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Proteome serological determination of tumor-associated antigens in melanoma.

Forgber M, Trefzer U, Sterry W, Walden P - PLoS ONE (2009)

Bottom Line: One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness.Similarly, enolase has been found deregulated in different cancers.With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venerology and Allergy, Charité Universitätsmedizin Berlin, Humboldt University, Berlin, Germany.

ABSTRACT
Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2-6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

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Peptide mass fingerprint spectra of antigens identified by mass spectrometry.Panel A shows the mass spectrum of the tryptic fragments for antigen spot number 9 identified as galectin-3, panel B the respective spectrum for antigen spot 1 identified as the actin filament capping protein MCP, panel C antigen spot 4 identified as the heat shock protein HSP60 and panel D antigen spot 28 identified as the elongation factor EF-Tu. The peptide mass fingerprint spectra for the other 17 antigen spots are provided with the supplementary materials indicated in Table 2 together with the statistics of the mass-spectrometric identification of the proteins. Asterisks indicate the tryptic fragment masses matched to the database sequences of the proteins.
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pone-0005199-g004: Peptide mass fingerprint spectra of antigens identified by mass spectrometry.Panel A shows the mass spectrum of the tryptic fragments for antigen spot number 9 identified as galectin-3, panel B the respective spectrum for antigen spot 1 identified as the actin filament capping protein MCP, panel C antigen spot 4 identified as the heat shock protein HSP60 and panel D antigen spot 28 identified as the elongation factor EF-Tu. The peptide mass fingerprint spectra for the other 17 antigen spots are provided with the supplementary materials indicated in Table 2 together with the statistics of the mass-spectrometric identification of the proteins. Asterisks indicate the tryptic fragment masses matched to the database sequences of the proteins.

Mentions: As examples for the mass-spectrometric identification of the antigens, the fingerprint mass spectra for spot number 9 identified as galectin-3, spot number 1 identified as the gelsolin-like actin filament-capping protein MCP, spot number 4 identified as heat shock protein 60 (HSP 60) and spot number 28 identified as the elongation factor EF-Tu are shown in Figure 4A, B, C and D, respectively. The spectra for the other antigens are provided as supplementary materials (Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17) as indicated in Table 2 together with the gene identifier numbers, protein-chemical parameter and the results of the mass-spectrometric identification for all antigens. In the cases where masses remained that could not be assigned to the identified proteins, a secondary peptide mass fingerprint analysis was done with these remaining masses. In no case could a second protein be identified indicating that the analyzed spots did not contain major contaminating proteins. The identified antigens can be grouped according to their cell biological functions into different categories. Three of the antigens are heat shock proteins (HSP60, HSP70 and HSP70 protein 9B), 7 are enzymes of the cellular metabolism (enolase I, dienoyl-CoA reductase, aldolase A, fumarate hydratase, aldose reductase, aconitase and lactate dehydrogenase), hnRNP-1 is a nuclear protein involved in RNA processing, EF-Tu an elongation factor in protein biosynthesis, MCP affiliated with the cytoskeleton and involved in cell migration, calumenin a calcium-binding protein involved in regulation of metabolic processes, VCP participates in the regulation of protein export and the organization of the Golgi apparatus, LAP3 and PSME1 are involved in protein metabolism, and galectin-3 is a lectin with specificity for galactose. The sequence coverage of the mass-spectrometric fingerprint analysis of HnRNP is somewhat low. However, HnRNP had been repeatedly identified from different gels with better sequence coverage so that we are sure of its correct identification. For all other antigens the sequence coverage is sufficiently high for unequivocal identification of the corresponding proteins. Galectin is extracellularly expressed and involved in various interactions with serum proteins, other cells and extracellular matrix, and variously implicated in cancer-related processes such as metastasis formation and invasiveness. Enolase I, although a cytosolic protein, has been reported to be exported and is found in the extracellular environment of some tumors. Of the identified antigens, galectin-3 and HSP60 most often induce antibody responses in melanoma patients followed by calumenin and enolase I. For HSP60 and calumenin seroreactivity was also detected in healthy donors tested.


Proteome serological determination of tumor-associated antigens in melanoma.

Forgber M, Trefzer U, Sterry W, Walden P - PLoS ONE (2009)

Peptide mass fingerprint spectra of antigens identified by mass spectrometry.Panel A shows the mass spectrum of the tryptic fragments for antigen spot number 9 identified as galectin-3, panel B the respective spectrum for antigen spot 1 identified as the actin filament capping protein MCP, panel C antigen spot 4 identified as the heat shock protein HSP60 and panel D antigen spot 28 identified as the elongation factor EF-Tu. The peptide mass fingerprint spectra for the other 17 antigen spots are provided with the supplementary materials indicated in Table 2 together with the statistics of the mass-spectrometric identification of the proteins. Asterisks indicate the tryptic fragment masses matched to the database sequences of the proteins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667248&req=5

pone-0005199-g004: Peptide mass fingerprint spectra of antigens identified by mass spectrometry.Panel A shows the mass spectrum of the tryptic fragments for antigen spot number 9 identified as galectin-3, panel B the respective spectrum for antigen spot 1 identified as the actin filament capping protein MCP, panel C antigen spot 4 identified as the heat shock protein HSP60 and panel D antigen spot 28 identified as the elongation factor EF-Tu. The peptide mass fingerprint spectra for the other 17 antigen spots are provided with the supplementary materials indicated in Table 2 together with the statistics of the mass-spectrometric identification of the proteins. Asterisks indicate the tryptic fragment masses matched to the database sequences of the proteins.
Mentions: As examples for the mass-spectrometric identification of the antigens, the fingerprint mass spectra for spot number 9 identified as galectin-3, spot number 1 identified as the gelsolin-like actin filament-capping protein MCP, spot number 4 identified as heat shock protein 60 (HSP 60) and spot number 28 identified as the elongation factor EF-Tu are shown in Figure 4A, B, C and D, respectively. The spectra for the other antigens are provided as supplementary materials (Figures S1, S2, S3, S4, S5, S6, S7, S8, S9, S10, S11, S12, S13, S14, S15, S16, S17) as indicated in Table 2 together with the gene identifier numbers, protein-chemical parameter and the results of the mass-spectrometric identification for all antigens. In the cases where masses remained that could not be assigned to the identified proteins, a secondary peptide mass fingerprint analysis was done with these remaining masses. In no case could a second protein be identified indicating that the analyzed spots did not contain major contaminating proteins. The identified antigens can be grouped according to their cell biological functions into different categories. Three of the antigens are heat shock proteins (HSP60, HSP70 and HSP70 protein 9B), 7 are enzymes of the cellular metabolism (enolase I, dienoyl-CoA reductase, aldolase A, fumarate hydratase, aldose reductase, aconitase and lactate dehydrogenase), hnRNP-1 is a nuclear protein involved in RNA processing, EF-Tu an elongation factor in protein biosynthesis, MCP affiliated with the cytoskeleton and involved in cell migration, calumenin a calcium-binding protein involved in regulation of metabolic processes, VCP participates in the regulation of protein export and the organization of the Golgi apparatus, LAP3 and PSME1 are involved in protein metabolism, and galectin-3 is a lectin with specificity for galactose. The sequence coverage of the mass-spectrometric fingerprint analysis of HnRNP is somewhat low. However, HnRNP had been repeatedly identified from different gels with better sequence coverage so that we are sure of its correct identification. For all other antigens the sequence coverage is sufficiently high for unequivocal identification of the corresponding proteins. Galectin is extracellularly expressed and involved in various interactions with serum proteins, other cells and extracellular matrix, and variously implicated in cancer-related processes such as metastasis formation and invasiveness. Enolase I, although a cytosolic protein, has been reported to be exported and is found in the extracellular environment of some tumors. Of the identified antigens, galectin-3 and HSP60 most often induce antibody responses in melanoma patients followed by calumenin and enolase I. For HSP60 and calumenin seroreactivity was also detected in healthy donors tested.

Bottom Line: One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness.Similarly, enolase has been found deregulated in different cancers.With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venerology and Allergy, Charité Universitätsmedizin Berlin, Humboldt University, Berlin, Germany.

ABSTRACT
Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2-6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins.

Show MeSH
Related in: MedlinePlus