Background: The aim of this study was to present the proteomic approach based on liquid phase isoelectric focusing fractionation coupled to nLC-MALDI-TOF/TOF-MS/MS analysis to characterize cerebrospinal fluid from control patients and those suffering from subarachnoid hemorrhage. The new perspective in characterization of this brain neuropathology are in constant demand to point a valuable panel of indicators which could improve the treatment outcome.
Methods: The cerebrospinal fluid samples were applied to a commercial liquid phase isoelectric focusing apparatus and separated into 10 fractions by pI. Further, the untargeted mass spectrometry investigations were performed with data dependent acquisition mode for full-scan MS analysis with subsequently MS/MS fragmentation by using nLC-MALDI-TOF/TOF-MS/MS.
Results: In total, the detection of 1664 and 2187 unique tryptic peptides provided biological evidence for 134 and 271 proteins in control and subarachnoid hemorrhage sample, respectively. The interpretation of liquid phase separation was performed by intersection analysis of two items between groups of ten fractions. The cumulative intersection exploration revealed the highest concentration of the detected components in the middle fractions of the focusing chamber, whereas the gradual dilution appeared on its extreme.
Conclusions: The employed strategy ensured overall screening of investigated material presenting the proteins abundance in the current state of analysis. Few proteins such as proenkephalin A, peroxiredoxin-6, cathepsin B, thrombospondin-1, glial fibrillary acidic protein and ? – spectrin were recognized as potential indicators, according to literature, pointing the possibility for its monitoring in further studies as panel of valuable biomarkers.
- Yuan X, Desiderio DM. Proteomics analysis of prefractionated human lumbar cerebrospinal fluid. Proteomics. 2005 Feb;5(2):541–550.
- Huhmer AF, Biringer RG, Amato H, et al. Protein analysis in human cerebrospinal fluid: Physiological aspects, current progress and future challenges. Dis Markers. 2006 Dec;22(1–2):3–26.
- Maurer MH, Haux D, Sakowitz OW, et al. Identification of early markers for symptomatic vasospasm in human cerebral microdialysate after subarachnoid hemorrhage: preliminary results of a proteome-wide screening. J Cereb Blood Flow Metab. 2007 Oct;27(10):1675–1683.
- Chen XL, Yu BJ, Chen MH. Circulating levels of neuropeptide proenkephalin A predict outcome in patients with aneurysmal subarachnoid hemorrhage. Peptides. 2014 Jun;56:111–115.
- Kuruppu S, Chou SH, Feske SK, et al. Soluble and catalytically active endothelin converting enzyme-1 is present in cerebrospinal fluid of subarachnoid hemorrhage patients. Mol Cell Proteomics: MCP. 2014 Apr;13(4):1091–1094.
- Sokol B, Wozniak A, Jankowski R, 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. 2015 Aug;24(8):1897–1904.
- Steinhoff RF, Karst DJ, Steinebach F, et al. Microarray-based MALDI-TOF mass spectrometry enables monitoring of monoclonal antibody production in batch and perfusion cell cultures. Methods. 2015 Jul;104:33–40.
- Hajduk J, Matysiak J, Kokot ZJ. Challenges in biomarker discovery with MALDI-TOF MS. Clin Chim Acta. 2016 Jul;458:84–98.
- Annesley TM. Ion supression in mass spectrometry. Clin Chem. 2003 Jul;49(7):1041–1044.
- Rabilloud T, Chevallet M, Luche S, et al. Two-dimensional gel electrophoresis in proteomics: Past, present and future. J Proteomics. 2010 Oct;73(11):2064–2077.
- Beranova-Giorgianni S. Proteome analysis by two-dimensional gel electrophoresis and mass spectrometry: strengths and limitations. Trac-Trend Anal Chem. 2003 May;22(5):273–281.
- Michel PE, Reymond F, Arnaud IL, et al. Protein fractionation in a multicompartment device using Off-Gel (TM) isoelectric focusing. Electrophoresis. 2003 Jan;24(1–2):3–11.
- Tomas R, Yan LS, Krenkova J, et al. Autofocusing and ESI-MS analysis of protein digests in a miniaturized multicompartment electrolyzer. Electrophoresis. 2007 Jul;28(13):2283–2290.
- Moreda-Pineiro A, Garcia-Otero N, Bermejo-Barrera P. A review on preparative and semi-preparative offgel electrophoresis for multidimensional protein/peptide assessment. Anal Chim Acta. 2014 Jul;836:1–17.
- Wall DB, Kachman MT, Gong SY, et al. Isoelectric focusing nonporous RP HPLC: A two-dimensional liquid-phase separation method for mapping of cellular proteins with identification using MALDI-TOF mass spectrometry. Anal Chem. 2000 Mar;72(6):1099–1111.
- Righetti PG, Castagna A, Herbert B, et al. How to bring the "unseen" proteome to the limelight via electrophoretic pre-fractionation techniques. Bioscience Rep. 2005 Apr;25(1–2):3–17.
- Wang HX, Kachman MT, Schwartz DR, et al. A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique. Electrophoresis. 2002 Sep;23(18):3168–3181.
- Hey J, Posch A, Cohen A, et al. Fractionation of complex protein mixtures by liquid-phase isoelectric focusing. Methods Mol Biol. 2008;424:225–239.
- Davidsson P, Paulson L, Hesse C, et al. Proteome studies of human cerebrospinal fluid and brain tissue using a preparative two-dimensional electophoresis approach prior to mass spectrometry. Proteomics. 2001 Mar;1(3):444–452.
- Amicon® Ultra-2 Pre-launch Centrifugal Filter Devices for volumes up to 2 mL User Guide [cited 13.07.2016]. Available from: http://www.biocenter.hu/pdf/amiconultra2.PDF.
- ReadyPrep™ 2-D Cleanup Kit Instruction Manual [cited 13.07.2016]. Available from: http://www.bio-rad.com/webroot/web/pdf/lsr/literature/MS4110143A.pdf.
- Shevchenko A, Wilm M, Vorm O, et al. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar;68(5):850–858.
- Oliveira BM, Coorssen JR, Martins-de-Souza D. 2DE: The Phoenix of Proteomics. J Proteomics. 2014 Jun;104:140–150.
- Shang TQ, Ginter JM, Johnston MV, et al. Carrier ampholyte-free solution isoelectric focusing as a prefractionation method for the proteomic analysis of complex protein mixtures. Electrophoresis. 2003 Jul;24(14):2359–2368.
- Liu H, Sadygov RG, Yates JR, 3rd. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal Chem. 2004;76(14):4193–4201.
- Elias JE, Haas W, Faherty BK, et al. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat Methods. 2005 Sep;2(9):667–675.
- Scherl A. Clinical protein mass spectrometry. Methods. 2015 Jun;81:3–14.
- Gao JB, Tang WD, Wang X, et al. Prognostic value of neuropeptide proenkephalin A in patients with severe traumatic brain injury. Peptides. 2014 Aug;58:42–46.
- Manevich Y, Hutchens S, Halushka PV, et al. Peroxiredoxin VI oxidation in cerebrospinal fluid correlates with traumatic brain injury outcome. Free Radic Biol Med. 2014 Jul;72:210–221.
- Yu ZQ, Jia Y, Chen G. Possible involvement of cathepsin B/D and caspase-3 in deferoxamine-related neuroprotection of early brain injury after subarachnoid haemorrhage in rats. Neuropathol Appl Neurobiol. 2014 Apr;40(3):270–283.
- Lad SP, Hegen H, Gupta G, et al. Proteomic biomarker discovery in cerebrospinal fluid for cerebral vasospasm following subarachnoid hemorrhage. J Stroke Cerebrovasc Dis. 2012 Jan;21(1):30–41.
- Shen YF, Wang WH, Yu WH, et al. The prognostic value of plasma thrombospondin-1 concentrations after aneurysmal subarachnoid hemorrhage. Clin Chim Acta. 2015 Aug;448:155–160.
- Nylen K, Csajbok LZ, Ost M, et al. Serum glial fibrillary acidic protein is related to focal brain injury and outcome after aneurysmal subarachnoid hemorrhage. Stroke. 2007 May;38(5):1489–1494.
- Dvorak F, Haberer I, Sitzer M, et al. Characterisation of the diagnostic window of serum glial fibrillary acidic protein for the differentiation of intracerebral haemorrhage and ischaemic stroke. Cerebrovac Dis. 2009 Jan;27(1):37–41.