Authors: Sravani Musunuri Kim Kultima Bernhard Clemens Richard Martin Ingelsson Lars Lannfelt Jonas Bergquist Ganna Shevchenko
Publish Date: 2014/11/22
Volume: 407, Issue: 4, Pages: 1041-1057
Abstract
Integral membrane proteins MPs such as transporters receptors and ion channels are of great interest because of their participation in various vital cellular functions including cell–cell interactions ion transport and signal transduction However studies of MPs are complicated because of their hydrophobic nature heterogeneity and low abundance Cloudpoint extraction CPE with the nonionic surfactant Triton X114 was performed to simultaneously extract and phase separate hydrophobic and hydrophilic proteins from Alzheimer’s disease AD and unaffected control brain tissue Quantitative proteomics analysis of temporal neocortex samples of AD patients and controls was performed using a shotgun approach based on stable isotope dimethyl labeling DML quantification technique followed by nanoLCMS/MS analysis A total of 1096 unique proteins were identified and quantified with 403 211/524 predicted as integral MPs with at least one transmembrane domain TMD found in the detergent phase and 10 80/798 in the detergentdepleted phase Among these 62 proteins were shown to be significantly altered pvalue 005 in AD versus control samples In the detergent fraction we found 10 hydrophobic transmembrane proteins containing up to 14 putative TMDs that were significantly up or downregulated in AD compared with control brains Changes in four of these proteins alphaenolase ENOA lysosomeassociated membrane glycoprotein 1 LAMP1 1433 protein gamma 1433G and sarcoplasmic/endoplasmic reticulum calcium ATPase2 AT2A2 were validated by immunoblotting Our results emphasize that separating hydrophobic MPs in CPE contributes to an increased understanding of the underlying molecular mechanisms in AD Such knowledge can become useful for the development of novel disease biomarkersThis research was supported by Uppsala Berzelii Technology Centre for Neurodiagnostics with financing from the Swedish Governmental Agency for Innovation Systems and the Swedish Research Council P297971 BCR was awarded a U4 longterm research travel grant and thereby partially funded by the German Academic Exchange Service DAAD The authors acknowledge Dr Levon Manukyan and Varun Maturi for technical assistance with Western blot analysis and Dr Denys Shevchenko for technical assistance with Fig 2
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