We use cookies to improve your experience. By continuing to browse this site, you accept our cookie policy.×

MS-based approaches to unravel the molecular complexity of proprotein-derived biomarkers and support their quantification: the examples of B-type natriuretic peptide and apelin peptides

    Cédric Mesmin

    CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, F-91191 Gif-sur-Yvette, France

    ,
    Margaux Renvoisé

    CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, F-91191 Gif-sur-Yvette, France

    ,
    François Becher

    CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, F-91191 Gif-sur-Yvette, France

    ,
    Eric Ezan

    CEA, iBEB, Service de Biochimie et Toxicologie Nucléaire, F-30207 Bagnols-sur-Cèze, France

    &
    François Fenaille

    * Author for correspondence

    CEA, iBiTec-S, Service de Pharmacologie et d’Immunoanalyse, F-91191 Gif-sur-Yvette, France.

    Published Online:https://doi.org/10.4155/bio.12.259

    The specific forms of described protein biomarkers that occur in human blood are not yet fully established. Even though B-type natriuretic peptide (BNP) and N-terminal proBNP are now well known markers of heart failure and other cardiac disorders, several studies yielded highly controversial results reporting various truncated, multimerized or modified forms in human blood. Similar discrepancies were observed for other biomarkers also originating from proproteins, such as the apelin peptides. The drawback of most of these studies is that they used methods with low resolving power, such as immunoassays after HPLC separation. MS-based techniques may be able to avoid such flaws. In this review, we discuss the usefulness of MS-based approaches for the characterization of circulating forms of peptide biomarkers that originate from a given proprotein. Two particular examples are discussed in detail: BNP-related peptides and some more putative biomarkers of heart failure, the apelin peptides.

    Papers of special note have been highlighted as: ▪ of interest ▪▪ of considerable interest

    References

    • Huttenhain R, Malmstrom J, Picotti P, Aebersold R. Perspectives of targeted mass spectrometry for protein biomarker verification. Curr. Opin. Chem. Biol.13(5–6),518–525 (2009).Crossref, Medline, CASGoogle Scholar
    • Carr SA, Anderson L. Protein quantitation through targeted mass spectrometry: the way out of biomarker purgatory? Clin. Chem.54(11),1749–1752 (2008).Crossref, Medline, CASGoogle Scholar
    • Anderson NL. The clinical plasma proteome: a survey of clinical assays for proteins in plasma and serum. Clin. Chem.56(2),177–185 (2010).Crossref, Medline, CASGoogle Scholar
    • Lopez MF, Rezai T, Sarracino DA et al. Selected reaction monitoring-mass spectrometric immunoassay responsive to parathyroid hormone and related variants. Clin. Chem.56(2),281–290 (2010).Crossref, Medline, CASGoogle Scholar
    • Chen CY, Asakawa A, Fujimiya M, Lee SD, Inui A. Ghrelin gene products and the regulation of food intake and gut motility. Pharmacol. Rev.61(4),430–481 (2009).Crossref, Medline, CASGoogle Scholar
    • Tatemoto K, Hosoya M, Habata Y et al. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor. Biochem. Biophys. Res. Commun.251(2),471–476 (1998).▪ First description of apelin, endogenous ligand of APJ.Crossref, Medline, CASGoogle Scholar
    • Sudoh T, Kangawa K, Minamino N, Matsuo H. A new natriuretic peptide in porcine brain. Nature332(6159),78–81 (1988).Crossref, Medline, CASGoogle Scholar
    • Kambayashi Y, Nakao K, Mukoyama M et al. Isolation and sequence determination of human brain natriuretic peptide in human atrium. FEBS Lett.259(2),341–345 (1990).Crossref, Medline, CASGoogle Scholar
    • Glenner GG, Wong CW. Alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun.120(3),885–890 (1984).Crossref, Medline, CASGoogle Scholar
    • 10  Masters CL, Simms G, Weinman NA et al. Amyloid plaque core protein in Alzheimer disease and Down syndrome. Proc. Natl Acad. Sci. USA82(12),4245–4249 (1985).Crossref, Medline, CASGoogle Scholar
    • 11  Kang J, Lemaire HG, Unterbeck A et al. The precursor of Alzheimer’s disease amyloid A4 protein resembles a cell-surface receptor. Nature325(6106),733–736 (1987).Crossref, Medline, CASGoogle Scholar
    • 12  Brewer HB, Ronan R Jr. Bovine parathyroid hormone: amino acid sequence. Proc. Natl Acad. Sci. USA67(4),1862–1869 (1970).Crossref, Medline, CASGoogle Scholar
    • 13  Prelli F, Castano E, Glenner GG, Frangione B. Differences between vascular and plaque core amyloid in Alzheimer’s disease. J. Neurochem.51(2),648–651 (1988).Crossref, Medline, CASGoogle Scholar
    • 14  Kojima M, Hosoda H, Date Y et al. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature402(6762),656–660 (1999).Crossref, Medline, CASGoogle Scholar
    • 15  Tatemoto K, Carlquist M, Mutt V. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature296(5858),659–660 (1982).Crossref, Medline, CASGoogle Scholar
    • 16  Tatemoto K. Neuropeptide Y: complete amino acid sequence of the brain peptide. Proc. Natl Acad. Sci. USA79(18),5485–5489 (1982).Crossref, Medline, CASGoogle Scholar
    • 17  Hoofnagle AN, Wener MH. The fundamental flaws of immunoassays and potential solutions using tandem mass spectrometry. J. Immunol. Methods347(1–2),3–11 (2009).▪ Good review summarizing the usefulness of MS-based approaches as an alternative or complement to immunoassays.Crossref, Medline, CASGoogle Scholar
    • 18  Portelius E, Mattsson N, Andreasson U, Blennow K, Zetterberg H. Novel aβ isoforms in Alzheimer’s disease-their role in diagnosis and treatment. Curr. Pharm. Des.17(25),2594–2602 (2011).Crossref, Medline, CASGoogle Scholar
    • 19  Halim A, Brinkmalm G, Ruetschi U et al. Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid beta-peptides in human cerebrospinal fluid. Proc. Natl Acad. Sci. USA108(29),11848–11853 (2011).Crossref, Medline, CASGoogle Scholar
    • 20  Mesmin C, Dubois M, Becher F, Fenaille F, Ezan E. Liquid chromatography/tandem mass spectrometry assay for the absolute quantification of the expected circulating apelin peptides in human plasma. Rapid Commun. Mass Spectrom.24(19),2875–2884 (2010).▪ First report describing the analysis of apelin peptides in human plasma using targeted MS.Crossref, Medline, CASGoogle Scholar
    • 21  Mair J. Biochemistry of B-type natriuretic peptide-where are we now? Clin. Chem. Lab. Med.46(11),1507–1514 (2008).Crossref, Medline, CASGoogle Scholar
    • 22  Mair J. Clinical significance of pro-B-type natriuretic peptide glycosylation and processing. Clin. Chem.55(3),394–397 (2009).Crossref, Medline, CASGoogle Scholar
    • 23  Nishikimi T, Kuwahara K, Nakao K. Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides. J. Cardiol.57(2),131–140 (2011).Crossref, MedlineGoogle Scholar
    • 24  Macht M. Mass spectrometric top-down analysis of proteins. Bioanalysis1(6),1131–1148 (2009).Link, CASGoogle Scholar
    • 25  Breuker K, Jin M, Han X, Jiang H, McLafferty FW. Top-down identification and characterization of biomolecules by mass spectrometry. J. Am. Soc. Mass Spectrom.19(8),1045–1053 (2008).Crossref, Medline, CASGoogle Scholar
    • 26  Seidler J, Zinn N, Boehm ME, Lehmann WD. De novo sequencing of peptides by MS/MS. Proteomics10(4),634–649 (2010).Crossref, Medline, CASGoogle Scholar
    • 27  Domon B, Aebersold R. Mass spectrometry and protein analysis. Science312(5771),212–217 (2006).Crossref, Medline, CASGoogle Scholar
    • 28  Ewles M, Goodwin L. Bioanalytical approaches to analyzing peptides and proteins by LC–MS/MS. Bioanalysis3(12),1379–1397 (2011).Link, CASGoogle Scholar
    • 29  Eyrich B, Sickmann A, Zahedi RP. Catch me if you can: mass spectrometry-based phosphoproteomics and quantification strategies. Proteomics11(4),554–570 (2011).Crossref, Medline, CASGoogle Scholar
    • 30  van den Broek I, Sparidans RW, Schellens JH, Beijnen JH. Quantitative bioanalysis of peptides by liquid chromatography coupled to (tandem) mass spectrometry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.872(1–2),1–22 (2008).▪▪ Excellent review describing the issues for the quantitative analysis of peptides by MS(/MS).Crossref, Medline, CASGoogle Scholar
    • 31  Domon B, Aebersold R. Options and considerations when selecting a quantitative proteomics strategy. Nat. Biotechnol.28(7),710–721 (2010).Crossref, Medline, CASGoogle Scholar
    • 32  Izrael-Tomasevic A, Phu L, Phung QT, Lill JR, Arnott D. Targeting interferon alpha subtypes in serum: a comparison of analytical approaches to the detection and quantitation of proteins in complex biological matrices. J. Proteome Res.8(6),3132–3140 (2009).Crossref, Medline, CASGoogle Scholar
    • 33  Kiernan UA, Addobbati R, Nedelkov D, Nelson RW. Quantitative multiplexed C-reactive protein mass spectrometric immunoassay. J. Proteome Res.5(7),1682–1687 (2006).Crossref, Medline, CASGoogle Scholar
    • 34  Anderson NL, Razavi M, Pearson TW et al. Precision of heavy-light peptide ratios measured by MALDI-TOF mass spectrometry. J. Proteome Res.11(3),1868–1878 (2012).Crossref, Medline, CASGoogle Scholar
    • 35  Mesmin C, Fenaille F, Ezan E, Becher F. MS-based approaches for studying the pharmacokinetics of protein drugs. Bioanalysis3(5),477–480 (2011).Link, CASGoogle Scholar
    • 36  Kalea AZ, Batlle D. Apelin and ACE2 in cardiovascular disease. Curr. Opin. Investig. Drugs11(3),273–282 (2010).Medline, CASGoogle Scholar
    • 37  Palazzuoli A, Gallotta M, Quatrini I, Nuti R. Natriuretic peptides (BNP and NT-proBNP): measurement and relevance in heart failure. Vasc. Health Risk Manag.6411–6418 (2010).Google Scholar
    • 38  Sudoh T, Minamino N, Kangawa K, Matsuo H. Brain natriuretic peptide-32: N-terminal six amino acid extended form of brain natriuretic peptide identified in porcine brain. Biochem. Biophys. Res. Commun.155(2),726–732 (1988).Crossref, Medline, CASGoogle Scholar
    • 39  Ueda S, Minamino N, Sudoh T, Kangawa K, Matsuo H. Regional distribution of immunoreactive brain natriuretic peptide in porcine brain and spinal cord. Biochem. Biophys. Res. Commun.155(2),733–739 (1988).Crossref, Medline, CASGoogle Scholar
    • 40  Hunt PJ, Yandle TG, Nicholls MG, Richards AM, Espiner EA. The amino-terminal portion of pro-brain natriuretic peptide (Pro-BNP) circulates in human plasma. Biochem. Biophys. Res. Commun.214(3),1175–1183 (1995).Crossref, Medline, CASGoogle Scholar
    • 41  Shimizu H, Masuta K, Aono K et al. Molecular forms of human brain natriuretic peptide in plasma. Clin. Chim. Acta316(1–2),129–135 (2002).Crossref, Medline, CASGoogle Scholar
    • 42  Shimizu H, Masuta K, Asada H, Sugita K, Sairenji T. Characterization of molecular forms of probrain natriuretic peptide in human plasma. Clin. Chim. Acta334(1–2),233–239 (2003).Crossref, Medline, CASGoogle Scholar
    • 43  Seferian KR, Tamm NN, Semenov AG et al. The brain natriuretic peptide (BNP) precursor is the major immunoreactive form of BNP in patients with heart failure. Clin. Chem.53(5),866–873 (2007).Crossref, Medline, CASGoogle Scholar
    • 44  Liang F, O’Rear J, Schellenberger U et al. Evidence for functional heterogeneity of circulating B-type natriuretic peptide. J. Am. Coll. Cardiol.49(10),1071–1078 (2007).Crossref, Medline, CASGoogle Scholar
    • 45  Semenov AG, Postnikov AB, Tamm NN et al. Processing of pro-brain natriuretic peptide is suppressed by O-glycosylation in the region close to the cleavage site. Clin. Chem.55(3),489–498 (2009).Crossref, Medline, CASGoogle Scholar
    • 46  Ala-Kopsala M, Magga J, Peuhkurinen K et al. Molecular heterogeneity has a major impact on the measurement of circulating N-terminal fragments of A- and B-type natriuretic peptides. Clin. Chem.50(9),1576–1588 (2004).Crossref, Medline, CASGoogle Scholar
    • 47  Seferian KR, Tamm NN, Semenov AG et al. Immunodetection of glycosylated NT-proBNP circulating in human blood. Clin. Chem.54(5),866–873 (2008).Crossref, Medline, CASGoogle Scholar
    • 48  Tateyama H, Hino J, Minamino N et al. Characterization of immunoreactive brain natriuretic peptide in human cardiac atrium. Biochem. Biophys. Res. Commun.166(3),1080–1087 (1990).Crossref, Medline, CASGoogle Scholar
    • 49  Hino J, Tateyama H, Minamino N, Kangawa K, Matsuo H. Isolation and identification of human brain natriuretic peptides in cardiac atrium. Biochem. Biophys. Res. Commun.167(2),693–700 (1990).Crossref, Medline, CASGoogle Scholar
    • 50  Tateyama H, Hino J, Minamino N et al. Concentrations and molecular forms of human brain natriuretic peptide in plasma. Biochem. Biophys. Res. Commun.185(2),760–767 (1992).Crossref, Medline, CASGoogle Scholar
    • 51  Hammerer-Lercher A, Halfinger B, Sarg B et al. Analysis of circulating forms of proBNP and NT-proBNP in patients with severe heart failure. Clin. Chem.54(5),858–865 (2008).Crossref, Medline, CASGoogle Scholar
    • 52  Niederkofler EE, Kiernan UA, O’Rear J et al. Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure. Circ. Heart Fail.1(4),258–264 (2008).▪▪ A quantitative MS immunoassay to capture endogenous B-type natriuretic peptide (BNP) peptides was developed using high-affinity antibodies. The results revealed rapid in vitro degradation of BNP-(1-32) in plasma, and showed that plasma collection should be performed in the presence of high protease inhibitor concentrations.Crossref, Medline, CASGoogle Scholar
    • 53  Seidler T, Pemberton C, Yandle T et al. The amino terminal regions of proBNP and proANP oligomerise through leucine zipper-like coiled-coil motifs. Biochem. Biophys. Res. Commun.255(2),495–501 (1999).Crossref, Medline, CASGoogle Scholar
    • 54  Crimmins DL. Human N-terminal proBNP is a monomer. Clin. Chem.51(6),1035–1038 (2005).Crossref, Medline, CASGoogle Scholar
    • 55  Crimmins DL, Kao JL. A glycosylated form of the human cardiac hormone pro B-type natriuretic peptide is an intrinsically unstructured monomeric protein. Arch. Biochem. Biophys.475(1),36–41 (2008).Crossref, Medline, CASGoogle Scholar
    • 56  Schellenberger U, O’Rear J, Guzzetta A et al. The precursor to B-type natriuretic peptide is an O-linked glycoprotein. Arch. Biochem. Biophys.451(2),160–166 (2006).Crossref, Medline, CASGoogle Scholar
    • 57  Semenov AG, Tamm NN, Seferian KR et al. Processing of pro-B-type natriuretic peptide: furin and corin as candidate convertases. Clin. Chem.56(7),1166–1176 (2010).Crossref, MedlineGoogle Scholar
    • 58  Lam CS, Burnett JC Jr, Costello-Boerrigter L, Rodeheffer RJ, Redfield MM. Alternate circulating pro-B-type natriuretic peptide and B-type natriuretic peptide forms in the general population. J. Am. Coll. Cardiol.49(11),1193–1202 (2007).Crossref, Medline, CASGoogle Scholar
    • 59  Miller WL, Phelps MA, Wood CM et al. Comparison of mass spectrometry and clinical assay measurements of circulating fragments of B-type natriuretic peptide in patients with chronic heart failure. Circ. Heart Fail.4(3),355–360 (2011).▪ Comparison of MS-based approaches and immunoassays for the measurement of circulating fragments of BNP.Crossref, Medline, CASGoogle Scholar
    • 60  Brandt I, Lambeir AM, Ketelslegers JM et al. Dipeptidyl-peptidase IV converts intact B-type natriuretic peptide into its des-SerPro form. Clin. Chem.52(1),82–87 (2006).Crossref, Medline, CASGoogle Scholar
    • 61  Ala-Kopsala M, Moilanen AM, Rysa J, Ruskoaho H, Vuolteenaho O. Characterization of molecular forms of N-terminal B-type natriuretic peptide in vitro. Clin. Chem.56(12),1822–1829 (2010).Crossref, Medline, CASGoogle Scholar
    • 62  Xu-Cai YO, Wu Q. Molecular forms of natriuretic peptides in heart failure and their implications. Heart96(6),419–424 (2010).Crossref, MedlineGoogle Scholar
    • 63  Hawkridge AM, Heublein DM, Bergen HR III et al. Quantitative mass spectral evidence for the absence of circulating brain natriuretic peptide (BNP-32) in severe human heart failure. Proc. Natl Acad. Sci. USA102(48),17442–17447 (2005).Crossref, Medline, CASGoogle Scholar
    • 64  Luckenbill KN, Christenson RH, Jaffe AS et al. Cross-reactivity of BNP, NT-proBNP, and proBNP in commercial BNP and NT-proBNP assays: preliminary observations from the IFCC committee for standardization of markers of cardiac damage. Clin. Chem.54(3),619–621 (2008).Crossref, Medline, CASGoogle Scholar
    • 65  Picotti P, Aebersold R. Selected reaction monitoring-based proteomics: workflows, potential, pitfalls and future directions. Nat. Methods9(6),555–566 (2012).Crossref, Medline, CASGoogle Scholar
    • 66  Rauh M. LC-MS/MS for protein and peptide quantification in clinical chemistry. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.883–884,59–67 (2012).Crossref, Medline, CASGoogle Scholar
    • 67  Michalski A, Damoc E, Hauschild JP et al. Mass spectrometry-based proteomics using Q Exactive, a high-performance benchtop quadrupole Orbitrap mass spectrometer. Mol. Cell Proteomics10(9),M111.011015 (2011).Crossref, MedlineGoogle Scholar
    • 68  Peterson AC, Russell JD, Bailey DJ, Westphall MS, Coon JJ. Parallel reaction monitoring for high resolution and high mass accuracy quantitative, targeted proteomics. Mol. Cell Proteomics (2012) (Epub ahead of print).MedlineGoogle Scholar
    • 69  Rochat B. Quantitative/qualitative analysis using LC–HRMS: the fundamental step forward for clinical laboratories and clinical practice. Bioanalysis4(14),1709–1711 (2012).Link, CASGoogle Scholar
    • 70  Pitkin SL, Maguire JJ, Bonner TI, Davenport AP. International union of basic and clinical pharmacology. LXXIV. Apelin receptor nomenclature, distribution, pharmacology, and function. Pharmacol. Rev.62(3),331–342 (2010).Crossref, Medline, CASGoogle Scholar
    • 71  Barnes G, Japp AG, Newby DE. Translational promise of the apelin–APJ system. Heart96(13),1011–1016 (2010).Crossref, Medline, CASGoogle Scholar
    • 72  Goetze JP, Rehfeld JF, Carlsen J et al. Apelin: a new plasma marker of cardiopulmonary disease. Regul. Pept.133(1–3),134–138 (2006).Crossref, Medline, CASGoogle Scholar
    • 73  Habata Y, Fujii R, Hosoya M et al. Apelin, the natural ligand of the orphan receptor APJ, is abundantly secreted in the colostrum. Biochim. Biophys. Acta1452(1),25–35 (1999).Crossref, Medline, CASGoogle Scholar
    • 74  Castan-Laurell I, Dray C, Attane C et al. Apelin, diabetes, and obesity. Endocrine40(1),1–9 (2011).Crossref, Medline, CASGoogle Scholar
    • 75  Charles CJ. Update on apelin peptides as putative targets for cardiovascular drug discovery. Exp. Opin. Drug Discovery6(6),633–644 (2011).Crossref, Medline, CASGoogle Scholar
    • 76  Kawamata Y, Habata Y, Fukusumi S et al. Molecular properties of apelin: tissue distribution and receptor binding. Biochim. Biophys. Acta1538(2–3),162–171 (2001).Crossref, Medline, CASGoogle Scholar
    • 77  De MN, Reaux-Le GA, El MS et al. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release. Proc. Natl Acad. Sci. USA101(28),10464–10469 (2004).Crossref, MedlineGoogle Scholar
    • 78  Földes G, Horkay F, Szokodi I et al. Circulating and cardiac levels of apelin, the novel ligand of the orphan receptor APJ, in patients with heart failure. Biochem. Biophys. Res. Commun.308(3),480–485 (2003).Crossref, Medline, CASGoogle Scholar
    • 79  Maguire JJ, Kleinz MJ, Pitkin SL, Davenport AP. [Pyr1]apelin-13 identified as the predominant apelin isoform in the human heart: vasoactive mechanisms and inotropic action in disease. Hypertension54(3),598–604 (2009).▪ First report identifying a molecular form of apelin by MS in human heart.Crossref, Medline, CASGoogle Scholar
    • 80  Aydin S. The presence of the peptides apelin, ghrelin and nesfatin-1 in the human breast milk, and the lowering of their levels in patients with gestational diabetes mellitus. Peptides31(12),2236–2240 (2010).Crossref, Medline, CASGoogle Scholar
    • 81  Hosoya M, Kawamata Y, Fukusumi S et al. Molecular and functional characteristics of APJ. Tissue distribution of mRNA and interaction with the endogenous ligand apelin. J. Biol. Chem.275(28),21061–21067 (2000).Crossref, Medline, CASGoogle Scholar
    • 82  Mesmin C, Fenaille F, Becher F, Tabet JC, Ezan E. Identification and characterization of apelin peptides in bovine colostrum and milk by liquid chromatography–mass spectrometry. J. Proteome Res.10(11),5222–5231 (2011).Crossref, Medline, CASGoogle Scholar
    • 83  Miettinen KH, Magga J, Vuolteenaho O et al. Utility of plasma apelin and other indices of cardiac dysfunction in the clinical assessment of patients with dilated cardiomyopathy. Regul. Pept.140(3),178–184 (2007).Crossref, Medline, CASGoogle Scholar
    • 84  Azizi M, Iturrioz X, Blanchard A et al. Reciprocal regulation of plasma apelin and vasopressin by osmotic stimuli. J. Am. Soc. Nephrol.19(5),1015–1024 (2008).Crossref, Medline, CASGoogle Scholar
    • 85  Chen MM, Ashley EA, Deng DX et al. Novel role for the potent endogenous inotrope apelin in human cardiac dysfunction. Circulation108(12),1432–1439 (2003).Crossref, Medline, CASGoogle Scholar
    • 86  Telejko B, Kuzmicki M, Wawrusiewicz-Kurylonek N et al. Plasma apelin levels and apelin/APJ mRNA expression in patients with gestational diabetes mellitus. Diabetes Res. Clin. Pract.87(2),176–183 (2010).Crossref, Medline, CASGoogle Scholar
    • 87  Semenov AG, Seferian KR, Tamm NN et al. Human pro-B-type natriuretic peptide is processed in the circulation in a rat model. Clin. Chem.57(6),883–890 (2011).Crossref, Medline, CASGoogle Scholar
    • 88  Tao Y, Lu Q, Jiang YR et al. Apelin in plasma and vitreous and in fibrovascular retinal membranes of patients with proliferative diabetic retinopathy. Invest. Ophthalmol. Vis. Sci.51(8),4237–4242 (2010).Crossref, MedlineGoogle Scholar
    • 89  Chong KS, Gardner RS, Morton JJ, Ashley EA, McDonagh TA. Plasma concentrations of the novel peptide apelin are decreased in patients with chronic heart failure. Eur. J. Heart Fail.8(4),355–360 (2006).Crossref, Medline, CASGoogle Scholar
    • 90  Weir RA, Chong KS, Dalzell JR et al. Plasma apelin concentration is depressed following acute myocardial infarction in man. Eur. J. Heart Fail.11(6),551–558 (2009).Crossref, Medline, CASGoogle Scholar
    • 91  Li Z, Bai Y, Hu J. Reduced apelin levels in stable angina. Intern. Med.47(22),1951–1955 (2008).Crossref, MedlineGoogle Scholar
    • 92  Francia P, Salvati A, Balla C et al. Cardiac resynchronization therapy increases plasma levels of the endogenous inotrope apelin. Eur. J. Heart Fail.9(3),306–309 (2007).Crossref, Medline, CASGoogle Scholar
    • 93  Boso M, Emanuele E, Politi P et al. Reduced plasma apelin levels in patients with autistic spectrum disorder. Arch. Med. Res.38(1),70–74 (2007).Crossref, Medline, CASGoogle Scholar
    • 94  Przewlocka-Kosmala M, Kotwica T, Mysiak A, Kosmala W. Reduced circulating apelin in essential hypertension and its association with cardiac dysfunction. J. Hypertens.29(5),971–979 (2011).Crossref, Medline, CASGoogle Scholar
    • 95  Ellinor PT, Low AF, Macrae CA. Reduced apelin levels in lone atrial fibrillation. Eur. Heart J.27(2),222–226 (2006).Crossref, Medline, CASGoogle Scholar
    • 101  ExPASy: Bioinformatics Research Portal. www.expasy.orgGoogle Scholar