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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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Related in: MedlinePlus

Seroreactivities of melanoma patients and healthy controls against antigens of the melanoma cell line M-NRT isolated by 2DE.Total protein extracts of M-NRT cells were separated with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots shown in Panels A through G were probed successively with 8 different patient sera each, blot H was probed with the sera of 9 healthy controls. The arrows indicated the antigens that could be assigned to protein spots in the silver-stained gel show with Figure 3. The numbering for the antigens is used throughout this report.
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pone-0005199-g002: Seroreactivities of melanoma patients and healthy controls against antigens of the melanoma cell line M-NRT isolated by 2DE.Total protein extracts of M-NRT cells were separated with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots shown in Panels A through G were probed successively with 8 different patient sera each, blot H was probed with the sera of 9 healthy controls. The arrows indicated the antigens that could be assigned to protein spots in the silver-stained gel show with Figure 3. The numbering for the antigens is used throughout this report.

Mentions: Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.


Proteome serological determination of tumor-associated antigens in melanoma.

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

Seroreactivities of melanoma patients and healthy controls against antigens of the melanoma cell line M-NRT isolated by 2DE.Total protein extracts of M-NRT cells were separated with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots shown in Panels A through G were probed successively with 8 different patient sera each, blot H was probed with the sera of 9 healthy controls. The arrows indicated the antigens that could be assigned to protein spots in the silver-stained gel show with Figure 3. The numbering for the antigens is used throughout this report.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005199-g002: Seroreactivities of melanoma patients and healthy controls against antigens of the melanoma cell line M-NRT isolated by 2DE.Total protein extracts of M-NRT cells were separated with an isoelectric focusing pH range of 3–10 in the first and SDS-PAGE in the second dimension, blotted onto nitrocellulose membranes and probed with the sera of the patients as listed in Table 1. Serum dilutions were 1/200. The blots shown in Panels A through G were probed successively with 8 different patient sera each, blot H was probed with the sera of 9 healthy controls. The arrows indicated the antigens that could be assigned to protein spots in the silver-stained gel show with Figure 3. The numbering for the antigens is used throughout this report.
Mentions: Total protein extract of the tumor cells were separated by SDS-PAGE, blotted onto nitrocellulose and probed with the sera of the melanoma patients (Panels A–C), of healthy control donors (Panel D, sera 102–110) or patients with cutaneous lymphoma (Panel E, sera 96–98), pancreas carcinoma (Panel E, serum 101) or visceral leishmaniasis (Panel E, sera 111–113). The numbers atop of each lane represent the number of the sera and are used throughout this report. For comparison, melanoma serum 7 was included in all blots. The Western blot analyses were done with the sera at a dilution of 1/6 in a multiple channel blotting/Western blot developing chamber for panels A–E. The letters underneath the lanes indicated the combinations of sera for the multiple probing of the 2D Western blots shown in Figure 2 and summarized in Table 1. Panel F: High-resolution 1D Western blot for comparison of the seroreactivities of melanoma patients and a healthy donor against the melanoma cell line M-NRT. In contrast to the blots shown in panels A–E, the sera were applied 1/200 to isolated lanes from SDS-PAGE in sealed plastic bags which results in a better definition of the bands when compared to blots from multiple blotting devices. Serum 18 is autologous to tumor cell line M-NRT, serum 7 is from a different patient and serum 103 from a healthy donor. The arrows to the left of the lanes indicated antigen bands that are shared between the patients and the healthy controls and occur in every blot. The arrows to the right of the lanes indicated prominent antigen that are detected only by the sera of melanoma patients.

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