The 70-KDa Heat Shock Protein Surface Plasmon Resonance Biosensor for Examination of Blood Plasma Proteome in Myelodysplastic Syndromes Subgroups
Authors: Pastva Ondrej , Chrastinova Leona , Bockova Marketa , Sacha Pavel , Suttnar Jiri , Kotlin Roman , Hlavackova Alzbeta , Cermak Jaroslav , Homola Jiri , Dyr Jan E. 
Published: October 2019

Abstract
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.