Quantitative determination of the effect of transcription factors TFEB and TFE3 on the expression of lysosomal proteins

研究溶酶体蛋白质的研究通常集中在单一蛋白质或几种具有代表性的蛋白质组上，这取决于它们在蛋白质印迹或qPCR实验中是否容易被检测到。使用这些成熟的方法，平行研究所有已证实的溶酶体蛋白是非常复杂的。此外，不可能确定它们的绝对量、拷贝数或这些蛋白质在溶酶体中的化学计量比。为了研究这一点，我们设计了人工合成的标准蛋白-所谓的QConCats(Pratt et.2006年)-该技术可以绝对定量来自任何鼠源样本的153个确认溶酶体蛋白中的144个。QConCats是一种人造蛋白质，由几种待定量的蛋白质中的胰蛋白酶多肽组成。QConCats在稳定的同位素标记的介质中表达，因此可以添加到样品中，从而允许对感兴趣的蛋白质进行绝对定量。胰酶消化后，QConCat释放同位素标记的多肽，每个标记的多肽都作为内源蛋白质产生的非标记多肽的内部标准。这允许对多肽进行绝对量化，从而对相关蛋白质进行绝对量化。这是使用在多反应监测(MRM)模式下工作的所谓的三重四极杆质谱仪来实现的。一旦建立，这些分析是高度可靠的，线性的，允许从高度复杂的混合物(整个细胞或组织裂解产物)中定量所需的多肽(因此蛋白质)，而无需事先浓缩或分级。我们生成并验证了12个重同位素标记的QConCats，每个QConCats包含12个溶酶体蛋白的代表性多肽，从而允许对144个目前已知的溶酶体蛋白进行绝对定量。我们称这些标准为溶酶体QConCats(LysoQCC)。该项目将确定这些LysoQCC可用于确定小鼠全细胞裂解物或组织中溶酶体蛋白的绝对量的条件。这项技术将被用来定量转录因子EB(TFEB)及其近亲TFE3对144个已证实的溶酶体蛋白表达水平的调节。TFEB的广泛转录后修饰(PTM)导致一系列TFEB亚型。我们将分析这些不同亚型的PTM，以及它们在多大程度上受到ERK、mTORC1、GSK3？和PKC信号转导通路的影响。基于LysoQCC对这些样品中溶酶体蛋白的定量，我们将进一步确定选定的翻译后修饰将如何影响TFEB控制的溶酶体蛋白的谱。

Studies investigating lysosomal proteins usually focus either on a single protein or on a representative group of several proteins, dependent on their ease of detection in Western blot or qPCR experiments. Using these well-established methods, it is very intricate to investigate all confirmed lysosomal proteins in parallel. Furthermore, it is impossible to determine their absolute amounts, their copy numbers or the stoichiometry at which these proteins are present in the lysosome. In order to investigate this, we designed synthetic standard proteins - so-called QConCats (Pratt et. al 2006) - which allow for absolute quantification of 144 out of 153 confirmed lysosomal proteins from any sample of mouse origin. QConCats are artificial proteins which consist of tryptic peptides from several proteins which are to be quantified. The QConCats are expressed in stable isotope labeled medium and can therefore be added to the sample allowing for absolute quantification of the proteins of interest. Upon tryptic digestion, the QConCat releases the isotope labelled peptides and each of them serves as internal standard for the non-labelled peptide produced by the endogenous protein. This allows to perform absolute quantification of the peptides, and therefore the correlating proteins. This is achieved using so-called triple quadrupole mass spectrometers operated in the multiple reaction monitoring (MRM) mode. Once established, these assays are highly robust, linear and allow to quantify the desired peptides (and therefore proteins) from highly complex mixtures (whole cell or tissue lysates) without prior enrichment or fractionation. We generated and verified 12 heavy isotope labelled QConCats containing representative peptides for 12 lysosomal proteins each, allowing for the absolute quantification of 144 currently known lysosomal proteins. We call these standards Lysosomal QConCats (LysoQCCs). The project will establish the conditions under which these LysoQCCs can be used to determine the absolute amounts of lysosomal proteins from murine whole cell lysates or tissues. The technique will be used to quantify the regulation of the expression levels of the 144 confirmed lysosomal proteins by the transcription factor EB (TFEB), which is considered as a master regulator of lysosomal biogenesis, and its close relative TFE3. Extensive posttranscriptional modifications (PTMs) of TFEB lead to an array of TFEB isoforms. We will analyze the PTMS of these various isoforms and in how far they are influenced by ERK, mTORC1, GSK3ß and PKC signal transduction pathways. Based on LysoQCC quantification of lysosomal proteins in these samples, we will furthermore determine how selected posttranslational modifications will affect the spectrum of lysosomal proteins controlled by TFEB.