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Introduction Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal stem cell disorders characterized by a high risk of evolution into acute myeloid leukemia (AML). Different processes are involved in its pathogenesis, such as (epi)genetic alterations and immunological dysfunctions. Till now, in plasma several protein markers for MDS were found. One of them is a misfolded receptor protein VEGFR-1 for which we have created a detection assay based on the surface plasmon resonance (SPR) (Pimkova K, Suttnar J, et al., Anal Bioanal Chem. 2012, 402, 381-387). An elevated level of circulating 70-kDa heat shock proteins (Hsp70) has been detected in the blood of patients with myeloid leukemia. The Hsp70 are ATP-dependent molecular chaperones which interacts with unfolded/misfolded and aggregated proteins, thus providing cytoprotective role against various cellular stresses. Hsp70 released from cancer cells can carry peptide antigens including ones specific for a tumour and contribute to the development of anti-cancer immune response. The goal of this study is to identify proteins in HSP70 complexes using SPR biosensor followed by mass spectroscopy (MS) in the blood plasma of patients from MDS subgroups. Methods A six-channel spectroscopic SPR biosensor for detection of protein comlexes in diluted blood plasma samples was used. Preincubated ATP-Hsp70 complex was immobilized on the sensor surface via covalent attachment by dispersionless microfluidics. The biosensor utilizes Hsp70 rate-limiting step of ATPase cycle, ADP/ATP exchange, which allows in an ATP-controlled manner binding of protein substrates and their elution with minimal non specificity. Eluted proteins were subsequently analyzed and identified by using LC-MS/MS (TripleTOF 5600). Patients of MDS (n=10) and MDS patients who progress to AML (n=5) were chosen. Results and Conclusion Using preincubated ATP-Hsp70 complex significant differences in SPR responses of MDS patients in comparison to healthy donors as well as differences among MDS patients were observed. The responses represented hundreds of proteins identified by mass spectrometry. Protein interaction networks were visualized and analyzed by bioinformatics tools which cluster proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. The identified proteins are known to be involved in several processes including ubiquitinylation, protein degradation, cell signaling, stress response, protein synthesis, and also regulation of the immune system. We used Power Graphs, a novel representation of (protein) networks which provided a valuable insight into the presence of protein complexes, their internal organization and relationships. Interaction networks also indicated possible proteins involved in immunomodulatory functions of MDS. The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Introduction Myelodysplastic syndromes are clonal hematopoetic stem cell disorders characterized by refractory cytopenias with displasia as a result of ineffective hematopoiesis. Approximately 30 percent of MDS cases progress to acute myeloid leukemia. Excess blasts are the strongest predictors for poor outcome and are associated with disease progression. Because of its heterogeneity and lack of molecular and protein markers that effectively monitor disease progression, clinical management of MDS patients is challenging. The concentrations of several proteins were found elevated during MDS disease. Their interaction with receptors/ligands is a part of signaling pathways that may play a crucial role in pathogenesis of the disease. Serum levels of vascular cell adhesion protein 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) were found higher in MDS patients in comparison with control group and were related to MDS severity. Alpha-2-HS-glycoprotein (FETUA) was analyzed in plasma of MDS patients. Leucine-rich alpha-2-glycoprotein (LRG) was found increased in RAEB-I and RAEB-II patients by proteomic studies. In this work we developed a protein chip for study of molecular interactions in plasma of MDS patients using surface plasmon resonance (SPR) imaging. Interacting proteins were analyzed and identified using LC-MS/MS. Methods Patients with diagnosis of refractory anemia and refractory anemia with ringed sideroblasts (RA/RARS, n=13), refractory cytopenia with multilineage dysplasia (RCMD, n=16), and refractory anemia with excess blasts (RAEB, n=15) as a subgroup of MDS, and MDS patients who progress to acute myeloid leukemia (AML, n=8) were chosen for this study. The high-resolution SPR imaging (SPRi) system with polarization contrast and internal referencing combined with dispersionless microfluidics was used to study protein interactions. Passive mixing structures were employed to further improve the sensing performance of this SPRi biosensor. The respective biomarkers (ICAM1, VCAM1, FETUA and LRG) were covalently attached to the functionalized sensor surface and interaction with their counterparts in MDS plasma samples were monitored. Interacting proteins were removed from the chip surface by diluted NaOH, treated by trypsin, analyzed by nanoLC-MS/MS (TripleTOF 5600) and identified using ProteinPilot database. Results and Conclusion Significant differences were observed between analyzed MDS subtypes for VCAM1, FETUA, and LRG (ANOVA: P = 0,00755, P = 0,0306 and P < 0.001 respectively). Using post hoc Tukey contrasts tests we ascertained that sensor responses were significantly higher in group RCMD and AML with respect to the healthy donors for immobilized VCAM1 and in group RA/RARS, RCMD, RAEB and AML with respect to the healthy donors for LRG. Moreover sensor responses were significantly higher in group AML with respect to the RA, RCMD or RAEB for immobilized LRG. Based on our results LRG seems to be a suitable marker of MDS progression. The identified proteins presented in MDS plasma samples unlike in healthy donor plasma are clusterin, protein S100-A8, serum amyloid A-1 protein, annexin A2, and zinc-alpha-2-glycoprotein among others. S100-A8 and clusterin have been connected with MDS or AML previously; it could be tested as a new protein marker of MDS by SPRi. The results show that SPR biosensors combined with nanoLC-MS/MS are a promising tool for improvement of the diagnosis in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.

Introduction The protein microarrays are becoming the leading technology in proteomic research area. They enable to implement both features of proteins that can be altered in disease as quantitative proteomics (levels in biological samples) as well as functional proteomics (determination of their selective interactions with other biomolecules). Protein microarray techniques such as sandwich immunoassays, antigen capture immunoassay and direct immunoassays use labeling and antibodies. However, these labels can interfere with the analyte binding site and thus affect protein activity. Using antibodies requires a prior knowledge of the studied proteins which is in the case of a heterogeneous disease with little knowledge in its proteomic field a huge disadvantage. Surface plasmon resonance (SPR) is a label-free and direct method allowing quantification as well as monitoring of protein-protein interactions simultaneously and in real time. Myelodysplastic syndrome (MDS) is a heterogeneous group of hematological malignancies. It affects pluripotent hematopoietic stem cell and is manifested by variety of clinical symptoms according to predominant involvement of development lineage. The high risk of MDS to transform into acute myeloid leukemia makes it a suitable model for study of biological processes leading to leukemia development. In this work, we use SPR imaging for simultaneous screening of blood plasma of MDS patients followed by mass spectrometry (MS) for identification of interacting partners and analysis of protein network properties. Proteins, whose levels are either elevated during MDS disease or interaction with their receptors/ligands is a part of signaling pathway, were employed. Method SPR imaging system with polarization contrast and internal referencing was combined with dispersionless microfluidics for parallel screening of blood plasma samples. Proteins involved in pathogenesis of MDS and their physiological counterparts were immobilized under flow to create 6x6 sensing spots. Specifically, these include integrin αMβ2 (LFA1), intercellular adhesion molecule 1 (ICAM1), integrin α4β1 (VLA4), vascular cell adhesion protein 1 (VCAM1) and cytotoxic T-lymphocyte protein 4 (CTLA4). The sensor surface functionalization was optimized with respect to its ability to provide a low-fouling sensing surface with biologically active receptors. Plasma samples of controls and MDS patients were flowed along the functionalized surface and differences in individual interactions were evaluated. Selected interacting partners were further identified using 2D-HPLC/ESI-MS/MS. Identified proteins were analyzed by String Networks and Power Graph Analysis. Results and Conclusion Significant differences in the protein profiles among different MDS groups of patients as well as relative to control healthy donors were observed using SPR imaging; tens of interacting proteins were identified by mass spectrometry. Protein interaction networks were explored through clustering of proteins into groups that share the same biological function, are similarly localized in the cell, or are known to be a part of a complex. Identified proteins are involved in several processes; regulation of immune system, ubiquitinylation and protein degradation, cell signaling, hemostasis, protein synthesis, cell adhesion, metastasis, and inhibition of blood coagulation. Using of Power Graphs, a novel representation of (protein) networks, provided valuable insight into the existence of protein complexes, their internal organization, and their relationships. Interaction networks also indicated possible pathways involved in MDS pathogenesis (especially Src tyrosine kinases). The results showed that SPR biosensors are a promising tool for the diagnosis and follow-up efficiency treatment in complex heterogeneous malignancies. Disclosures No relevant conflicts of interest to declare.