In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The... Show moreIn this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC–MS/MS. From a Mascot database search in combination with “semiTrypsin” tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37°C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of “unexpected” peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a “single-step” MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Show less
In this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The... Show moreIn this study temperature-dependent instability of the cTnI subunit of the three-protein complex NIST SRM2921 was demonstrated using a mass spectrometric tryptic peptide mapping approach. The results were compared to the cTnI subunit obtained as a protein standard from Calbiochem with identical amino acid sequence. Both the three-protein complex from NIST as well as the cTnI subunit were incubated at elevated temperatures and then evaluated with respect to the primary sequence. The corresponding peptide maps were analyzed using LC–MS/MS. From a Mascot database search in combination with "semiTrypsin" tolerance it was found that two peptide backbone cleavages had occurred in subunit cTnI in NIST SRM2921 material upon incubation at 37◦C, namely between amino acids at 148/149 and 194/195. The Calbiochem standard did not show increased levels of "unexpected" peptides in tryptic peptide maps. One of the two peptide backbone cleavages could also be monitored using a "single-step" MALDI-MS approach, i.e. without the need for peptide separation. The amount of degradation appeared rather constant in replicate temperature-instability experiments. However, for accurate quantification internal labelled standards are needed. Show less
Smit, N.; Musson, R.; Romijn, F.; Rossum, H. van; Pelt, J. van 2010
Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has... Show moreCalcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450 kJ m(-2) of UVA-1 (340-400 nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, gamma-interferon, IL-4 and IL-10 that are controlled by the Ca(2+)-Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors. Show less
Smit, N.; Musson, R.; Romijn, F.; Rossum, H. van; Pelt, J. van 2010
Calcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has... Show moreCalcineurin (Cn) is the target of immunosuppressive drugs used for maintenance therapy of transplant patients. UV radiation is also known to be immunosuppressive and, like the Cn inhibitors, UV has been shown to positively influence various inflammatory skin diseases. Recently, Cn activity has been demonstrated in skin and skin cell cultures. In the present study we have investigated the effects of UV(A-1) irradiation on Cn activity in skin. In total skin we found a significant reduction in Cn activity after exposure to 450kJm−2 of UVA-1 (340–400nm). In repeated experiments cultures of fibroblasts and keratinocytes also showed dose-dependent and selective reduction in Cn activity after UVA-1 irradiation. UVB irradiation caused a decrease in the Cn activity of one of two fibroblast cultures and was ineffective in keratinocytes. In Jurkat cells and PBMC UVA-1 reduced Cn activity and also the production of cytokines such as interleukin (IL)-2, -interferon, IL-4 and IL-10 that are controlled by the Ca2+–Cn pathway. These results indicate that UV(A-1) irradiation may lead to inactivation of Cn in the skin and thus suppress the skin immune system in a similar fashion to the Cn inhibitors. Show less
