- Email: [email protected]
Proteomics in BAL: improved technique may yield more fish
Paola Rottoli1
Proteomics in BAL: improved technique may yield more fish
Abstract There has been growing interest in proteomics in the last ten years. Its fields of application are still emerging and include application to a wide spectrum of lung diseases. In this context the proteomic approach has been directed especially to the study of broncholveolar lavage (BAL) protein composition, with the aim of obtaining insights into the pathogenetic mechanisms of lung diseases and of identifying possible disease-specific biomarkers. Proteomic analysis is complex and produces a great mass of data, even more than genomics.Many techniques are available today for
Proteomics provides much information on gene products and post-translational modifications, it is therefore complementary to genomics and enables protein composition in body fluids, cells and tissues to be investigated under different conditions1,2. Proteomic analysis is complex and produces a great mass of data, even more than genomics. It is really like an ocean with many different fishes, or proteins, to catch! There has been growing interest in proteomics in the last ten years following the genomic era1-4. Its fields of application are still
1
a proteomic approach and they can be applied to BAL. Each has advantages and disadvantages. Critical points to be considered before planning research in this field are: appropriate experimental design, reproducible methods and suitable statistical analysis. This means a multidisciplinary approach, exploiting molecular biology, clinical and bioinformatics expertise, is needed. Here we report a summary of recent literature on proteomics in BAL and of our experience in this field. Key-words : Bronchoalveolar lavage (BAL), proteomics, lung disease
emerging and include the study of a wide spectrum of lung diseases : interstitial lung diseases, asthma, occupational lung diseases, acute lung injury, lung cancer, cystic fibrosis, chronic lung rejection, etcs.2,3,5-8. In this context the proteomic approach has been directed especially to the study of broncholveolar lavage (BAL) protein composition, with the aim of obtaining insights into the pathogenetic mechanisms and of identifying possible disease-specific biomarkers9-15. Many techniques are available today for a proteomic approach and some of them have
Respiratory Diseases Section, Dept. of Clinical Medicine and Immunology, Siena University – Siena – Italy. ([email protected])
R E V I S T A
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 41
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
ben applied to BAL analysis. Each has advantages and disadvantages1,2,4,5,14-16. Their complexity and rapid expansion call for great care and competence, because production of useless data is to be avoided and subsequent confirmation and validation of results must be done. The need to develop guidelines to ensure high quality data required by this type of approach has been recently pointed out17.Critical points to be considered before planning research in this field are: appropriate experimental design, reproducible methods and suitable statistical analysis. This means a multidisciplinary approach, exploiting molecular biology, clinical and bioinformatics expertise, is needed. Experimental protocol must include not only appropriate techniques but also provide the number of samples needed to obtain meaningful data. The distribution of results (normal or skewed) must be carefully determined, and if necessary transformed, before statistical analysis is applied. This aspect is crucial for differential display of proteins and for identifying biomarkers, because correct statistical analysis makes it possible to determine whether differences in protein expression in groups of diseases are significant or whether they are only an effect of variability of the sample5,18-19. The state of the art of BAL proteomics has been the topic of some recent reviews underlining the growing interest in this field1,15,16,19. We also recently wrote a review on the application of proteomics to BAL analysis considering all the methodological aspects and the studies performed in various lung diseases20. Most of the data available so far regards protein composition in BAL fluid, the cellular component has been less investigated1,20. S 42
R E V I S T A
P O R T U G U E S A
BAL fluid has some limitations, that should be considered, for proteomic analysis, they are: high salt concentrations, dilution and low protein concentration, abundance of plasma high Mw proteins, but they can be at least partially corrected by appropriate expedients according to the experimental design and the techniques used (dyalisis, concentration of the sample, removal of high Mw proteins,etc).Therefore sample preparation can introduce significant analytical variability and the study completeness also depends on correct sample processing2,5,15,17,20-22. The groups of Lenz, Lindahl, Wattiez, Grunewald and ours were among the first to work in this field and to give a relevant contribute to the identification of BAL proteins with the aims to realise a database and/ or to characterize disease specific protein patterns7,9-14. Our experience with this technique began around 1996 with the collaboration with the Proteomic Laboratory of the Molecular Biology Dept. in Siena University. We studied serum and BAL from patients with sarcoidosis (S) pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF), three interstitial lung diseases (ILD) characterized by different pathogenesis and evolution9,13,23. Proteins were separated by high resolution 2D-electrophoresis (2D-E) and then identified by gel matching, immunoblotting and especially mass spectrometry ( MALDI TOF MS)13. 2D- electrophoresis application to BAL provides much information on protein composition and allows simultaneous display of proteins present in alveolar spaces, but to obtain suitable data a particular attention must be given to appropriate sample processing and loading and to the correct staining of the gel. In two-dimensional electrophoreD E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
sis (2-DE), polypeptides are first separated according to their isoelectric point and then by their molecular weight. After staining, the 2D-E gels from different samples can be compared (differential display proteomics), for identifying disease specific protein patterns20,21. Therefore we assessed carefully the method to obtain reproducible and comparable results13,24,25 (Fig. 1). According to our experience the most interesting findings were in BAL, where we found huge amount of plasma and locally produced proteins (800-1000 spots /gel). 85 proteins were identified, 38 for the first time in BAL. They had different origins (plasma derived, locally secreted, or products of cell damage and proteolytic activity) and functions (e.g. antiprotease, anti- or proinflammatory, anti-
Fig. 1 – Silver stained 2D-Electrophoresis gel from BAL of an patient with IPF. 45 ìg of BAL sample were loaded . The BALF sample was dialysed against water, lyophilised and dissolved in lysis buffer so that 100 ml contained 45 mg proteins. Two-dimensional gel electrophoresis (2D-E) was performed as described by us (13,24).Electrophoretogram images were obtained with a computing densitometer (Molecular Dynamics 300S) and processed with the Melanie IV computer system . Quantitative variations in proteins were expressed as relative volumes of spots (% volume).
R E V I S T A
oxidant, cell growth or coagulation related or unknown ). The percentage volumes of the identified spots were calculated and compared among sarcoidosis, SSc and IPF, then groups of proteins expressed in significantly different amount were found13,24. Therefore the three diseases were characterized by different protein profile in line with their different pathogenesis. An increase in plasma proteins was expecially found in S compared to IPF, while in IPF a greater abundance of a group of low molecular weight proteins was present compared to S. SSc showed a profile with characteristics intermediate between the two other diseases, there were up and down regulated proteins in different proportions. We have also compared BAL protein percentage volumes in the three ILD with those of a control group and significant differences were found in some protein expression (paper in preparation ). We identified groups of proteins of interest that we are now investigating by easier and faster methods, such as ELISA, in a greater number of cases, for possible biomarkers of disease. They are low molecular weight proteins with various, not completely known, functions, potentially involved in pulmonary fibrosis pathogenetic mechanisms, especially in fibroblast proliferation and activation, tissue remodeling, oxidant-antioxidant and protease-antiprotease systems13,24. MIF is one of these proteins and ELISA confirmed that it is significantly increased in IPF compared to controls (paper ready to be submitted). The role of the imbalance between protease/ antiprotease and oxidant/antioxidant in tissue damage is well known, so we also investigated oxidative stress by a proteomic approach. The identification of protein targets of oxidation by proteomic analysis (2-DE
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 43
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
combined with immunoblotting with specific antibodies for carbonyl groups) suggested that carbonylation involved a limited number of carbonylation-sensitive proteins (mainly albumin, AAT, IgG heavy chains and complement components) in the three diseases and controls, whereas a greater number of oxidized proteins with various functions was found only in IPF26. Therefore proteomics allows to identify in BAL proteins that underwent structural changes locally, loosing their original function and in some circumstances acquiring a new one with a possible role in the pathogenesis of the disease. This could happen for oxidized, fragmented or polymerized proteins that we have found in BAL in ILD13,23,26. Sarcoidosis BAL has been also studied by other techniques such as narrow range pH gradients that allow to better analyze regions of interest14,27, or by SELDI TOF MS that seems to facilitate the discovery of disease related protein profiles28 Surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy enabled determination of protein patterns in sarcoidosis BAL fluid and allowed detection of protein patterns linked to a particular disease course28. Proteomic analysis of BAL has been also applied to BAL of patients with obstructive lung diseases such as COPD and asthma15,22,29,30. It was performed in allergic asthmatic patients after segmental allergen challenge using affinity depletion of six abundant BAL proteins, SDS-PAGE separation, HPLC combined with mass spectrometry for protein identification and semiquantitation (SAC).By these methods a huge number of proteins was identified, some of which were expressed differently in challenged asthmatic patients versus controls29. This wide dataS 44
R E V I S T A
P O R T U G U E S A
base of BAL proteins may be useful to identify new diagnostic markers of asthma and to complete the BAL protein composition. A proteomic approach has been applied to the study of fibroblasts in BAL from patients with mild asthma compared to control subjects Differential expression patterns between the fibroblasts in the two groups in motility-regulating proteins were identified by two-dimensional electrophoresis and mass spectrometry30. Some researchers from this group have developed and validated novel technology applications that can be used to characterize the patterns of global protein expression in tissue and biofluids in either gel-based systems or by automated multidimensional nanocapillary liquid chromatography22. Moreover very recently applying a technology toolbox consisting of replicate 2-dimensional gel separations, image annotation, and mass spectrometry identification, they found protein expression patterns associated with progression of COPD in BAL of smokers31. In conclusion, BAL proteomics has passed fom the pioneering stage to a high level of optimization in sample preparation and in the use of new techniques to separate and identify proteins. They include advanced MS and gel electrophoresis, bioinformatics, automation and miniaturization, that permit high sample throughput analysis with improved sensitivity and reproducibility. Therefore a proteomic approach applied to the study of BAL can help to characterize the complex alveolar microenvironment, important for a better understanding of pathogenetic mechanisms, and to identify groups of disease-related proteins, useful for the future identification of diagnostic and prognostic markers, or for new advances in therapy. D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
References 1. Bini L, Pallini V, Hochstrasser DF, Sanchez JC.-Proteomics 2007 May;7(10):1561-1563. 2. Hirsch J, Hansen KC, Burlingame AL, Matthay MA. – Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L1-23 3. Noel-Georis, I., Bernard, A., Falmagne, P., Wattiez, R.J.- Chromatogr B Analyt Technol Biomed Life Sci 2002, 771, 221-236 4. Marko-Varga G., Lindberg H., Lofdahl C.-G., Jonsson P. et Al.-J. Proteome Res. 2005, 4, 1200-1212 5. Bowler RP, Ellison MC, Reisdorph N.Proteomics in pulmonary medicine-Chest 2006 Aug;130(2):567-74. 6. Penque D.- Expert Rev Proteomics 2007; 2:199-209 7. Wattiez, R., Hermans, C., Cruyt, C., Bernard, A., Falmagne, P. Electrophoresis 2000;21: 2703-2712. 8. Rottoli, P, Bargagli, E., Curr Opin Pulm Med 2003; 9:418-425 9. Magi B, Perari MG, Bini L, Fossi A, Pallini V, Rottoli P.- Medicina Toracica. 2001; 23 :113-117. 10. Lenz, A.G., Meyer, B., Costabel, U., Maier, K.- Electrophoresis 1993, 14, 242-244. 11. Lindahl, M., Ekstrom T, Sorensen J, Tagesson C.Thorax 1996, 51, 1028-1035 12. Lindahl, M., Stahlbom, Tagesson, C.- Electrophoresis 1999, 20, 881-890 13. Magi, B., Bini, L., Perari, M.G., Fossi, Rottoli P,et al.Electrophoresis 2002; 23:3434-3444. 14. Sabounchi-Schutt, F, Astrom, J., Hellman, U., Eklund, A., Grunewald, J.- Eur Respir J 2003, 21, 414-420 15. Crameri R.- Allergy 2005;60: 1227-1237 16. Wattiez, R., Falmagne, P. J.- Chromatogr. 2005; 815,:169-178
R E V I S T A
17. Wilkins, M.R., Appel, R.D., Van Eyk, J.E., Chung, M.C.M., et al. -Proteomics 2006; 6: 4-8 18. Hunt SM, Thomas MR, Sebastian LT, Pedersen SK, Harcourt RL, Sloane AJ, Wilkins MR.-J Proteome Res 2005 May-Jun;4(3):809-819 19. Houtman, R., van den Worm, E. J. Chromatogr. B 2005, 815: 285-294 20. Magi B, Bargagli E, Bini L, Rottoli P. Proteomics 2006,6(23):6354-69 21. Righetti PG, Campostrini, N., Pascali, J., Hamdan, M., Astner, H. Eur. J. Mass. Spectrom. )2004; 10: 335-348. 22. Plymoth A, Lofdahl CG, Ekberg-Jansson A, Dahlback M et al.- Proteomics. 2003; 3: 962-972 23. Magi B, Rottoli P,Bini L, Perari MG, Pallini V, Vagliasindi M.- Sarcoidosis 1997 Sept; 14 (1s):19 24. Rottoli P, Magi B, Perari MG, Liberatori S, et al.Proteomics 2005;5:1423-1430 25. Bjellquist,B, Pasquali, C, Ravier, F, Sanchez JC, Hochstrasser DF.-Electrophoresis 1993,14:1357-1365 26. Rottoli P, Magi B, Cianti, R., Bargagli, E. et al. Proteomics 2005,5 : 2612-2618 27. Sabounchi-Schutt F, Mikko M, Eklund A., Grunewald J, Astrom, J- Sarcoidosis Vasc Diffuse Lung Dis 2004;21: 187-190 28. Kriegova, E., Melle, C., Kolek, V., Hutyrova, B. et al.Am J Respir Crit Care Med. 2006 May 15;173(10):1145-54. 29. Wu, J., Kobayashi, M., Sousa, E.A., Lieu, W. et al.Mol. Cell. Proteomics 2005 ; 4 : 1251-126 30. Larsen K, Tufvesson E, Malmström J, Mörgelin M et al.Am J Respir Crit Care Med.2004 Nov15;170(10):1049-56. 31. Plymoth A, Löfdahl CG, Ekberg-Jansson A, Dahlbäck M, et al. – Clin Chem. 2007, Apr;53(4):636-44
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 45
Proteomics in BAL: improved technique may yield more fish
Abstract There has been growing interest in proteomics in the last ten years. Its fields of application are still emerging and include application to a wide spectrum of lung diseases. In this context the proteomic approach has been directed especially to the study of broncholveolar lavage (BAL) protein composition, with the aim of obtaining insights into the pathogenetic mechanisms of lung diseases and of identifying possible disease-specific biomarkers. Proteomic analysis is complex and produces a great mass of data, even more than genomics.Many techniques are available today for
Proteomics provides much information on gene products and post-translational modifications, it is therefore complementary to genomics and enables protein composition in body fluids, cells and tissues to be investigated under different conditions1,2. Proteomic analysis is complex and produces a great mass of data, even more than genomics. It is really like an ocean with many different fishes, or proteins, to catch! There has been growing interest in proteomics in the last ten years following the genomic era1-4. Its fields of application are still
1
a proteomic approach and they can be applied to BAL. Each has advantages and disadvantages. Critical points to be considered before planning research in this field are: appropriate experimental design, reproducible methods and suitable statistical analysis. This means a multidisciplinary approach, exploiting molecular biology, clinical and bioinformatics expertise, is needed. Here we report a summary of recent literature on proteomics in BAL and of our experience in this field. Key-words : Bronchoalveolar lavage (BAL), proteomics, lung disease
emerging and include the study of a wide spectrum of lung diseases : interstitial lung diseases, asthma, occupational lung diseases, acute lung injury, lung cancer, cystic fibrosis, chronic lung rejection, etcs.2,3,5-8. In this context the proteomic approach has been directed especially to the study of broncholveolar lavage (BAL) protein composition, with the aim of obtaining insights into the pathogenetic mechanisms and of identifying possible disease-specific biomarkers9-15. Many techniques are available today for a proteomic approach and some of them have
Respiratory Diseases Section, Dept. of Clinical Medicine and Immunology, Siena University – Siena – Italy. ([email protected])
R E V I S T A
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 41
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
ben applied to BAL analysis. Each has advantages and disadvantages1,2,4,5,14-16. Their complexity and rapid expansion call for great care and competence, because production of useless data is to be avoided and subsequent confirmation and validation of results must be done. The need to develop guidelines to ensure high quality data required by this type of approach has been recently pointed out17.Critical points to be considered before planning research in this field are: appropriate experimental design, reproducible methods and suitable statistical analysis. This means a multidisciplinary approach, exploiting molecular biology, clinical and bioinformatics expertise, is needed. Experimental protocol must include not only appropriate techniques but also provide the number of samples needed to obtain meaningful data. The distribution of results (normal or skewed) must be carefully determined, and if necessary transformed, before statistical analysis is applied. This aspect is crucial for differential display of proteins and for identifying biomarkers, because correct statistical analysis makes it possible to determine whether differences in protein expression in groups of diseases are significant or whether they are only an effect of variability of the sample5,18-19. The state of the art of BAL proteomics has been the topic of some recent reviews underlining the growing interest in this field1,15,16,19. We also recently wrote a review on the application of proteomics to BAL analysis considering all the methodological aspects and the studies performed in various lung diseases20. Most of the data available so far regards protein composition in BAL fluid, the cellular component has been less investigated1,20. S 42
R E V I S T A
P O R T U G U E S A
BAL fluid has some limitations, that should be considered, for proteomic analysis, they are: high salt concentrations, dilution and low protein concentration, abundance of plasma high Mw proteins, but they can be at least partially corrected by appropriate expedients according to the experimental design and the techniques used (dyalisis, concentration of the sample, removal of high Mw proteins,etc).Therefore sample preparation can introduce significant analytical variability and the study completeness also depends on correct sample processing2,5,15,17,20-22. The groups of Lenz, Lindahl, Wattiez, Grunewald and ours were among the first to work in this field and to give a relevant contribute to the identification of BAL proteins with the aims to realise a database and/ or to characterize disease specific protein patterns7,9-14. Our experience with this technique began around 1996 with the collaboration with the Proteomic Laboratory of the Molecular Biology Dept. in Siena University. We studied serum and BAL from patients with sarcoidosis (S) pulmonary fibrosis associated with systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF), three interstitial lung diseases (ILD) characterized by different pathogenesis and evolution9,13,23. Proteins were separated by high resolution 2D-electrophoresis (2D-E) and then identified by gel matching, immunoblotting and especially mass spectrometry ( MALDI TOF MS)13. 2D- electrophoresis application to BAL provides much information on protein composition and allows simultaneous display of proteins present in alveolar spaces, but to obtain suitable data a particular attention must be given to appropriate sample processing and loading and to the correct staining of the gel. In two-dimensional electrophoreD E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
sis (2-DE), polypeptides are first separated according to their isoelectric point and then by their molecular weight. After staining, the 2D-E gels from different samples can be compared (differential display proteomics), for identifying disease specific protein patterns20,21. Therefore we assessed carefully the method to obtain reproducible and comparable results13,24,25 (Fig. 1). According to our experience the most interesting findings were in BAL, where we found huge amount of plasma and locally produced proteins (800-1000 spots /gel). 85 proteins were identified, 38 for the first time in BAL. They had different origins (plasma derived, locally secreted, or products of cell damage and proteolytic activity) and functions (e.g. antiprotease, anti- or proinflammatory, anti-
Fig. 1 – Silver stained 2D-Electrophoresis gel from BAL of an patient with IPF. 45 ìg of BAL sample were loaded . The BALF sample was dialysed against water, lyophilised and dissolved in lysis buffer so that 100 ml contained 45 mg proteins. Two-dimensional gel electrophoresis (2D-E) was performed as described by us (13,24).Electrophoretogram images were obtained with a computing densitometer (Molecular Dynamics 300S) and processed with the Melanie IV computer system . Quantitative variations in proteins were expressed as relative volumes of spots (% volume).
R E V I S T A
oxidant, cell growth or coagulation related or unknown ). The percentage volumes of the identified spots were calculated and compared among sarcoidosis, SSc and IPF, then groups of proteins expressed in significantly different amount were found13,24. Therefore the three diseases were characterized by different protein profile in line with their different pathogenesis. An increase in plasma proteins was expecially found in S compared to IPF, while in IPF a greater abundance of a group of low molecular weight proteins was present compared to S. SSc showed a profile with characteristics intermediate between the two other diseases, there were up and down regulated proteins in different proportions. We have also compared BAL protein percentage volumes in the three ILD with those of a control group and significant differences were found in some protein expression (paper in preparation ). We identified groups of proteins of interest that we are now investigating by easier and faster methods, such as ELISA, in a greater number of cases, for possible biomarkers of disease. They are low molecular weight proteins with various, not completely known, functions, potentially involved in pulmonary fibrosis pathogenetic mechanisms, especially in fibroblast proliferation and activation, tissue remodeling, oxidant-antioxidant and protease-antiprotease systems13,24. MIF is one of these proteins and ELISA confirmed that it is significantly increased in IPF compared to controls (paper ready to be submitted). The role of the imbalance between protease/ antiprotease and oxidant/antioxidant in tissue damage is well known, so we also investigated oxidative stress by a proteomic approach. The identification of protein targets of oxidation by proteomic analysis (2-DE
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 43
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
combined with immunoblotting with specific antibodies for carbonyl groups) suggested that carbonylation involved a limited number of carbonylation-sensitive proteins (mainly albumin, AAT, IgG heavy chains and complement components) in the three diseases and controls, whereas a greater number of oxidized proteins with various functions was found only in IPF26. Therefore proteomics allows to identify in BAL proteins that underwent structural changes locally, loosing their original function and in some circumstances acquiring a new one with a possible role in the pathogenesis of the disease. This could happen for oxidized, fragmented or polymerized proteins that we have found in BAL in ILD13,23,26. Sarcoidosis BAL has been also studied by other techniques such as narrow range pH gradients that allow to better analyze regions of interest14,27, or by SELDI TOF MS that seems to facilitate the discovery of disease related protein profiles28 Surface-enhanced laser desorption/ionization-time-of-flight mass spectroscopy enabled determination of protein patterns in sarcoidosis BAL fluid and allowed detection of protein patterns linked to a particular disease course28. Proteomic analysis of BAL has been also applied to BAL of patients with obstructive lung diseases such as COPD and asthma15,22,29,30. It was performed in allergic asthmatic patients after segmental allergen challenge using affinity depletion of six abundant BAL proteins, SDS-PAGE separation, HPLC combined with mass spectrometry for protein identification and semiquantitation (SAC).By these methods a huge number of proteins was identified, some of which were expressed differently in challenged asthmatic patients versus controls29. This wide dataS 44
R E V I S T A
P O R T U G U E S A
base of BAL proteins may be useful to identify new diagnostic markers of asthma and to complete the BAL protein composition. A proteomic approach has been applied to the study of fibroblasts in BAL from patients with mild asthma compared to control subjects Differential expression patterns between the fibroblasts in the two groups in motility-regulating proteins were identified by two-dimensional electrophoresis and mass spectrometry30. Some researchers from this group have developed and validated novel technology applications that can be used to characterize the patterns of global protein expression in tissue and biofluids in either gel-based systems or by automated multidimensional nanocapillary liquid chromatography22. Moreover very recently applying a technology toolbox consisting of replicate 2-dimensional gel separations, image annotation, and mass spectrometry identification, they found protein expression patterns associated with progression of COPD in BAL of smokers31. In conclusion, BAL proteomics has passed fom the pioneering stage to a high level of optimization in sample preparation and in the use of new techniques to separate and identify proteins. They include advanced MS and gel electrophoresis, bioinformatics, automation and miniaturization, that permit high sample throughput analysis with improved sensitivity and reproducibility. Therefore a proteomic approach applied to the study of BAL can help to characterize the complex alveolar microenvironment, important for a better understanding of pathogenetic mechanisms, and to identify groups of disease-related proteins, useful for the future identification of diagnostic and prognostic markers, or for new advances in therapy. D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
Proteomics in BAL: improved technique may yield more fish Paola Rottoli
References 1. Bini L, Pallini V, Hochstrasser DF, Sanchez JC.-Proteomics 2007 May;7(10):1561-1563. 2. Hirsch J, Hansen KC, Burlingame AL, Matthay MA. – Am J Physiol Lung Cell Mol Physiol. 2004 Jul;287(1):L1-23 3. Noel-Georis, I., Bernard, A., Falmagne, P., Wattiez, R.J.- Chromatogr B Analyt Technol Biomed Life Sci 2002, 771, 221-236 4. Marko-Varga G., Lindberg H., Lofdahl C.-G., Jonsson P. et Al.-J. Proteome Res. 2005, 4, 1200-1212 5. Bowler RP, Ellison MC, Reisdorph N.Proteomics in pulmonary medicine-Chest 2006 Aug;130(2):567-74. 6. Penque D.- Expert Rev Proteomics 2007; 2:199-209 7. Wattiez, R., Hermans, C., Cruyt, C., Bernard, A., Falmagne, P. Electrophoresis 2000;21: 2703-2712. 8. Rottoli, P, Bargagli, E., Curr Opin Pulm Med 2003; 9:418-425 9. Magi B, Perari MG, Bini L, Fossi A, Pallini V, Rottoli P.- Medicina Toracica. 2001; 23 :113-117. 10. Lenz, A.G., Meyer, B., Costabel, U., Maier, K.- Electrophoresis 1993, 14, 242-244. 11. Lindahl, M., Ekstrom T, Sorensen J, Tagesson C.Thorax 1996, 51, 1028-1035 12. Lindahl, M., Stahlbom, Tagesson, C.- Electrophoresis 1999, 20, 881-890 13. Magi, B., Bini, L., Perari, M.G., Fossi, Rottoli P,et al.Electrophoresis 2002; 23:3434-3444. 14. Sabounchi-Schutt, F, Astrom, J., Hellman, U., Eklund, A., Grunewald, J.- Eur Respir J 2003, 21, 414-420 15. Crameri R.- Allergy 2005;60: 1227-1237 16. Wattiez, R., Falmagne, P. J.- Chromatogr. 2005; 815,:169-178
R E V I S T A
17. Wilkins, M.R., Appel, R.D., Van Eyk, J.E., Chung, M.C.M., et al. -Proteomics 2006; 6: 4-8 18. Hunt SM, Thomas MR, Sebastian LT, Pedersen SK, Harcourt RL, Sloane AJ, Wilkins MR.-J Proteome Res 2005 May-Jun;4(3):809-819 19. Houtman, R., van den Worm, E. J. Chromatogr. B 2005, 815: 285-294 20. Magi B, Bargagli E, Bini L, Rottoli P. Proteomics 2006,6(23):6354-69 21. Righetti PG, Campostrini, N., Pascali, J., Hamdan, M., Astner, H. Eur. J. Mass. Spectrom. )2004; 10: 335-348. 22. Plymoth A, Lofdahl CG, Ekberg-Jansson A, Dahlback M et al.- Proteomics. 2003; 3: 962-972 23. Magi B, Rottoli P,Bini L, Perari MG, Pallini V, Vagliasindi M.- Sarcoidosis 1997 Sept; 14 (1s):19 24. Rottoli P, Magi B, Perari MG, Liberatori S, et al.Proteomics 2005;5:1423-1430 25. Bjellquist,B, Pasquali, C, Ravier, F, Sanchez JC, Hochstrasser DF.-Electrophoresis 1993,14:1357-1365 26. Rottoli P, Magi B, Cianti, R., Bargagli, E. et al. Proteomics 2005,5 : 2612-2618 27. Sabounchi-Schutt F, Mikko M, Eklund A., Grunewald J, Astrom, J- Sarcoidosis Vasc Diffuse Lung Dis 2004;21: 187-190 28. Kriegova, E., Melle, C., Kolek, V., Hutyrova, B. et al.Am J Respir Crit Care Med. 2006 May 15;173(10):1145-54. 29. Wu, J., Kobayashi, M., Sousa, E.A., Lieu, W. et al.Mol. Cell. Proteomics 2005 ; 4 : 1251-126 30. Larsen K, Tufvesson E, Malmström J, Mörgelin M et al.Am J Respir Crit Care Med.2004 Nov15;170(10):1049-56. 31. Plymoth A, Löfdahl CG, Ekberg-Jansson A, Dahlbäck M, et al. – Clin Chem. 2007, Apr;53(4):636-44
P O R T U G U E S A
D E
P N E U M O L O G I A
Vol XIII Suplemento 2 Outubro 2007
S 45