Mass spectrometry analysis of redox forms of High-Mobility Group Box-1 Protein in cerebrospinal fluid: initial experience.

Agata Światły; Norbert Wąsik; Joanna Hajduk; Eliza Matuszewska; Paweł Dereziński; Bartosz Sokół; Roman Jankowski; Zenon Kokot; Jan Matysiak

doi:10.20883/jms.311

Authors

Agata Światły Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0002-3603-0207
Norbert Wąsik Department of Neurosurgery, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0003-4588-7787
Joanna Hajduk Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0003-4654-8373
Eliza Matuszewska Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0002-5765-2603
Paweł Dereziński Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0002-6066-1260
Bartosz Sokół Department of Neurosurgery, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0003-4814-2000
Roman Jankowski Poznan University of Medical Sciences https://orcid.org/0000-0002-2368-4578
Zenon Kokot Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0003-4950-9759
Jan Matysiak Department of Inorganic and Analytical Chemistry, Poznan University of Medical Sciences, Poland https://orcid.org/0000-0002-9993-1504

DOI:

https://doi.org/10.20883/jms.311

Keywords:

HMGB1 protein, cerebrospinal fluid, MALDI, mass spectrometry, top-down sequencing, redox

Abstract

Introduction. High-mobility group box 1 (HMGB1) is an alarmin with proinflammatory potential determined by redox status of the cysteines at position 23 and 45. It may also play a role as a biomarker in biological fluids. The aim of this study was the identification of different HMGB1 redox forms in cerebrospinal fluid (CSF) obtained from subarachnoid hemorrhage patients.

Material and Methods. 6 CSF samples were collected from aneurysmal subarachnoid haemorrhage patients. Commercially available HMGB1 isoforms served as a positive control. Immunoprecipitation and electrophoretic isolation of HMGB1 protein were performed, then both CSF and control were analyzed using mass spectrometry technique. To distinguish between fully reduced (thiol group at C23 and C45) and disulfide (disulfide bond connecting C23 and C45) HMGB1 forms, top-down sequencing of the spectra was performed.

Results. Top-down sequencing analysis allowed to distinguish between HMGB1 isoforms only in commercially available standard without preceding immunoprecipitation and electrophoresis. MALDI spectra differ i.e. on the fully reduced HMGB1 spectrum fragmentation occurs before and beyond C22, which is not present on the disulfide HMGB1 spectrum. Analysis of HMGB1 isolated from CSF obtained from subarachnoid hemorrhage patients gave no results.

Conclusions. Top-down sequencing enables to distinguish between redox forms of HMGB1. Electrophoresis and tryptic digestion cannot precede mass spectrometry analysis of redox forms of HMGB1 due to the reduction of disulfide bonds during these processes. Preferred method of isolation of HMGB1 for direct analysis using top-down sequencing mustn’t include protein digestion or degradation.

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