Development of a nano-liquid chromatography on chip tandem mass spectrometry method for high-sensitivity hepcidin quantitation

Houbart, Virginie; Cobraiville, Gaël; Lecomte, Frédéric; Debrus, Benjamin; Hubert, Philippe; Fillet, Marianne

doi:10.1016/j.chroma.2011.10.030

Cited by 36 publications

(15 citation statements)

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“…Extraction procedure, calibration standard preparation, chromatographic and MS parameters were previously described by Houbart et al [17]. Briefly, serum samples were thawed at room temperature and centrifuged during 10 min at 13,400 rpm.…”

Section: Methodsmentioning

confidence: 99%

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

et al. 2016

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BackgroundNonunion is a failure of healing following a bone fracture. Its physiopathology remains partially unclear and the discovery of new mediators could promote the understanding of bone healing.MethodsThirty-three atrophic nonunion (NU) patients that failed to demonstrate any radiographic improvement for 6 consecutive months were recruited for providing serum samples. Thirty-five healthy volunteers (HV) served as the control group. Proteomics studies were performed using SELDI-TOF–MS and 2D-DIGE approaches, associated or not with Proteominer® preprocessing, to highlight biomarkers specific to atrophic nonunion pathology. Peak intensities were analyzed by two statistical approaches, a nonparametric Mann–Whitney U tests (univariate approach) and a machine-learning algorithm called extra-trees (multivariate approach). Validation of highlighted biomarkers was performed by alternative approaches such as microfluidic LC–MS/MS, nephelometry, western blotting or ELISA assays.ResultsFrom the 35 HV and 33 NU crude serum samples and Proteominer® eluates, 136 spectra were collected by SELDI-TOF–MS using CM10 and IMAC-Cu2+ ProteinChip arrays, and 665 peaks were integrated for extra-trees multivariate analysis. Accordingly, seven biomarkers and several variants were identified as potential NU biomarkers. Their levels of expression were found to be down- or up-regulated in serum of HV vs NU. These biomarkers are inter-α-trypsin inhibitor H4, hepcidin, S100A8, S100A9, glycated hemoglobin β subunit, PACAP related peptide, complement C3 α-chain. 2D-DIGE experiment allowed to detect 14 biomarkers as being down- or up-regulated in serum of HV vs NU including a cleaved fragment of apolipoprotein A-IV, apolipoprotein E, complement C3 and C6. Several biomarkers such as hepcidin, complement C6, S100A9, apolipoprotein E, complement C3 and C4 were confirmed by an alternative approach as being up-regulated in serum of NU patients compared to HV controls.ConclusionTwo proteomics approaches were used to identify new biomarkers up- or down-regulated in the nonunion pathology, which are involved in bone turn-over, inflammation, innate immunity, glycation and lipid metabolisms. High expression of hepcidin or S100A8/S100A9 by myeloid cells and the presence of advanced glycation end products and complement factors could be the result of a longstanding inflammatory process. Blocking macrophage activation and/or TLR4 receptor could accelerate healing of fractured bone in at-risk patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1019-1) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

et al. 2016

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“…High hepcidin levels are indicative of the ACD and of IRIDA. Mass spectrometry assays are based on the identification of the 25-amino acid form of hepcidin in plasma (93)(94)(95)(96)(97). This form of hepcidin binds to FPN1 and targets FPN1 for degradation in the lysosome (5).…”

Section: Development Of Assays To Quantify Hepcidinmentioning

confidence: 99%

Iron regulation by hepcidin

Zhao

Zhang²,

Enns³

2013

J. Clin. Invest.

190

158

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Discovery and function of hepcidin in iron homeostasisHepcidin is a key peptide hormone that regulates iron homeostasis in chordates. Hepcidin was initially characterized as an antimicrobial peptide in a mass spectrometry-based search for cysteine-rich defensin-like peptides in blood (1) and in urine (2). However, both groups showed that hepcidin displays only a weak antimicrobial activity. Further, unlike defensins, which vary in sequence among species, hepcidin is highly conserved from zebrafish to humans. Shortly after hepcidin was described, subtractive hybridization studies comparing the livers of normal and iron overloaded mice established a connection between iron loading and increased hepcidin mRNA (3, 4). The fundamental insight into hepcidin's role in iron homeostasis came in 2004 with the discovery that hepcidin acts to lower iron in the blood by binding to and downregulating the iron transporter, ferroportin (FPN1) (5). FPN1 is the only known transporter that is responsible for the efflux of iron from cells. Downregulation of FPN1 by hepcidin in splenic and hepatic macrophages decreases the ability of macrophages to export recycled iron from senescent rbcs, which constitute the primary source of iron in the plasma. In addition, a high concentration of hepcidin in the blood decreases the transport of iron out of intestinal epithelial cells, further limiting iron in the blood. Control of hepcidin expressionHepcidin is synthesized, processed to an active form, and secreted predominantly by hepatocytes (6, 7). The expression of hepcidin is mediated through the bone morphogenetic protein (BMP) and JAK2/STAT3 signaling pathways (Figure 1). Under nonpathological conditions, iron levels in the body upregulate hepcidin expression. Although the underlying mechanisms are poorly understood, recent studies have documented the essential roles of hemojuvelin (HJV), hereditary hemochromatosis protein (HFE), transferrin receptor 2 (TfR2), and matriptase-2 (MT2, encoded by the gene TMPRSS6) in the process of hepcidin regulation in humans and animal models as well as of BMP6, neogenin, and BMP receptors (ActRIIA/ALK2/ALK3) in animal models (8)(9)(10)(11)(12)(13)(14)(15)(16)(17).Intact BMP signaling is essential for hepcidin expression. The canonical BMP-signaling pathway is initiated upon BMP binding to a BMP receptor complex on the cell surface, which activates the receptor kinase to phosphorylate the cytoplasmic proteins SMAD1, SMAD5, and SMAD8. These phosphorylated, receptor-regulated SMADs then form transcription factor complexes with SMAD4, consisting of receptor-regulated SMADs and SMAD4, that translocate into the nucleus to induce the transcription of target genes such as hepcidin (18). In mice, liver-specific disruption of SMAD4 or the BMP receptors ALK2 or ALK3 markedly decreased hepcidin expression, resulting in iron overload (15,19).BMP6. At least 20 BMPs are expressed in mammals. In vitro studies reveal that BMP2, -4, -5, -6, -7, and -9 can robustly induce BMP signaling and markedly increase hepcidin expres...

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“…Using microfluidic chip-based technology, incorporating an enrichment column, a six-port valve, a separation column, and a nano electrospray tip allows the coupling of miniaturized separation with a nano ESI source (30). This design results in lower dead volumes and fewer connections compared with classical nano LC equipment, thereby leading to increased sensitivity and robustness by less peak broadening and dilution effects during transfer of the sample to the LC column and later to the LC-MS interface (31).…”

mentioning

confidence: 99%

Quantitative profiling of endocannabinoids and related N-acylethanolamines in human CSF using nano LC-MS/MS

Kantae

Ogino

Noga

et al. 2017

Journal of Lipid Research

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The endocannabinoid system has emerged as a crucial regulator of synaptic communication in the CNS (1-3). Dysregulation of this system is implicated in various neurological and psychiatric disorders, such as neuroinflammation, stroke, brain trauma, anxiety, and depression (4-8). The endocannabinoid system consists of endogenous lipid messengers (endocannabinoids) that activate two distinct cannabinoid (CB) (CB 1 and CB 2 ) receptors and the en-

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Development of a nano-liquid chromatography on chip tandem mass spectrometry method for high-sensitivity hepcidin quantitation

Cited by 36 publications

References 38 publications

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

Biomarkers of inflammation and innate immunity in atrophic nonunion fracture

Iron regulation by hepcidin

Quantitative profiling of endocannabinoids and related N-acylethanolamines in human CSF using nano LC-MS/MS

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