我们非常感谢阿兰特西尔宝贵的建议，讨论和技术支持。我们承认在Concordia大学生物应用质谱杰出服务中心。这项工作得到了CIHR和加拿大NSERC资助。 VIT是一个新的CIHR调查和Concordia大学基因组学研究主席，细胞生物学和老化。

材料和方法<ol><li>酵母菌株和生长条件。 丰富YEPD培养基中生长的野生型菌株BY4742（MATαhis3Δ1leu2Δ0lys2Δ0ura3Δ0）（1％酵母提取物，2％蛋白胨，2％葡萄糖） 。细胞培养在30 ° C旋转摇晃锥形瓶在200 RPM“瓶体积/中等音量”5:1的比例。 </li><li>脂质提取的酵母细胞储存 <ol><li>文化是收获的细胞50毫升离心5分钟在3000 XG。 </li><li>用冷水冲洗两次。 <ol style=" list-style-type:lower-alpha;"><li>悬浮颗粒在25毫升冰水</li><li>在离心沉淀细胞，5分钟在3000 XG </li></ol></li><li>悬浮颗粒和转移到Eppendorf管</li><li>球离心，16000 XG 2分钟，取出使用上清于-80 ° C，直到和冻结。 （我们使用异丙醇的烧杯中，也可以用液态氮保存在-80冷冻） </li></ol></li><li>试剂 自Sigma - Aldrich生物技术级氯仿和甲醇。游离脂肪酸和甘油三酯均购自Larodan（瑞典马尔默）。获得各种种磷脂 - 包括磷脂，磷脂，磷脂酰肌醇，磷脂酰丝氨酸，磷脂酸，并cardiolipins - 阿凡提极性脂（雪花石膏，AL，美国）。从费舍尔高速离心管玻璃与聚四氟乙烯内衬的盖子。 </li></ol>脂质提取这是一个布莱和代尔8所述的协议的修改。提取血脂所有的操作，应使用玻璃移液管或注射器，塑料将创建一个大的背景信号，如果他们接触到，以氯仿。确切的卷是不是只要关键氯仿的比例为1:2:0.8 -甲醇-在第3步中的H 2 O和2:2:1.8为氯仿-甲醇- H 2 O在第7步是保守的。 <ol><li>冷冻细胞重悬在1.6毫升的冰冷的蒸馏水H 2 O </li><li> 1.6毫升细胞悬液转移到高速的玻璃离心管。 </li><li>加入6毫升氯仿和甲醇（1:2）混合和0.8毫升细胞悬液的玻璃珠。 </li><li>涡细胞悬液，用玻璃珠1分钟的2倍。 </li><li>加入2毫升氯仿，轻轻混匀。 </li><li>孵育5分钟室温搅拌偶尔。 </li><li>加入2毫升的蒸馏水H 2 O，轻轻混匀。 </li><li>孵育5分钟室温搅拌偶尔。 </li><li>在3000 XG在室温下5分钟离心。 </li><li>整个液相收集到一个新的高速玻璃离心管。 </li><li>从第9步的细胞沉淀，加入3.2毫升氯仿。 </li><li>涡1分钟两次。 </li><li>在3000 XG在室温下5分钟离心。 </li><li>从第10步液相添加上清。 </li><li>在3000 XG在室温下5分钟离心。 </li><li>倒掉上层（水）相和较低的阶段（有机）转移到一个新的高速玻璃离心管。 </li><li>在3000 XG在室温下5分钟离心。 </li><li>整个上清转移到一个玻璃小瓶，干燥的氮气保护下。 </li><li>脂质膜溶解于500μL氯仿和储存在-20 ° C </li></ol>质谱分析血脂血脂标准的股票组合中的氯仿应事先准备好的，按表1，以及原液的甲醇 - 氯仿（1:1）0.1％（V / V）氢氧化铵。 <ol><li>注射前，结合10μL在表1提供的血脂标准混合样品10μL。在200μL1:1的氯仿：甲醇与0.1％NH 4 OH。 <ul><li>样本比例的标准是可以改变的需要。 </li></ul></li><li>解决使用一个微团的Q - TOF质谱仪配备与1μL/ min的流速在纳米电源经营血脂。 <ul><li>从仪器仪表精确的仪器参数会有所不同。一个微团配备一个纳米电源2 Q - TOF（沃特斯，米尔福德，马萨诸塞州，美国）的设置，请参阅表2。虽然我们已采取的Q - TOF质谱仪的一个类型的高分辨率的优势，它不是一种强制性要求。 </li></ul></li><li>收购后的质谱是平滑的，减去背景和中心，然后高峰列表导出到Excel。每个脂质类的峰值，然后归到他们的内部标准。根据不同的应用，它可能需要做进行进一步处理后deisotoping和反褶积等。 </li></ol> 表1。内部血脂标准，其浓度标准的组合，并为他们的分析MS模式。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td>脂质类</td><td>标准链的构成</td><td>标准的质量</td><td>浓度（微克/毫升） </td><td> MS模式</td></tr><tr><td>磷脂酸</td><td> 14:0 / 14:0 </td><td> 591.40 </td><td> 100 </td><td>负面</td></tr><tr><td>磷脂酰乙醇胺</td><td> 14:0 / 14:0 </td><td> 634.45 </td><td> 200 </td><td>负面</td></tr><tr><td>磷脂</td><td> N / A </td><td> N / A </td><td> N / A </td><td>负面</td></tr><tr><td>磷脂</td><td> 14:0 / 14:0 </td><td> 622.37 </td><td> 40 </td><td>负面</td></tr><tr><td>心磷脂</td><td> 4x14：0 </td><td> 619.92 </td><td> 100 </td><td>负面</td></tr><tr><td>游离脂肪酸</td><td> 19:0 </td><td> 297.28 </td><td> 100 </td><td>负面</td></tr><tr><td>卵磷脂</td><td> 14:0 / 14:0 </td><td> 650.48 </td><td> 100 </td><td>积极的</td></tr><tr><td>甘油三酯</td><td> 13:0 / 13:0 / 13:0 </td><td> 698.63 </td><td> 200 </td><td>积极的</td></tr></tbody></table> 表2。一个微团Q - TOF 2（沃特斯，米尔福德，马萨诸塞州，美国）的仪器设置配备纳米电源。 <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td></td><td>流量</td><td>锥孔电压</td><td>毛细管电压</td><td>碰撞气体</td></tr><tr><td>正离子模式</td><td> 1μl/min </td><td> -28 v </td><td> 3.0 KV </td><td> 10 </td></tr><tr><td>负离子模式</td><td> 1μl/min </td><td> 30 V </td><td> -3.2 KV </td><td> 10 </td></tr></tbody></table>

<ol>
	<li>Cao, H., Gerhold, K., Mayers, J. R., Wiest, M. M., Watkins, S. M., Hotamisligil, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+lipokine,+a+lipid+hormone+linking+adipose+tissue+to+systemic+metabolism.">Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.</a> Cell. 134, 933-944 (2008).</li><li>Claypool, S. M., Oktay, Y., Boontheung, P., Loo, J. A., Koehler, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiolipin+defines+the+interactome+of+the+major+ADP/ATP+carrier+protein+of+the+mitochondrial+inner+membrane.">Cardiolipin defines the interactome of the major ADP/ATP carrier protein of the mitochondrial inner membrane.</a> J. Cell Biol. 182, 937-950 (2008).</li><li>Guo, T., Gregg, C., Boukh-Viner, T., Kyryakov, P., Goldberg, A., Bourque, S., Banu, F., Haile, S., Milijevic, S., San, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+signal+from+inside+the+peroxisome+initiates+its+division+by+promoting+the+remodeling+of+the+peroxisomal+membrane.">A signal from inside the peroxisome initiates its division by promoting the remodeling of the peroxisomal membrane.</a> J. Cell Biol. 177, 289-303 (2007).</li><li>Russell, S. J., Kahn, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+regulation+of+ageing.">Endocrine regulation of ageing.</a> Nat. Rev. Mol. Cell Biol. 8, 681-691 (2007).</li><li>Czabany, T., Athenstaedt, K., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+storage+and+degradation+of+neutral+lipids+in+yeast.">Synthesis, storage and degradation of neutral lipids in yeast.</a> Biochim. Biophys. Acta. 1771, 299-309 (2007).</li><li>Br&amp;uuml, g. g. e. r., Erben, B., Sandhoff, G., Wieland, R., &amp;amp, F. T., Lehmann, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+biological+membrane+lipids+at+the+low+picomole+level+by+nano-electrospray+ionization+tandem+mass+spectroscopy.">Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectroscopy.</a> Proc. Natl. Acad. Sci. USA. 94, 2339-2344 (1997).</li><li>Schneiter, R., Br&#252;gger, B., Sandhoff, R., Zellnig, G., Leber, A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+tandem+mass+spectrometry+(ESI-MS/MS)+analysis+of+the+lipid+molecular+species+composition+of+yeast+subcellular+membranes+reveals+acyl+chain-based+sorting/remodeling+of+distinct+molecular+species+en+route+to+the+plasma+membrane.">Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane.</a> J. Cell Biol. 146, 741-754 (1999).</li><li>Bligh, E. G., Dyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+of+total+lipid+extraction+and+purification.">A rapid method of total lipid extraction and purification.</a> Can. J. Biochem. Physiol. 37, 911-917 (1959).</li></ol>

这种方法使酵母lipidome使用现成的，廉价的材料的快速定量评估。该方法允许大部分diacylglycerols，ergosterols和ergosteryl酯的例外在酵母细胞中发现的脂质物种的鉴定，虽然这些脂质可取代氢氧化锂，氢氧化铵确定。所描述的方法可低至微克/毫升的浓度脂质识别和量化，与浓度的线性超过2〜3个数量级（脂质种而定）的蔓延。虽然市售磷酸肌醇的适当的标准，本磷脂不同的分子形式可以使用其他磷脂?...

a quantitative assessment of the yeast lipidome using electrospray ionization mass spectrometry

脂质是生物分子的主要类别之一，并发挥重要作用膜动力学，能量存储，和信令<sup&gt; 1-4</sup&gt;。芽殖酵母<em&gt;酿酒酵母</em&gt;，一种基因和生物化学来操作带注释的基因组，很简单lipidome的单细胞真核生物，是一个有价值的模型，研究在多细胞真核生物中各种脂质物种的生物功能<sup&gt; 2,3,5</sup&gt;。<em&gt; S。酵母</em与链长，主要是16或18个碳原子和零或一个不饱和度&gt; 10血脂的主要类别<sup&gt; 6,7</sup&gt;。现有脂质的鉴定和定量的方法 - 如高效液相色谱法，薄层色谱法，荧光显微镜，气相色谱仪由MS - 已经非常成熟，但灵敏度低，不够独立的各种脂类分子形式，要求前脂质derivitization分析，或可相当费时。在这里，我们提出了我们的实验方法的详细说明，以解决这些固有的局限性，所使用的整个酵母细胞中的脂类补充的识别和定量调查扫描ESI / MS。所描述的方法不需要复杂的脂质混合物从酵母细胞中提取的色谱分离，从而大大加快了数据采集的过程。这种方法能够在低克/毫升的浓度脂质识别和量化，并已成功地应用于评估整个酵母细胞及其纯化细胞器lipidomes。血脂，使用这种方法血脂分析的整个酵母细胞中提取需要两到三个小时。它只有五到十分钟，上运行的Q - TOF质谱光谱仪配备了一个纳米电源每个样本提取和干燥血脂。

Watch this Scientific Journal Video about A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry at JoVE.com

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

我们描述了一个新的定量确定使用调查的扫描电喷雾电离质谱（ESI / MS）酵母大量的脂质物种lipidomics方法。这种方法超过目前可用的方法脂质识别和量化的能力来解决血脂，灵敏度和速度不同的分子形式。

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

a-quantitative-assessment-yeast-lipidome-using-electrospray

Research

JoVE Journal

Biology

11.6K 次观看.  Concordia University. 我们描述了一个新的定量确定使用调查的扫描电喷雾电离质谱（ESI / MS）酵母大量的脂质物种lipidomics方法。这种方法超过目前可用的方法脂质识别和量化的能力来解决血脂，灵敏度和速度不同的分子形式。

视频: A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI样品制备：超薄层的方法

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

科研

生物学

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

加上质谱肽免疫亲和富集的蛋白质定量

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein interactions, protein conformational changes, protein dynamics and protein-ligand interactions. H/D exchange on the backbone amide positions has been utilized to measure the deuteration rates of the micro-regions in a protein by mass spectrometry1,2,3. The resolution of this method depends on pepsin digestion of the deuterated protein of interest into peptides that normally range from 3-20 residues. Although the resolution of H/D exchange measured by mass spectrometry is lower than the single residue resolution measured by the Heteronuclear Single Quantum Coherence (HSQC) method of NMR, the mass spectrometry measurement in H/D exchange is not restricted by the size of the protein4. H/D exchange is carried out in an aqueous solution which maintains protein conformation. We provide a method that utilizes the MALDI-TOF for detection2, instead of a HPLC/ESI (electrospray ionization)-MS system5,6. The MALDI-TOF provides accurate mass intensity data for the peptides of the digested protein, in this case protein kinase Pak2 (also called &gamma;-Pak). Proteolysis of Pak 2 is carried out in an offline pepsin digestion. This alternative method, when the user does not have access to a HPLC and pepsin column connected to mass spectrometry, or when the pepsin column on HPLC does not result in an optimal digestion map, for example, the heavily disulfide-bonded secreted Phospholipase A2 (sPLA2). Utilizing this method, we successfully monitored changes in the deuteration level during activation of Pak2 by caspase 3 cleavage and autophosphorylation7,8,9.

MALDI-TOF mass spectrometry was successfully utilized to monitor the amide hydrogen/deuterium exchange in protein kinase Pak2 activation.

酰胺氢/氘交换和MALDI - TOF质谱PAK2激活的质谱分析

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

利用同位素标记的质谱剖析巯基氧化还原蛋白质

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

一个协议，用于基于蛋白质 - 蛋白质相互作用的鉴定<sup&gt; 15</sup&gt; N代谢标记，免疫沉淀法，的定量质谱法和亲和调制

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

捕获复合质谱 - 一个强大的工具来确定新型C-二GMP效应蛋白

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.

Proteome characterization of ocular microvascular beds is pivotal for in-depth understanding of many ocular pathologies in humans. This study demonstrates an effective, rapid, and robust method for protein extraction and sample preparation from small blood vessels employing the porcine short posterior ciliary arteries as model vessels for mass-spectrometry-based proteomics analyses.

基于性光谱的眼微血管蛋白质组学样品制备

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly ...