Elements from DNA microarray analysis, such as sample labeling and micro-spotting of capture reagents, have been successfully adapted to multiplex measurements of soluble cytokines. Application in cell biology is hampered by the lack of mono-specific antibodies and the fact that many proteins occur in complexes. Here, we incorporated a principle from Western blotting and resolved protein size as an additional parameter. Proteins from different cellular compartments were labeled and separated by size exclusion chromatography into 20 fractions. All were analyzed with replicate antibody arrays. The elution profiles of all antibody targets were compiled to color maps that resemble Western blots with bands of antibody reactivity across the size separation range (670 -10 kDa). A new solid phase designed for processing in microwell plates was developed to handle the large number of samples. Antibodies were bound to protein G-coupled microspheres surface-labeled with 300 combinations of four fluorescent dyes. Fluorescence from particle color codes and the protein label were measured by high-speed flow cytometry. Cytoplasmic protein kinases were detected as bands near predictable elution points. For proteins with atypical elution characteristics or multiple contexts, two or more antibodies were used as internal references of specificity. Membrane proteins eluted near the void volume, and additional bands corresponding to intracellular forms were detected for several targets. Elution profiles of cyclin-dependent kinases (cdks), cyclins, and cyclindependent kinase inhibitors, were compatible with their occurrence in complexes that vary with the cell cycle phase and subcellular localization. A two-dimensional platform circumvents the need for mono-specific capture antibodies and extends the utility of antibody array analysis to studies of protein complexes. Molecular & Cellular Proteomics 8:245-257, 2009.

Extracorporeal photopheresis (ECP) is an immunomodulatory alternative for treatment of graft versus host disease (GVHD). The blood is then separated into its various components through apheresis; buffy coat cells are thereafter treated with 8-methoxypsoralen before exposure to ultraviolet light and finally reinfused into the patient. There is a general agreement that this treatment has an anti-GVHD effect, but the mechanisms of action behind this effect are only partly understood. However, altered maturation of dendritic cells (DC) and thereby indirect modulation of T-cell reactivity seems to be one important mechanism together with DC-presentation of antigens derived from apoptotic donor T cells and induction of regulatory T cells. The treatment has been best studied in patients with chronic GVHD (both pediatric and adult patients), but most studies are not randomized and it is difficult to know whether the treatment is more effective than the alternatives. The clinical studies of ECP in adults with acute GVHD are few and not randomized; it is not possible to judge whether this treatment should be a preferred second- or third-line treatment. There is no evidence for increased risk of leukemia relapse or suppression of specific graft versus leukemia reactivity by this treatment, so specific antileukemic immunotherapy may still be possible. Thus, even though the treatment seems effective in patients with GVHD, further clinical (especially randomized) as well as biological studies with careful standardization of the treatment are needed before it is possible to conclude how ECP should be used in acute and chronic GVHD.

Leukemia is a complex disease pathologically manifested at the DNA, mRNA and protein level. Understanding leukemia pathogenesis is prevalently focused on mutations at the DNA (or mRNA) level, however the functional consequences of these changes on cellular machineries are not fully clarified. Since proteome analysis provides link between gene sequence and cellular physiology, proteomics can contribute to elucidate mechanism of disease and response to treatment. Moreover some alterations are manifested only at the protein level including subcellular localisation, post-translational modification (e.g. phosphorylation), protein cleavage or protein-protein interactions. Performing large-scale protein analysis of primary leukemia samples requires the development of more effective proteomic approaches as well as new analytical strategies. Here, we present novel microsphere-based antibody array format with automatical analysis tool that can follow changes in expression and post-translational modification of leukemia associated proteins with regards to intracellular localisation and protein cleavage in primary childhood acute leukemia (AL). Size Exclusion Chromatography-Microsphere-based Affinity Proteomics (SEC-MAP) is a set of 1728 populations of fluorescently-labeled microbeads, each carrying an antibody against respective human protein. Native cellular proteins (and their complexes) are isolated using detergents, labeled with biotin and subjected to size exclusion chromatography to obtain 24 molecular weight fractions. The fractions are incubated with SEC-MAP microbeads and the antibody-protein binding is detected using fluorescently-labeled streptavidin by flow cytometry. Flow cytometer resolves color-code of each microbead population and reads the amount of bound protein. The signals from 24 size fractions are combined and protein binding is detected as protein reactivity peaks similar to bands on western blot. The analysis is performed using automatic software created in R. It allows for automatic processing of fcs files as well as advanced follow-up analysis including quality control, normalisation, protein peaks recognition and clustering of results We have examined the expression of cytoplasmic (n=980) and membrane (n=769) proteins in 69 primary samples of AL obtained at diagnosis according to the Institutional Ethics Committee Giudelines. For the normalisation of protein expression we have used Loess normalisation commonly used in mRNA profiling studies. Due to ability of SEC-MAP to separate proteins according their molecular weight we have identified not only the expression of proteins but also the size that corresponds to its monomeric or multimeric presence and furthermore could serve as a control of proteolysis. We have revealed the sensitivity to proteolysis of 4 standard house-keeping proteins (Akt, Abl, β-actin and β2-microglobulin). Abl and Akt proved to be better controls of proteolysis. Detected with SEC-MAP or western blot, β-actin and β2-microglobulin, unlike Abl and Akt, have not been found in their cleaved forms in the proteolytically digested samples. Thus we have identified proteolysis in 12 samples which have been subsequently excluded from the analysis. So far we have identified 44 proteins (including CD markers distinguishing lineage specificity e.g. CD22, CD3, CD33) which have been differentially expressed in different subtypes of AL (B-cell precursor acute lymphoblastic leukemia, BCP-ALL, n=35), T-cell acute lymphoblastic leukemia (T-ALL, n=9) and acute myeloid leukemia (AML, n=13) (Multiple Testing Procedures - Bioconductor Package multtest, p<0.05). We have verified the expression using flow cytometry or western blot. From non-CD markers, we have found e.g. BLNK, DBN1, PAX5, PTK2 overexpressed in BCP-ALL, EIF5A, LAT, SH2D1A, SSEA4 overexpressed in T-ALL and CEBPA, CTBP2, GLUD1, LCP(pY145) and PTPN6 overexpressed in AML. In summary, SEC-MAP proved to be strong proteomic tool capable of identifying leukemia phenotype as well as providing novel insights into the protein expression and post-translational modification of primary childhood AL. Moreover it can bring complementary information about proteolysis not captured by planar arrays (western blot) which can significantly affect proteomic results. Supported by GAUK 596912, IGA NT13462, IGA NT12397, P302/12/G101, UNCE 204012, 00064203. Disclosures: No relevant conflicts of interest to declare.