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Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

Teranishi Y, Hur JY, Welander H, Frånberg J, Aoki M, Winblad B, Frykman S, Tjernberg LO - J. Cell. Mol. Med. (2010)

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.

View Article: PubMed Central - PubMed

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.

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γ-secretase components were identified from LC-MS/MS analysis. Captured sample from rat brain in the absence of L-685,458 was analysed by LC-MS/MS. (A) Total ion chromatogram acquired from mass-to-charge ratio (m/z) 200 to m/z 1800. (B) Extracted ion chromatogram for m/z 1016.5 corresponding to the tryptic peptide AAVQELSGSILTSEDPEER from PS1 (red line), and for m/z 762.4 corresponding to the tryptic peptide LVQSQALPPSSLQR from nicastrin (blue line). (C) MS/MS spectrum of LVQSQALPPSSLQR and AAVQELSGSILTSEDPEER. The series of y-ions correspond to peptide fragments with an intact C-terminus (i.e. y7 = PPSSLGR), while the b-ions correspond to peptide fragments with an intact N-terminus (i.e. b7 = LVQSQAL).
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fig03: γ-secretase components were identified from LC-MS/MS analysis. Captured sample from rat brain in the absence of L-685,458 was analysed by LC-MS/MS. (A) Total ion chromatogram acquired from mass-to-charge ratio (m/z) 200 to m/z 1800. (B) Extracted ion chromatogram for m/z 1016.5 corresponding to the tryptic peptide AAVQELSGSILTSEDPEER from PS1 (red line), and for m/z 762.4 corresponding to the tryptic peptide LVQSQALPPSSLQR from nicastrin (blue line). (C) MS/MS spectrum of LVQSQALPPSSLQR and AAVQELSGSILTSEDPEER. The series of y-ions correspond to peptide fragments with an intact C-terminus (i.e. y7 = PPSSLGR), while the b-ions correspond to peptide fragments with an intact N-terminus (i.e. b7 = LVQSQAL).

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.


Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

Teranishi Y, Hur JY, Welander H, Frånberg J, Aoki M, Winblad B, Frykman S, Tjernberg LO - J. Cell. Mol. Med. (2010)

γ-secretase components were identified from LC-MS/MS analysis. Captured sample from rat brain in the absence of L-685,458 was analysed by LC-MS/MS. (A) Total ion chromatogram acquired from mass-to-charge ratio (m/z) 200 to m/z 1800. (B) Extracted ion chromatogram for m/z 1016.5 corresponding to the tryptic peptide AAVQELSGSILTSEDPEER from PS1 (red line), and for m/z 762.4 corresponding to the tryptic peptide LVQSQALPPSSLQR from nicastrin (blue line). (C) MS/MS spectrum of LVQSQALPPSSLQR and AAVQELSGSILTSEDPEER. The series of y-ions correspond to peptide fragments with an intact C-terminus (i.e. y7 = PPSSLGR), while the b-ions correspond to peptide fragments with an intact N-terminus (i.e. b7 = LVQSQAL).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4373488&req=5

fig03: γ-secretase components were identified from LC-MS/MS analysis. Captured sample from rat brain in the absence of L-685,458 was analysed by LC-MS/MS. (A) Total ion chromatogram acquired from mass-to-charge ratio (m/z) 200 to m/z 1800. (B) Extracted ion chromatogram for m/z 1016.5 corresponding to the tryptic peptide AAVQELSGSILTSEDPEER from PS1 (red line), and for m/z 762.4 corresponding to the tryptic peptide LVQSQALPPSSLQR from nicastrin (blue line). (C) MS/MS spectrum of LVQSQALPPSSLQR and AAVQELSGSILTSEDPEER. The series of y-ions correspond to peptide fragments with an intact C-terminus (i.e. y7 = PPSSLGR), while the b-ions correspond to peptide fragments with an intact N-terminus (i.e. b7 = LVQSQAL).
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.

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.

View Article: PubMed Central - PubMed

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.

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Related in: MedlinePlus