Biochemical mechanisms of platelet function 

Biochemistry of normal and pathological human erythrocytes

Biochemistry of extracellular matrix

Functional analysis of human pathological variants of erythrocyte enzymes

Proteomics of COPD

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Investigators
Cesare Balduini, Mauro Torti, Ilaria Canobbio, Gianni Guidetti, Silvia Catricalà, Lina Cipolla, Alessandra Consonni

Overview
Platelets are involved in many physiological processes, mainly coagulation and clot retraction, but also inflammation, immunitary response, atherosclerosis and recently they have been implicated in neurodegenerative disorders. Defective platelet activation results in hemorrhagic diseases or at the contrary may cause thromboembolic pathologies which represent the principal cause of death in industrialized countries. Understanding the molecular mechanisms of platelet activation is important not only for the comprehension of the above mentioned physiological processes but also for designing new and specific drugs as a tool for the treatment of pathological conditions.

Our research group is interested in investigating the molecular mechanism of platelet activation. Platelet activation and aggregation-mediated by several agonists is analyzed in wealthy and pathological condition, and signalling transduction pathways are analyzed using specific pharmacological inhibitors, knock-out mice, and cellular models. The laboratory provides the know how and the devices for the study of platelet activation.

Integrin a₂b₁ and a_IIb_b3 cross-talk

The aim of this investigation is the study of integrin a₂b₁-mediated platelet adhesion and activation.  We focused our attention on the cross-talk between integrins a₂b₁ and a_IIbb₃. Platelet adhesion to several integrin a₂b₁-specific ligands leads, through an inside-out signalling, to integrin a_IIbb₃ activation which promotes the binding of fibrinogen to the adherent platelets. Integrins cross-talk requires phospholipase Cg2 activation and subsequent activation of the small GTPase Rap1b. Downstream integrin a₂b₁, phospholipase Cg2 activation is regulated in a peculiar manner, different from the typical and well-defined phospholipase Cg activation pathway mediated by other platelet receptors. Indeed, recent evidences show that integrin a₂b₁-mediated phospholipase Cg2 activation is simultaneously and independently regulated by Src-kinases and the small Rac GTPase.

Analysis of APP expression and metabolism in human platelets

A non correct metabolism of amyloid precursor protein (APP) is the principal cause of Alzheimher disease. Accumulation of Ab amyloidogenic peptide derived from APP in neuritic plaques impairs correct neuronal function. It has been demonstrated that APP and APP metabolism-involved secretases are expressed in human platelets. This research project investigates the expression of APP in platelets of healthy subjects and its metabolism following platelet stimulation with different agonists, in order to delineate the transduction pathways that promote Ab production and accumulation. This studies will further be extended to Alzheimer patients.

P2Y12-mediated activation of human platelets

Human platelets present on the membrane surface three purinergic receptors: the ionotropic receptor P2X1, a calcium ion channel, and the seven spanning metabotropic receptors P2Y1 and P2Y12. The P2Y12  receptor activation, target of antithrombotic drugs highly used in prophylaxis of heart attack, is necessary to evoke a complete and irreversible platelet aggregation. The study of P2Y12-mediated activation of human platelets is under investigation in our laboratory for many years. Our study is now focused in particular on the activation mechanism of protein kinase C (PKC) induced by P2Y receptors activation in human platelets. Moreover, signalling transduction will be investigated in P2Y12 and P2Y1 stably transfected 1321N1 astrocytoma cell lines. This study could be remarkable to understand the advantages and the limits of drugs against P2Y12 receptor.

Project funding

PRIN, MURST, CNR, FAR, Progetto di Ateneo, CARIPLO, Telethon.

Selected publications 2006-2009
Paganini S, Guidetti GF, Catricala S, Trionfini P, Panelli S, Balduini C, Torti M.
Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins.
Biochimie, 88:285–295, 2006.

Canobbio I, Stefanini L, Guidetti GF, Balduini C, Torti M.
A new role for FcgIIA in the potentiation of human platelet activation induced by weak stimulation.
Cell Signal, 18:861–870, 2006.

Greco F, Ciana A, Pietra D, Balduini C, Minetti G, Torti M.
Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation
Biochim Biophys Acta, 1763:330–335, 2006.

Bernardi B, Guidetti GF, Campus F, Crittenden JR, Graybiel AM, Balduini C, Torti M.
The small GTPase Rap1b regulates the cross-talk between platelet integrin a₂b₁ and integrin a_IIbb₃.
Blood, 107:2728–2735, 2006.

Noris P, Guidetti GF, Conti V, Ceresa IF, Di Pumpo M, Pecci A, Torti M, Savoia A, Balduini CL.
Autosomal dominant thrombocytopenias with reduced expression of glycoprotein Ia.
Thromb Haemost, 95:483-489, 2006.

Reineri S, Bertoni A, Sanna E, Baldassarri S, Sarasso C, Zanfa M, Canobbio I, Torti M, Sinigaglia F.
Membrane lipid rafts coordinate estrogen-dependent signaling in human platelets.
Biochim Biophys Acta, 1773:273–278, 2007.

Mancini F, Rigacci S, Berti A, Balduini C, Torti M.
The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgRIIA-mediated cell activation.
Blood, 110:1871–1878, 2007.

Ghilotti M, Lova P, Balduini C, Torti M.
Epinephrine induces intracellular Ca²⁺ mobilization in thrombin-desensitized platelets: a role for GPIb-IX-V.
Platelets, 18:135–142, 2007.

Canobbio I, Trionfini P, Guidetti GF, Balduini C, Torti M.
Targeting of the small GTPase Rap2b, but not Rap1b, to lipid rafts is promoted by palmitoylation at Cys176 and Cys177 and is required for efficient protein activation in human platelets.
Cell Signal, 20:1662-1670, 2008.

Guidetti GF, Lova P, Bernardi B, Campus F, Baldanzi G, Graziani A, Balduini C, Torti M.
The Gi-coupled P2Y12 receptor regulates diacylglycerol-mediated signaling in human platelets.
J Biol Chem, 283:28795-28805, 2008.

Guidetti GF, Bernardi B, Consonni A, Rizzo P, Gruppi C, Balduini C, Torti M.
Integrin alpha2beta1 induces phosphorylation dependent and independent activation of phospholipase Cgamma2 in platelets: role of Src kinase and Rac GTPase.
J Thromb Haemost, in press PubMed PMID: 19422462, 2009.

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Biochemistry of normal and pathological human erythrocytes (ITALIAN)

Investigators
Giampaolo Minetti, Annarita Ciana, Cesare Achilli, Cesare Balduini

Research topics
As they age in vivo, human erythrocytes undergo dehydration, decrease in surface area extension, and alterations in the antigenic properties of the membrane. These events lead, after approximately 120 days of circulatory life, to the removal of the aged erythrocyte, probably by activation of an auto-immune mechanism involving binding of auto-antibodies to neo-antigens on the cell surface (anti: band3, alpha-galactosides, and other, still undefined epitopes ) and the activation of the complement. The reduced extension of the membrane surface area in old erythrocytes is likely due to loss of micro-vesicles. Erythrocytes can be induced to release micro- and nano-vesicles by a variety of stimuli, among which the activation of the complement appears to play a role in vivo. We study the vesiculation process, during which, at the molecular level, several modifications in the interactions between proteins of the membrane-skeleton and the integral membrane proteins of the lipid bilayer occur. The anchoring of the membrane-skeleton to the membrane is dependent on “vertical” interactions between spectrin oligomers (the main component of the membrane skeleton) and band 3, the principal integral protein, mediated by ankyrin, and between glycophorin C and spectrin/protein 4.1 complexes. We are characterizing the components of these various structures in normal, pathological and stored human erythrocytes. We also investigate the role of the  modification of band 3 by tyrosine phosphorylation, which is studied in relation to the stability of the membrane, to the redox state of the cell and to the control of ion transport and of intracellular pH (in collaboration with Prof. I. Bernhardt, Saarland University). Another research topic is the characteriziation of the low molecular weight G-proteins in erythrocytes of different age and in the micro-vesicles. We have studied the modifications erythrocytes undergo during storage, as a function of cell age.

Erythrocytes are endowed with enzymatic and non-enzymatic antioxidant systems,  and with repair systems for oxidized lipids and proteins: glutathione (GSH) catalase, superoxide dismutase, GSH-reductase, GSH-peroxidase, GSH-S-transferase and peroxiredoxin (Prx). The list of antioxidant components of the erythrocyte does not include enzymes capable of reducing methionine sulfoxide (Met-SO) residues in proteins. The number of  methionine-sulfoxide-reductase (Msr) activities that are being described is constantly increasing. The research conducted in our laboratory led us to conclude that human erythrocytes lack any detectable Msr activity. During this study, a method was proposed to assay Msr activity, based on reversed-phase-HPLC separation of DABS-derivatives of Met and Met-SO, which has been successfully  adopted by several authors ever since. While studying Msr activity from human neutrophils, we observed a stereospecific reduction of only one of the two sulfoxides of L-Met. Our original suspect that two classes of Msr with opposite stereospecificity might exist in nature, is now confirmed and supported by several reports in the recent literature. The characterization of the Msr endowment of human blood cells is in progress in our laboratory. Since March 2004, the group is involved in the CellPROM project, of the 6th Framework Programme of the European Commission. The project, which involves 27 academic and industrial researchers from 12 countries for a period of four years, aims at developing an automated device for the non-invasive "reprogramming" of individual cells on an industrial scale, by interaction of the cells with nanostructured surfaces (Nanolandscapes).

Research funds
MURST; FAR; Fondi Comitato Biotecnologie e Biologia Molecolare; Integrated Project CellPROM of the 6th Framework Programme of the European Commission.

Relevant publications
Minetti G, Balduini C, Brovelli A. 
Reduction of DABS-L-methionine-dl-sulfoxide by protein methionine sulfoxide reductase from polymorphonuclear leukocytes: stereospecificity towards the l-sulfoxide.
Ital J Biochem, 43:273–283, 1994.

Minetti G, Ciana A, Profumo A, Zappa M, Vercellati C, Zanella A,. Arduini A, Brovelli A.
Cell age-related monovalent cations content and density changes in stored human erythrocytes
Biochim Biophys Acta, 1527:149–155, 2001.

Brovelli A, Minetti G.
Red Cell Ageing. In: Red Cell Membrane Transport in Health and Disease, I. Bernhardt, J.C. Ellory, eds.,
Springer-Verlag, Berlin, pp 673–690 (chapter 29), 2003.

Minetti G, Ciana A. 
New and old integral proteins of the human erythrocyte membrane.
Blood, 101:3751, 2003.

Minetti G, Ciana A, Balduini C. 
Differential sorting of tyrosine kinases and phosphotyrosine phosphatases acting on band 3 during vesiculation of human erythrocytes.
Biochem J, 377:489–497, 2004.

Ciana A, Minetti G, Balduini C.
Phosphotyrosine phosphatases acting on band 3 in human erythrocytes of different ages: PTP1B processing during cell ageing.
Bioelectrochemistry, 62:169–173, 2004.

Ciana A, Balduini C, Minetti G.
Detergent-resistant membranes in human erythrocytes and their connection to the membrane-skeleton.
J Biosci, 30:317–328, 2005.

Greco F, Ciana A, Pietra D, Balduini C, Minetti G, Torti M.
Rap2, but not Rap1 GTPase is expressed in human red blood cells and is involved in vesiculation
Biochim Biophys Acta, 1763:330–335, 2006.

Arduini A, Minetti G, Ciana A, Seppi C, Brovelli A, Profumo A, Vercellati C, Zappa C, Zanella A, Dottori S, Bonomini M.
Cellular properties of human erythrocytes preserved in saline–adenine–glucose–mannitol in the presence of L-carnitine
Am J Hematol, 82:31–40, 2007.

Achilli C, Ciana A, Rossi A, Balduini C, Minetti G.
Neutrophil granulocytes uniquely express, among human blood cells, high levels of Methionine-sulfoxide-reductase enzymes.
J Leukoc Biol, 83:181–189, 2008.

Crepaldi Domingues C, Ciana A, Buttafava A, Balduini C, de Paula E, Minetti G.
Resistance of human erythrocyte membranes to Triton X-100 and C₁₂E₈.
J Membr Biol, 227:39-48, 2009.

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Investigators
M. Enrica Tira, Ruggero Tenni, Cristian Gruppi.

Research topics
The research is pointed to the study of interactions between small leucine-rich proteoglycans (SLRP) and fibrillar collagens, both key components of extracellular matrices in connective tissues. Proteoglycans decorin and biglycan (two CS/DS SLRPs) and fibromodulin (a KS SLRP) are now investigated in their relations with collagens; these PGs, of recombinant origin or extracted from bovine tendon as described previously, are characterized and tested for binding with type I and II collagens. At the same time type I and II collagen peptides were purified after treatment with CNBr, analyzed for purity and structure.  Collagens and peptides were used for binding assays with biotinylated proteoglycans. Collagenous samples were than chemically modified for N-methylation or N-acetylation on Lys and Hyl residues, to verify the importance of these residues in triple helix stability or binding with proteoglycans.

 Main results

Using CNBr peptides from type I and II collagens in solid phase assays (ELISA) with biotinylated proteoglycans we found that:

- decorin, biglycan and fibromodulin have multiple binding site on collagen type I, with different affinity; 

- decorin and fibromodulin bind probably in different regions within the same peptide at the N-terminal of the molecule of collagen type II;

- bindings have ionic character and requires the triple helical conformation of collagenous samples;

- chemical modifications of collagens and peptides revealed the importance of ionic (positively charged) residues in the binding and in the thermal stability of the triple helices.

Project funding

The researche is supported by grants from: M.U.R.S.T. 40% e 60%, C.N.R.; “Progetto giovani ricercatori”.

Relevant publications 2003-2007
Guidetti GF, Greco F, Bertoni A, Giudici C, Viola M, Tenni R, Tira ME, Balduini C,Torti M.
Platelet interaction with CNBr peptides from type II collagen via integrin a₂b₁
Biochim Biophys Acta, 1640:43–51, 2003.

Giudici C, Viola M, Tira ME, Forlino A, Tenni R.
Molecular stability of chemically modified collagen triple helices
FEBS Lett, 547:170–176, 2003.

Guidetti GF, Bartolini B, Bernardi B, Tira ME, Berndt MC, Balduini C, Torti M.
Binding of von Willebrand factor to the small proteoglycan decorin.
FEBS Lett, 574:95–100, 2004. 

Tenni R, Sonaggere M, Viola M, Bartolini B, Tira ME, Rossi A, Orsini E, Ruggeri A, Ottani V.
Self-aggregation of fibrillar collagens I and II involves lysine side chains.
Micron, 37:640–647, 2006.

Raspanti M, Viola M, Sonaggere M, Tira ME, Tenni R.
Collagen fibril structure is affected by collagen concentration and decorin.
Biomacromolecules, 8:2087–2091, 2007. 

Viola M, Bartolini B, Sonaggere M, Giudici C, Tenni R, Tira ME.
Fibromodulin interactions with type I and II collagens.
Connect Tissue Res, 48:141–148, 2007. 

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Functional analysis of human pathological variants of erythrocyte enzymes (ITALIAN)

Investigators
Giovanna Valentini, Laurent R. Chiarelli, Simone M. Morera

Research projects
The research unit carries out studies concerning structure-function relationships of proteins. The research unit has a long standing experience in the expression, purification, and characterization of proteins as well as in protein engineering. Starting from the last ten years, the research has been focused on the biochemical characterization of human defective enzymes accountable for hereditary non-spherocytic hemolytic anemia. The main aim of these studies is to understand the molecular bases of enzymatic deficiencies. The enzymes are produced with genetic engineering techniques, purified to homogeneity and characterized. The comparison of the molecular, kinetic and thermostability properties of the mutant forms with those of the wild-type enzymes allows to point out  the molecular alterations caused by mutations  and correlate the genotype with the clinical phenotype. Enzymes under investigation are mutant forms of pyruvate kinase type R, pyrimidine 5'-nucleotidase type 1, adenylate kinase type 1 and, recently, of phosphoglycerate kinase type 1. Main results have been published on peer-reviewed international journals and serve as valuable tools to understanding and assist with diagnosis and genetic counseling.

Relevant publications 2005-2008
Chiarelli LR, Bianchi P, Fermo E, Galizzi A, Iadarola P, Mattevi A, Zanella A, Valentini G.
Functional analysis of pyrimidine 5'-nucleotidase mutants causing nonspherocytic hemolytic anemia.
Blood, 105:3340-3345, 2005.

Fermo E, Bianchi P, Chiarelli LR, Cotton F, Vercellati C, Writzl K, Baker K, Hann I, Rodwell R, Valentini G, Zanella A.
Red cell pyruvate kinase deficiency: 17 new mutations of the PK-LR gene
Br J Haematol, 129:839-846, 2005

Zanella A, Fermo E, Bianchi P, Valentini G.
Red cell pyruvate kinase deficiency: molecular and clinical aspects.
Br J Haematol 130:11-25, 2005.

Azzalin A, Del Vecchio I, Chiarelli LR, Valentini G, Comincini S, Ferretti L.
Absence of Interaction between Doppel and GFAP, Grb2, PrPC Proteins in Human Tumor Astrocytic Cells
Anticancer Res 25:4369-4374, 2005

Chiarelli LR, Fermo E, Zanella A, Valentini G.
Hereditary erythrocyte pyrimidine 5’-nucleotidase deficiency: A biochemical, genetic and clinical overview.
Hematology, 11:67-72, 2006.

Chiarelli LR, Fermo E, Abrusci P, Bianchi P, Dellacasa CM, Galizzi A, Zanella A, Valentini G.
Two new mutations of the P5’N-1 gene found in Italian patients with hereditary hemolytic anemia: the molecular basis of the red cell enzyme disorder.
Haematologica, 91:1244-1247, 2006.

Zanella A, Bianchi P, Fermo E, Valentini G.
Hereditary pyrimidine 5'-nucleotidase deficiency: from genetics to clinical manifestations.
Br J Haematol, 133:113-123, 2006

Comincini S, Chiarelli LR, Zelini I, Del Vecchio I, Azzalin A, Arias A, Ferrara V, Rognoni P, Dipoto A, Nano R, Valentini G, Ferretti L.
Nuclear mRNA retention and aberrant doppel protein expression in human astrocytic tumor cells.
Oncology Report, 16:1325-1332, 2006.

Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G.
Pyruvate kinase deficiency: The genotype-phenotype association
Blood rev, 21:217-231, 2007.

Abrusci P, Chiarelli LR, Galizzi A, Fermo E, Bianchi P, Zanella A, Valentini G.
Erythrocyte adenylate kinase deficiency: characterization of recombinant mutant forms and relationship with nonspherocytic hemolytic anemia.
Exp Hematol, 35:1182-1189, 2007.

Chiarelli LR, Morera SM, Galizzi A, Fermo E, Zanella A, Valentini G.
Molecular basis of pyrimidine 5′-nucleotidase deficiency caused by 3 newly identified missense mutations (c187T>C, c469G>C and c740T>C) and a tabulation of known mutations.
Blood Cells Mol Dis, 40:295-301, 2008.

Cappelletti D, Chiarelli LR, Pasquetto MV, Stivala S, Valentini G, Scotti C.
Helicobacter pyloril-asparaginase: a promising chemotherapeutic agent.
Biochem Biophys Res Commun, 377:1222-1226, 2008.

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Proteomics of COPD (ITALIAN)

Investigators
Paolo Iadarola, Maurizia Valli, Simona Viglio, Fabio Ferrari, Marco Fumagalli.

Research project
Although several million people in the world are currently diagnosed with chronic obstructive pulmonary disease (COPD), research progress in this field has been very slow. Owing to the urgent need for additional research, and to overcome the gaps in the current state of knowledge, a group of scientists, convened to discuss potential directions for future investigations in COPD, have indicated specific recommendations are the efforts involving non-invasive methods for identifying new biomarkers of COPD. Aim of our research is to investigate the proteomics of COPD. The overall hypothesis is that unique polypeptide markers can be detected in sputum, blood, urine and exhaled breath condensate of chronic bronchitis and emphysema subjects. These markers may play roles in disease pathophysiology thus being potential targets for advanced therapies. Furthermore they may change with disease activity and offer prognostic information. These polypeptides can be separated, quantified and sequenced by liquid chromatography mass spectrometry, bidimensional electrophoresis and SELDI-TOF. The presence, absence, or polymorphism of specific polypeptides can be detected and the expression of these markers can be used to test specific hypothesis of COPD pathogenesis.
Our group collaborates with: Department of Pulmonology, University Medical Center, Leiden, The Netherlands; Georgetown University Proteomics Laboratory, Washington, DC, USA; Laboratory of Biochemistry and Genetics, Institute of Respiratory Disease, IRCCS San Matteo Hospital Foundation, Pavia; Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Ferrara; Dipartimento di Fisiopatologia e Medicina Sperimentale, Università di Siena.

Main results
Analyzing the proteome of a biological fluid is a daunting task if the goal is to detect specific changes in response to the stimulus of a pathophysiological event. To investigate the EBC proteome of controls and COPD patients, we have applied orthogonal techniques with the aim of increasing analytical rigor through multiple validation of results.
Cytokeratins have been observed as the major components of all samples from patients investigated. In particular we identified cytokeratin 1, cytokeratin 9 anda cytokeratin 10. On the contrary, neither 2-DE or HPLC have been able to detect these analytes in any of EBC from controls. On the light of these results we can hypothesize that the presence of cytokeratins in EBC may serve as an indicator of lung damage.
We also observed a series of low-molecular weight proteins that  were tentatively identified as IFN-γ; IL-2 and IL-15. These cytokines are well-known indicators of the inflammatory situation of the airway. Numerous cytokines (mainly determined by immunochemical methods) have been previously reported in EBC of individuals with different lung diseases. The fact that IFN-g; IL-2 and IL-15 could be evidenced in EBC of each single patients but not in that of controls, even though they were pooled, led us to hypothesize that their level was much higher in EBC of the formers. We have reasons to believe that, in case cytokines/cytokeratins may actually serve as an indicator of lung injury, their calculated level in single patients could be correlated with clinical parameters such as lung density score.
We can assume that the identification of these biomarkers represents the first step towards the interpretation of multiple mechanisms leading to the onset and progression of lung disease.

Project funding
The researchers are supported by grants Programma Strategico del Ministero della Sanità “Miglioramento della valutazione diagnostica e prognostica della BPCO mediante nuovi indici funzionali e biologici”; alfa1-International Registry; FAR.

Relevant publications 2005-2008
Stolk J, Veldhuisen B, Annovazzi L, Zanone C, Versteeg EM, van Kuppevelt TH, Nieuwenhuizen W, Iadarola P, Berden JH, Luisetti M.
Short-term variability of biomarkers of proteinase activity in patients with emphysema associated with type Z alpha-1-antitrypsin deficiency.
Respir Res, 7:20, 2006.

Viglio S, Annovazzi L, Conti B, Genta I, Perugini P, Zanone C, Casado B, Cetta G, Iadarola P. 
The role of emerging techniques in the investigation of prolidase deficiency: from diagnosis to the development of a possible therapeutical approach.
J Chromatogr B Analyt Technol Biomed Life Sci, 832:1–8, 2006.

Boschetto P, Quintavalle S, Zeni E, Leprotti S, Potena A, Ballerin L, Papi A, Palladini G, Luisetti M, Annovazzi L, Iadarola P, De Rosa E, Fabbri LM, Mapp CE.
Association between markers of emphysema and more severe chronic obstructive pulmonary disease.
Thorax, 61:1037–1042, 2006.

Di Poto C, Iadarola P, Salvini R, Passadore I, Cereda C, Ceroni M, Bardoni AM.
Optimizing separation efficiency of 2-DE procedures for visualization of different superoxide dismutase forms in a cellular model of amyotrophic lateral sclerosis.
Electrophoresis, 28:4340–4347, 2007.

Di Poto C, Iadarola P, Bardoni AM, Passadore I, Giorgetti S, Cereda C, Carrì MT, Ceroni M, Salvini R.
2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis.
Electrophoresis, 28:4320–4329, 2007.

Casado B, Iadarola P, Pannell LK, Luisetti M, Corsico A, Ansaldo E, Ferrarotti I, Boschetto P, Baraniuk JN.
Protein Expression in Sputum of Smokers and Chronic Obstructive Pulmonary Disease Patients: A Pilot Study by CapLC-ESI-Q-TOF.
J Proteome Res, 6:4615–4623, 2007.

Viglio S, Annovazzi L, Luisetti M, Stolk J, Casado B, Iadarola P.
Progress in the methodological strategies for the detection in real samples of desmosine and isodesmosine, two biological markers of elastin degradation.
J Sep Sci, 30:202–213, 2007.

Iadarola P, Ferrari F, Fumagalli M, Viglio S.
Determination of amino acids by micellar EKC: Recent advances in method development and novel applications to different matrices.
Electrophoresis, 29:224–236, 2008.

Luisetti M, Ma S, Iadarola P, Stone PJ, Viglio S, Casado B, Lin YY, Snider GL, Turino GM.
Desmosine as a biomarker of elastin degradation in COPD: current status and future directions.
Eur Respir J, 32:1146-1157, 2008.

Casado B, Iadarola P, Luisetti M, Kussmann M.
Proteomics-based diagnosis of chronic obstructive pulmonary disease: the hunt for new markers.
Expert Rev Proteomics, 5:693-704, 2008.

Fumagalli M, Dolcini L, Sala A, Stolk J, Fregonese L, Ferrari F, Viglio S, Luisetti M, Iadarola P.
Proteomic analysis of exhaled breath condensate from single patients with pulmonary emphysema associated to alpha1-antitrypsin deficiency.
J Proteomics, 71:211-221, 2008.

Casado B, Iadarola P, Pannell LK.
Preparation of nasal secretions for proteome analysis.
Methods Mol Biol. 2008; 425: 77-87.

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