Development of novel mass spectrometry approaches that enable phosphoproteomic analysis in tandem with cellular localisation

作为细胞信号传导的关键介质，磷酸化仍然是生物学问题的主要目标。鉴定和定量参与细胞进展、代谢、生长和疾病的蛋白质的磷酸化状态对于阐明细胞功能至关重要。该项目的最初目的是扩展最近的发展，这些发展已经证明，使用钛基质和同量异位素化学标记（TMT）的磷酸肽富集组合允许从总细胞裂解物中鉴定10，000至15，000个磷酸肽（1，2）。这与hyperLOPIT方法（通过同位素标记定位细胞器蛋白质，（3-5），其将生物化学细胞器分级分离与定量质谱分析相结合，将使细胞器水平的蛋白质组学分析成为可能。虽然科学和实验上具有挑战性，但设想将这些方法结合起来提供一种新的方法是现实的，该方法将增加磷酸蛋白质组学分析的细胞分辨率。研究的第二个目标将是将这些方法应用于探测疾病相关的细胞类型和对化合物治疗的反应。这将利用阿斯利康的疾病专业知识和丰富的注释探针化合物。通过靶向蛋白质组学策略（选择反应监测）和抗体方法进一步测试从这些分析中产生的假设，以使用荧光显微镜直接检测蛋白候选物的磷酸化状态及其在细胞内的定位。最后，利用阿斯利康对疾病相关组织的访问，可以在小鼠/人样品上验证靶向蛋白候选物的表达。(1)在混合四极杆质量过滤器/线性离子阱/轨道阱质谱仪上评估多重定量磷酸肽分析。Erickson BK，Jedrychowski MP，McAlister GC，Everley RA，Kunz R，Gygi SP. Anal Chem.（2015）. (2)人胚胎干细胞和神经干细胞膜蛋白质组、磷酸化蛋白质组和唾液酸组的综合定量比较。Melo-Braga MN，Schulz M，Liu Q，Swistowski A，Palmisano G，Engholm-Keller K，Jakobsen L，Zeng X，Larsen MR. Mol Cell Proteomics.（2014年）。(3)小鼠多能干细胞空间蛋白质组草图。Christoforou A，Mulvey CM，Breckels LM，Geladaki A，Hurrell T，海沃德PC，Naake T，Gatto L，Viner R，Martinez阿里亚斯A，Lilley KS. Nat Commun. 2016. (4)使用hyperLOPIT进行空间蛋白质组的高分辨率映射。Mulvey CM和等人Nature Protocols 12（6）：1110-1135. doi：10.1038/nprot.2017.026（2017）。(5)人类蛋白质组的亚细胞图谱。Thul，PJ等人，Science 356（6340）. pii：eaal3321。doi：10.1126/science.aal3321（2017）.

As a key mediator of cellular signalling, phosphorylation remains a principal target for biological question. Identifying and quantifying the phosphorylation state of proteins involved in cell progression, metabolism, growth and disease is critical for the elucidation of cellular function. The initial aim of the project with be to extend recent developments that have demonstrated that the combination of phospho-peptides enrichment using titanium matrices and isobaric chemical labelling (TMT) allows the identification of 10,000 to 15,000 phosphopeptides from total cell lysate (1, 2). This coupled with the hyperLOPIT methodology (Localisation of Organelle proteins by isotope tagging, (3-5), which combines a biochemical organelle fractionation with a quantitative mass spectrometry analysis, will enable proteomic analysis at the organelle level. Whilst scientifically and experimentally challenging it is realistic to envisage the combination of these methods to deliver a novel approach which will add cellular resolution to phosphoproteomic analysis.The second goal of the research will be the application of these methods to probe disease relevant cell types and response to compound treatment. This will leverage AstraZeneca's disease expertise and wealth of annotated probe compounds. Hypotheses generated from these analyses with be further tested by, targeted proteomic strategies (Selected Reaction Monitoring) and antibody methods to directly characterise the phosphorylation state of the protein candidates and their localisation within the cell using fluorescence microscopy. Finally, exploiting AstraZeneca's access to disease relevant tissues, the expression of targeted protein candidates can be validated on mouse/human samples.(1) Evaluating multiplexed quantitative phosphopeptide analysis on a hybrid quadrupole mass filter/linear ion trap/orbitrap mass spectrometer. Erickson BK, Jedrychowski MP, McAlister GC, Everley RA, Kunz R, Gygi SP. Anal Chem. (2015).(2) Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells. Melo-Braga MN, Schulz M, Liu Q, Swistowski A, Palmisano G, Engholm-Keller K, Jakobsen L, Zeng X, Larsen MR. Mol Cell Proteomics. (2014).(3) A draft map of the mouse pluripotent stem cell spatial proteome. Christoforou A, Mulvey CM, Breckels LM, Geladaki A, Hurrell T, Hayward PC, Naake T, Gatto L, Viner R, Martinez Arias A, Lilley KS. Nat Commun. 2016.(4) Using hyperLOPIT to perform high-resolution mapping of the spatial proteome. Mulvey CM and et al Nature Protocols 12(6):1110-1135. doi: 10.1038/nprot.2017.026 (2017).(5) A subcellular map of the human proteome. Thul, PJ et al, Science 356(6340). pii: eaal3321. doi: 10.1126/science.aal3321 (2017).