have been brought to the clinic in the form of multicenter studies. It is anticipated that proteomic analysis of clinical samples collected and stored during the completion of these and other clinical studies will generate new lines of investigation in the laboratory and foster new therapeutic strategies for GVHD and pulmonary dysfunction in the future. Biology of Blood and Marrow Transplantation 2011 17, S101-S108DOI: (10.1016/j.bbmt.2010.11.025) Copyright © 2011 Terms and Conditions