Mass spectrometry analysis of redox forms of High-Mobility Group Box-1 Protein in cerebrospinal fluid: initial experience.
Keywords:HMGB1 protein, cerebrospinal fluid, MALDI, mass spectrometry, top-down sequencing, redox
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.
Goodwin GH, Sanders C, Johns EW, Electrophoresis P. A New Group of Chromatin‑Associated Proteins with a High Content of Acidic and Basic Amino Acids. Eur J Biochem. 1973;38(1):14–9.
Fang P, Schachner M, Shen Y‑Q. HMGB1 in development and diseases of the central nervous system. Mol Neurobiol. 2012 Jun;45(3):499–506.
Wang H, Bloom O, Zhang M, Vishnubhakat JM, Ombrellino M, Che J, et al. HMG-1 as a late mediator of endotoxin lethality in mice. Science. 1999 Jul;285(5425):248–51.
Goldstein RS, Gallowitsch‑Puerta M, Yang L, Rosas‑Ballina M, Huston JM, Czura CJ, et al. Elevaed High‑Mobility Group Box 1 levels in patients with cerebral and myocardial ischemia. Shock. 2006 Jun;25(6):571–4.
Qiu J, Nishimura M, Wang Y, Sims JR, Qiu S, Savitz SI, et al. Early release of HMGB-1 from neurons after the onset of brain ischemia. J Cereb Blood Flow Metab. 2008 May;28(5):927–38.
Kokkola R, Sundberg E, Ulfgren A‑K, Palmblad K, Li J, Wang H, et al. High mobility group box chromosomal protein 1: a novel proinflammatory mediator in synovitis. Arthritis Rheum. 2002 Oct;46(10):2598–603.
Levy RM, Mollen KP, Prince JM, Kaczorowski DJ, Vallabhaneni R, Liu S, et al. Systemic inflammation and remote organ injury following trauma require HMGB1. Am J Physiol Regul Integr Comp Physiol. 2007 Oct;293(4):R1538–44.
Lotze MT, Tracey KJ. High‑mobility group box 1 protein (HMGB1): nuclear weapon in the immune arsenal. Nat Rev Immunol. 2005 Apr;5(4):331–42.
Magna M, Pisetsky DS. The role of HMGB1 in the pathogenesis of inflammatory and autoimmune diseases. Mol Med. 2014 Jan;20(c):138–46.
Sokół B, Woźniak A, Jankowski R, Jurga S, Wąsik N, Shahid H, et al. HMGB1 Level in Cerebrospinal Fluid as a Marker of Treatment Outcome in Patients with Acute Hydrocephalus Following Aneurysmal Subarachnoid Hemorrhage. J Stroke Cerebrovasc Dis. W.B. Saunders; 2015 Aug;24(8):1897–904.
Yang H, Lundbäck P, Ottosson L, Erlandsson‑Harris H, Venereau E, Bianchi ME, et al. Redox modification of cysteine residues regulates the cytokine activity of high mobility group box-1 (HMGB1). Mol Med. 2012 Jan;18(8):250–9.
Antoine DJ, Harris HE, Andersson U, Tracey KJ, Bianchi ME. A systematic nomenclature for the redox states of high mobility group box (HMGB) proteins. Mol Med. 2014 Jan;20:135–7.
Janko C, Filipović M, Munoz LE, Schorn C, Schett G, Ivanović‑Burmazović I, et al. Redox modulation of HMGB1-related signaling. Antioxid Redox Signal. 2014 Mar;20(7):1075–85.
Tang D, Billiar TR, Lotze MT. A Janus tale of two active high mobility group box 1 (HMGB1) redox states. Mol Med. 2012 Jan;18:1360–2.
Immunoprecipitation Protocol. https://www.sigmaaldrich.com/life‑science/cell‑biology/antibodies/antibodies‑application/protocols/immunoprecipitation.html#method_a
Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver‑stained polyacrylamide gels. Anal Chem. 1996;68(5):850–8.
Schiraldi M, Raucci A, Muñoz LM, Livoti E, Celona B, Venereau E, et al. HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4. J Exp Med. 2012;209(3):551–63.
Urbonaviciute V, Fürnrohr BG, Meister S, Munoz L, Heyder P, De Marchis F, et al. Induction of inflammatory and immune responses by HMGB1–nucleosome complexes: implications for the pathogenesis of SLE. J Exp Med. The Rockefeller University Press; 2008 Dec 22;205(13):3007–18.
Harlow E. Antibodies: A Laboratory Manual. 1988. 469 p.
Roque ACA, Lowe CR. Affinity Chromatography BT — Affinity Chromatography: Methods and Protocols. In: Zachariou M, editor. Totowa, NJ: Humana Press; 2008. p. 1–23.
Dunham WH, Mullin M, Gingras AC. Affinity‑purification coupled to mass spectrometry: Basic principles and strategies. Vol. 12, Proteomics. 2012. p. 1576–90.
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Copyright (c) 2019 Agata Światły, Norbert Wąsik, Joanna Hajduk, Eliza Matuszewska, Paweł Dereziński, Bartosz Sokół, Roman Jankowski, Zenon Kokot, Jan Matysiak
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