Affinity pulldown of γ-secretase and associated proteins from human and rat brain.
Bottom Line: After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin.Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain.We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.
Affiliation: The Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden.Show MeSH
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Mentions: The samples were eluted with RapiGest (a detergent which is suitable for tryptic digestion of membrane proteins) supplemented with reducing agent, the SA beads were removed, and the samples were incubated with trypsin at 37°C overnight. RapiGest was removed according to the manufacturer’s instructions and the tryptic peptides were desalted and concentrated by using ZipTips. The peptides were extracted from the ZipTips by ACN and the samples were dried by vacuum centrifugation. The samples were diluted in water supplemented with 0.2% FA, and injected into the LC-MS/MS system. For the unbiased identification of GSAPs, we used nanoflow liquid chromatography coupled online to an electrosprayionization-ion trap mass spectrometer. The small volumes used in the LC-system and the high sensitivity of the mass spectrometer allow the identification of proteins at around one fmol. The peptides were eluted by a water/ACN gradient over 2 hrs. The masspectrometer was set to subject the five highest peaks in each MS-scan to MS/MS analysis. The cycle time was around 1 sec., and 6000 MS/MS spectra were collected in one analysis. A typical total ion chromatogram is shown in Fig. 3A.
Affiliation: The Karolinska Institutet (KI) Dainippon Sumitomo Pharma Alzheimer Center (KASPAC), KI-Alzheimer's Disease Research Center, Department of Neurobiology, Care Sciences and Society, Karolinska Institutet, Novum, Huddinge, Sweden.