Acute‐phase proteins investigation based on lectins affinity capture prior to 2‐DE separation: Application to serum from multiple sclerosis patients

A major cause of disability in secondary progressive multiple Background sclerosis (SPMS) is progressive brain atrophy, whose pathogenesis is not fully understood. The objective of this study was to identify protein biomarkers of brain atrophy in SPMS.: We used surface-enhanced laser desorption-ionization time-of-flight Methods mass spectrometry to carry out an unbiased search for serum proteins whose concentration correlated with the rate of brain atrophy, measured by serial MRI scans over a 2-year period in a well-characterized cohort of 140 patients with SPMS. Protein species were identified by liquid chromatography-electrospray ionization tandem mass spectrometry.: There was a significant (p<0.004) correlation between the rate of Results brain atrophy and a rise in the concentration of proteins at 15.1 kDa and 15.9 kDa in the serum. Tandem mass spectrometry identified these proteins as alpha-haemoglobin and beta-haemoglobin, respectively. The abnormal concentration of free serum haemoglobin was confirmed by ELISA (p<0.001). The serum lactate dehydrogenase activity was also highly significantly raised (p<10 ) in patients with secondary progressive multiple sclerosis.: The results are consistent with the following hypothesis. In Conclusions progressive multiple sclerosis, low-grade chronic intravascular haemolysis releases haemoglobin into the serum; the haemoglobin is subsequently translocated into the central nervous system (CNS) across the damaged blood-brain barrier. In the CNS, the haemoglobin and its breakdown products, including haem and iron, contribute to the neurodegeneration and consequent brain atrophy seen in progressive disease. We postulate that haemoglobin is a source of the iron whose deposition along blood vessels in multiple sclerosis plaques is associated with neurodegeneration. Amendments from Version 1We thank the reviewers for their comments and suggestions. In the revised version of the paper (v.2), we have taken into account the points raised by each set of reviewers. The main changes are the following:-clarification of points of methodology (e.g. sampling; use of Top 12 protein depletion columns) -more cautious wording on possible therapy, and to make clear the principle that we have not proved causality or claim that free serum haemoglobin is the sole correlate of brain atrophy in SPMS -clearer and fairer representation of the results reported in previous publications -addition of 8 papers to the bibliography, citing -as suggested -both old work (on erythrocyte fragility) and very recent work -answering specific points concerning the normal total blood haemoglobin concentration and the kinetics of neurodegeneration -significance values for pairwise statistical tests in Figure 3.

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“…Robotti et al . 28 identified an alteration in the ratio of isoforms of haptoglobin (which bind free haemoglobin) in MS.…”

Section: Discussionmentioning

confidence: 99%

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Lewin¹,

Hamilton²,

Witkover³

et al. 2016

Wellcome Open Res

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“…2-DE experiments were carried out in triplicates according to Robotti et al . [ 16 ] and performed after conclusion of the CSF sample collection. An amount of 100 μg total proteins from each sample was loaded for each 2-DE gel.…”

Section: Methodsmentioning

confidence: 99%

Vitamin D Binding Protein Isoforms and Apolipoprotein E in Cerebrospinal Fluid as Prognostic Biomarkers of Multiple Sclerosis

Perga

Albo²,

Lis³

et al. 2015

PLoS ONE

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BackgroundMultiple sclerosis (MS) is a multifactorial autoimmune disease of the central nervous system with a heterogeneous and unpredictable course. To date there are no prognostic biomarkers even if they would be extremely useful for early patient intervention with personalized therapies. In this context, the analysis of inter-individual differences in cerebrospinal fluid (CSF) proteome may lead to the discovery of biological markers that are able to distinguish the various clinical forms at diagnosis.MethodsTo this aim, a two dimensional electrophoresis (2-DE) study was carried out on individual CSF samples from 24 untreated women who underwent lumbar puncture (LP) for suspected MS. The patients were clinically monitored for 5 years and then classified according to the degree of disease aggressiveness and the disease-modifying therapies prescribed during follow up.ResultsThe hierarchical cluster analysis of 2-DE dataset revealed three protein spots which were identified by means of mass spectrometry as Apolipoprotein E (ApoE) and two isoforms of vitamin D binding protein (DBP). These three protein spots enabled us to subdivide the patients into subgroups correlated with clinical classification (MS aggressive forms identification: 80%). In particular, we observed an opposite trend of values for the two protein spots corresponding to different DBP isoforms suggesting a role of a post-translational modification rather than the total protein content in patient categorization.ConclusionsThese findings proved to be very interesting and innovative and may be developed as new candidate prognostic biomarkers of MS aggressiveness, if confirmed.

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“…Each precipitate was dissolved in 30 ul of 0.5 M TEAB and mixed with 70 ul of isopropanol. The protein were labeled with iTRAQ reagent (AB SCIEX, Framingham, MA, USA) 113, 114, 115, 116, 117, 118, 119 and 121, respectively14. iTRAQ Labeling procedure was followed by validation in individual patients.…”

Section: Methodsmentioning

confidence: 99%

Plasma Proteomic Study in Pulmonary Arterial Hypertension Associated with Congenital Heart Diseases

Zhang

Hou

Wang

et al. 2016

Sci Rep

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Pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH) has serious consequence and plasma protein profiles in CHD-PAH are unknown. We aimed to reveal the differential plasma proteins in 272 CHD patients with or without PAH. Various types of CHD-PAH were studied. Differential plasma proteins were first detected by iTRAQ proteomic technology and those with significant clinical relevance were selected for further ELISA validation in new cohort of patients. Among the 190 differential plasma proteins detected by iTRAQ, carbamoyl-phosphate synthetase I (CPSI, related to urea cycle and endogenous nitric oxide production) and complement factor H-related protein 2 (CFHR2, related to complement system and coagulant mechanism) were selected for further ELISA validation in new cohort of 152 patients. Both CPSI and CFHR2 were down-regulated with decreased plasma levels (p < 0.01). Thus, we for the first time in CHD-PAH patients identified a large number of differential plasma proteins. The decreased CPSI expression in CHD-PAH patients may reveal a mechanism related to endogenous nitric oxide and the decrease of CFHR2 protein may demonstrate the deficiency of the immune system and coagulation mechanism. The findings may open a new direction for translational medicine in CHD-PAH with regard to the diagnosis and progress of the disease.

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Acute‐phase proteins investigation based on lectins affinity capture prior to 2‐DE separation: Application to serum from multiple sclerosis patients

Cited by 19 publications

References 57 publications

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Free serum haemoglobin is associated with brain atrophy in secondary progressive multiple sclerosis

Vitamin D Binding Protein Isoforms and Apolipoprotein E in Cerebrospinal Fluid as Prognostic Biomarkers of Multiple Sclerosis

Plasma Proteomic Study in Pulmonary Arterial Hypertension Associated with Congenital Heart Diseases

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