脂肪酸定量磷酸化刺激酿酒酵母</em
Summary
描述了一个定量的磷酸化过程，使用cryolysis，尿素solubilziation，HILIC分馏和富集磷酸化肽的iMac。
Abstract
这个协议描述的增长和刺激，与同位素重工业和轻工业的酿酒酵母细胞，脂肪酸油酸。细胞是地面使用球磨机研磨机和解决方案所带来的尿素增溶产生的grindate的一个cryolysis程序。此过程允许在新陈代谢处于非活动状态的细胞裂解，维护的磷酸化和防止细胞裂解过程中的磷酸化的方向调整。以下还原，烷基化，胰蛋白酶消化的蛋白质，样品脱盐C18柱和样品的复杂性，降低了使用亲水作用色谱（HILIC）分馏。 HILIC列优先保留，这是适合用于磷酸化的亲水性分子。磷酸化肽往往比非磷酸化的同行色谱个人资料后洗脱。分馏后，使用固定的金属色谱仪，依赖基于电荷的亲和力磷酸浓缩磷酸化是丰富。在此过程结束后的样品准备质谱定量分析。
Protocol
细胞生长和媒体 1.0然后将两个1公升文化的一个最小的酵母培养基（0.17％酵母无氮基硫酸铵或氨基酸，0.5％的硫酸铵）的种子细胞，在100 毫升到外径600富媒体BY4742Δarg4Δlys1过夜的一个单菌落含有充分补充氨基酸，辅以20毫克/ L的同位素正常或重精氨酸（13 6 15 N 4的 ; Isotec）和赖氨酸（13 C 6 H 15 N 2; Isotec）。 </li…
Discussion
这种方法将产生一个可定量分析的磷酸化的浓缩。在这个协议中许多参数可以改变，但要记住的最重要的方面是保护您的磷酸。定量细胞的制备，可在许多不同的方式实现，例如，如果你可以没有标签在体内，如作为ICAT标记你的细胞[3]或iTRAC工作[4]可以被使用后提取到不同的标签两种文化的量化目的。
最常见的方法进行分馏凝胶SDS – PAGE [5，6]分馏，SCX色谱法[7]，并基?…
Acknowledgements
我们要感谢丰富的罗杰斯博士与质谱分析和有益的讨论，提供技术援助和Drs。罗布莫里茨，杰夫Ranish，哈米德有益的讨论Mirzai。
这项工作是由美国国立卫生研究院的卓越中心。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Isotec™ L-Arginine-13C6,15N4 | Sigma-Aldrich | 608033 | ||
| Isotec™ L-Lysine-15N2 | Sigma-Aldrich | 609021 | ||
| GIBCO™ Phosphate-Buffered Saline (PBS) 7.4 (10X) liquid | Invitrogen | 70011044 | ||
| SigmaFAST Protease Inhibitor Tablets | Sigma-Aldrich | S8820 | ||
| Pierce Halt Phosphatase Inhibitor Cocktail | Thermo Scientific | 78420 | ||
| Retsch PM100 Ball Mill Grinder | Retsch | 20.540.0001 | ||
| Retsch 125 ml Stainless Steel Grinding Jar | Retsch | 01.462.0148 | ||
| Ultramicrospin C18 column | The Nest Group | SUM SS10 | ||
| Sep-Pak Vac 500 mg C18 | Waters | WAT043395 | ||
| TSK-Gel Amide-80 4.6 mm x 25 cm analytical column | TOSOH BioSciences | 13071 | This is the HILIC chromatographic column. | |
| Guard column 4.6 mm ID x1 cm | TOSOH BioSciences | 19021 | We recommend this guard column to extend the life of your HILIC column. | |
| PHOS-Select™ Iron Affinity Gel | Sigma-Aldrich | P9740 | This is the IMAC resin. Aliquot and store. |
References
- Albuquerque, C. P. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol Cell Proteomics. 7 (7), 1389-1396 (2008).
- McNulty, D. E., Annan, R. S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection. Mol Cell Proteomics. 7 (5), 971-980 (2008).
- Gygi, S. P. Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol. 17 (10), 994-999 (1999).
- Ross, P. L. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 3 (12), 1154-1169 (2004).
- Gruhler, A. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway. Mol Cell Proteomics. 4 (3), 310-327 (2005).
- Wilson-Grady, J. T., Villen, J., Gygi, S. P. Phosphoproteome analysis of fission yeast. J Proteome Res. 7 (3), 1088-1097 (2008).
- Villen, J., Gygi, S. P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry. Nat Protoc. 3 (10), 1630-1638 (2008).
- Jensen, S. S., Larsen, M. R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid Commun Mass Spectrom. 21 (22), 3635-3645 (2007).
- Larsen, M. R. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol Cell Proteomics. 4 (7), 873-886 (2005).
- Pinkse, M. W. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal Chem. 76 (14), 3935-3943 (2004).
- Tao, W. A. Quantitative phosphoproteome analysis using a dendrimer conjugation chemistry and tandem mass spectrometry. Nat Methods. 2 (8), 591-598 (2005).
- Zhou, H., Watts, J. D., Aebersold, R. A systematic approach to the analysis of protein phosphorylation. Nat Biotechnol. 19 (4), 375-378 (2001).
- Bodenmiller, B. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat Methods. 4 (3), 231-237 (2007).
Tags
Quantitative PhosphoproteomicsFatty Acid StimulationSaccharomyces CerevisiaeGrowth And StimulationOleateIsotopically Heavy And Light CellsCryolysis ProcedureBall Mill GrinderUrea SolubilizationMetabolically Inactive StatePhosphorylation PreservationPreventing ReorientationCell LysisReductionAlkylationTrypsin DigestionDesalting On C18 ColumnsSample Complexity ReductionFractionation Using Hydrophilic Interaction Chromatography (HILIC)Phosphopeptides Elution ProfileImmobilized Metal ChromatographyCharge-based AffinitiesMass Spectrometry Analysis
Cite This Article
Saleem, R. A., Aitchison, J. D. Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae. J. Vis. Exp. (32), e1474, doi:10.3791/1474 (2009).