Name: a mass spectrometry-based approach to identify phosphoprotein phosphatases and their interactors
Uploaded: 2022-04-29T14:00:00
Description: Watch this Scientific Journal Video about A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors at JoVE.com

A.N.K.感谢NIH R33 CA225458和R35 GM119455的支持。我们感谢凯滕巴赫和格伯实验室的有益讨论。

注意：PIB的产生是按照Moorhead等人的描述完成的，其中1mg微囊藻毒素和约6 mL琼脂糖偶联产生具有高达5mg / mL17的结合能力的PIB。
1. 样品制备
注意：PIB-MS的典型起始量是每种情况1mg的总蛋白。对于该实验，使用大约2.5×106 个HeLa细胞来提取1mg蛋白质。该计算应针对实验18中使用的每个细胞系或组织进行。...

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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+reprogramming+of+the+kinome+in+response+to+targeted+MEK+inhibition+in+triple-negative+breast+cancer.">Dynamic reprogramming of the kinome in response to targeted MEK inhibition in triple-negative breast cancer.</a> Cell. 149 (2), 307-321 (2012).</li><li>Cooper, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+multiplexed+kinase+inhibitor+beads+to+study+kinome+adaptations+in+drug-resistant+leukemia.">Application of multiplexed kinase inhibitor beads to study kinome adaptations in drug-resistant leukemia.</a> PLoS ONE. 8 (6), 66755 (2013).</li><li>Lyons, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+chemical+proteomic+strategy+for+profiling+phosphoprotein+phosphatases+from+yeast+to+humans.">A quantitative chemical proteomic strategy for profiling phosphoprotein phosphatases from yeast to humans.</a> Molecular and Cellular Proteomics. 17 (12), 2448-2461 (2018).</li><li>Nasa, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+kinase+and+phosphatase+profiling+reveal+that+CDK1+phosphorylates+PP2Ac+to+promote+mitotic+entry.">Quantitative kinase and phosphatase profiling reveal that CDK1 phosphorylates PP2Ac to promote mitotic entry.</a> Science Signaling. 13 (648), (2020).</li><li>Swingle, M., Ni, L., Honkanen, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+inhibitors+of+ser/thr+protein+phosphatases:+specificity,+use+and+common+forms+of+abuse.">Small-molecule inhibitors of ser/thr protein phosphatases: specificity, use and common forms of abuse.</a> Methods in Molecular Biology. 365, 23-38 (2007).</li><li>Moorhead, G. B. G., Haystead, T. A. J., MacKintosh, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+use+of+the+protein+phosphatase+affinity+matrices+microcystin-sepharose+and+microcystin-biotin-sepharose.">Synthesis and use of the protein phosphatase affinity matrices microcystin-sepharose and microcystin-biotin-sepharose.</a> Methods in Molecular Biology. 365, 39-45 (2007).</li><li>Brauer, B. L., Wiredu, K., Mitchell, S., Moorhead, G. B., Gerber, S. A., Kettenbach, A. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Affinity-based+profiling+of+endogenous+phosphoprotein+phosphatases+by+mass+spectrometry.">Affinity-based profiling of endogenous phosphoprotein phosphatases by mass spectrometry.</a> Nature Protocols. 16 (10), 4919-4943 (2021).</li><li>Hughes, C. S., Moggridge, S., M&#252;ller, T., Sorensen, P. H., Morin, G. B., Krijgsveld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-pot,+solid-phase-enhanced+sample+preparation+for+proteomics+experiments.">Single-pot, solid-phase-enhanced sample preparation for proteomics experiments.</a> Nature Protocols. 14 (1), 68-85 (2019).</li><li>Rappsilber, J., Mann, M., Ishihama, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+micro-purification,+enrichment,+pre-fractionation+and+storage+of+peptides+for+proteomics+using+StageTips.">Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.</a> Nature Protocols. 2 (8), 1896-1906 (2007).</li><li>Zecha, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TMT+labeling+for+the+masses:+A+robust+and+cost-efficient,+in-solution+labeling+approach.">TMT labeling for the masses: A robust and cost-efficient, in-solution labeling approach.</a> Molecular and Cellular Proteomics. 18 (7), 1468-1478 (2019).</li><li>Elias, J. 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PIB-MS是一种化学蛋白质组学方法，用于在单次分析中定量分析来自各种样品来源的PPPome。使用激酶抑制剂磁珠已经做了很多工作来研究运动组以及它在癌症和其他疾病状态下如何变化10，11，12，13。然而，对PPPome的研究却落后了。我们预计这种方法能够填补这一空白，并阐明细胞脱磷酸化的调节。在?...

蛋白质磷酸化控制大多数细胞过程，包括但不限于对DNA损伤的反应，生长因子信号传导以及通过有丝分裂1，2，3的传代。在哺乳动物细胞中，大多数蛋白质在某个时间点在一个或多个丝氨酸，苏氨酸或酪氨酸残基处磷酸化，磷酸盐和磷酸尿嘧啶约占所有磷酸化位点的98%2，3。虽然激酶已经在细胞信号传导中得到了广泛的研究，但PPP在动态细胞过程调节中的作用仍在出现。
磷酸化动力学由激酶和磷酸酶之间的动态相互作用控制。在哺乳动物细胞中，有400多种蛋白激酶催化丝氨酸/苏氨酸磷酸化。这些位点中超过90%由磷酸蛋白磷酸酶（PPP）脱磷酸化，磷蛋白磷酸酶是一种由PP1，PP2A，PP2B，PP4-7，PPT和PPZ2，3组成的酶的小家族。PP1和PP2A负责细胞2，3，4内大部分磷酸盐水和磷酸尿嘧啶去磷酸化。激酶和磷酸酶之间的数量显着差异以及体外PPP催化亚基缺乏特异性，导致人们相信激酶是磷酸化的主要决定因素2，3。然而，多项研究表明，磷酸酶通过形成多聚体全酶5，6，7，8，9来建立底物特异性。例如，PP1是一种异源二聚体，由催化亚基组成，并且在给定时间，150多个调节亚基6，7，8中的一个。相反，PP2A是由脚手架（A），调节（B）和催化（C）亚基2，3，9形成的异源三聚体。PP2A调节亚基有四个不同的家族（B55，B56，PR72和纹状体），每个家族都有多个基因，剪接变体和定位模式2，3，9。PPP的多聚体性质填补了激酶和PPP催化亚基数量的空白。然而，它为研究PPP信令带来了分析挑战。为了全面分析PPP信号传导，研究细胞或组织内的各种全息酶至关重要。通过使用激酶抑制剂微球（称为多重抑制剂磁珠或激蛋白），在研究人类基质组方面取得了巨大进展，这是一种化学蛋白质组学策略，其中激酶抑制剂固定在磁珠上，质谱法用于鉴定富集激酶及其相互作用子10，11，12，13。
我们已经建立了类似的方法来研究PPP生物学。该技术涉及使用带有固定化非选择性PPP抑制剂（称为微囊藻毒素-LR（MCLR）的磷酸酶抑制剂珠（PIB）的磁珠14，15）对PPP催化亚基的亲和力捕获。与其他需要内源性标记或表达可能改变蛋白质活性或定位的外源性PPP亚基的方法不同，PIB-MS允许在给定时间点或特定处理条件下富集内源性PPP催化亚基，其相关的调节和支架亚基以及来自细胞和组织的相互作用蛋白质（称为PPPome）。MCLR 在纳摩尔浓度下抑制 PP1、PP2A、PP4-6、PPT 和 PPZ，使 PIB 在富集16 方面非常有效。该方法可以缩放以用于从细胞到临床样品的任何起始材料。在这里，我们详细描述了使用PIB和质谱（PIB-MS）来有效地捕获，鉴定和量化内源性PPPome及其修饰状态。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63805/63805fig1v2.jpg"> 图 1：PIB-MS 协议的可视化摘要。 在PIB-MS实验中，样品可以以各种形式获得，从细胞到肿瘤。在PPP富集之前收集，裂解和均质样品。为了富集PPP，裂解物与PIB一起孵育，有或没有PPP抑制剂，例如MCLR。然后洗涤PIB，并在变性条件下洗脱PPP。通过SP3蛋白富集，胰蛋白酶消化和脱盐去除去垢剂，制备样品进行质谱分析。然后，可以在质谱分析之前选择性地对样品进行TMT标记。 <a href="https://www.jove.com/files/ftp_upload/63805/63805fig1v2large.jpg" target="_blank">请点击此处查看此图的大图。</a>
PIB-MS涉及细胞或组织的裂解和澄清，裂解物与PIB的孵育，洗脱以及通过蛋白质印迹或基于质谱的方法分析洗脱液（图1）。添加游离 MCLR 可用作区分特定 PIB 粘合剂和非特异性相互作用子的对照。对于大多数应用，无标记方法可用于直接鉴定洗脱液中的蛋白质。在需要更精确地定量或鉴定低丰度物种的情况下，可以使用串联质量标签（TMT）标签进行进一步处理，以增加覆盖范围并减少输入。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Acetonitrile (ACN)</td>
			<td>Honeywell</td>
			<td>AH015-4</td>
			<td>CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Anhydrous Acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004-100ML</td>
			<td>CAUTION: ACN is flammable and toxic; wear gloves, and work in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>model no. 5424</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta-glycerophosphoric acid, disodium salt pentahydrate</td>
			<td>Acros Organics</td>
			<td>410991000</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>model no. 5810 R 15 amp version</td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher Scientific</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce tissue grinder</td>
			<td>Fisherbrand Pellet Pestles</td>
			<td>12-141-363</td>
			<td></td>
		</tr>
		<tr>
			<td>Empore solid phase extraction disk, C18</td>
			<td>CDS Analytical</td>
			<td>76333-132</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22363204</td>
			<td>CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.</td>
		</tr>
		<tr>
			<td>Eppendorf tubes, 2 mL</td>
			<td>Eppendorf</td>
			<td>22363352</td>
			<td>CRITICAL: Other tubes may leach polymer into sample, contaminating the analysis.</td>
		</tr>
		<tr>
			<td>Extraction plate manifold</td>
			<td>Waters</td>
			<td>WAT097944</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes, 50 mL</td>
			<td>VWR</td>
			<td>21008</td>
			<td></td>
		</tr>
		<tr>
			<td>Generic blunt end needle and plunger</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Generic magnetic separation rack</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen chloride (HCl)</td>
			<td>VWR Chemicals BDH</td>
			<td>BDH3028</td>
			<td>CAUTION: HCl is corrosive; wear gloves and work in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Hydroxylamine solution 50% (wt/vol)</td>
			<td>Sigma-Aldrich</td>
			<td>467804</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator, 65 &#176;C</td>
			<td>VWR</td>
			<td>model no. 1380FM</td>
			<td></td>
		</tr>
		<tr>
			<td>Koptec Pure Ethanol, 200 Proof</td>
			<td>Decon Labs</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol for HPLC (MeOH)</td>
			<td>Sigma-Aldrich</td>
			<td>34860-4L-R</td>
			<td>CAUTION: MeOH is flammable and toxic; wear gloves, and work in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Microcystin LR (MCLR)</td>
			<td>Cayman Chemical</td>
			<td>10007188</td>
			<td>CAUTION: MCLR is toxic; wear gloves when handling and avoid skin contact.</td>
		</tr>
		<tr>
			<td>PBS, 1&#215; without calcium and magnesium, pH 7.4 &#177; 0.1</td>
			<td>Corning</td>
			<td>&#160;21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>pH test strips, such as MilliporeSigma MColorpHast pH test strips and indicator papers</td>
			<td>Fisher Scientific</td>
			<td>M1095310001</td>
			<td></td>
		</tr>
		<tr>
			<td>PIBs</td>
			<td></td>
			<td></td>
			<td>For protocol for the generation of PIBs, see Moorhead et al., 2007.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tips, 10 &#956;L</td>
			<td>Eppendorf</td>
			<td>22491504</td>
			<td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td>
		</tr>
		<tr>
			<td>Pipette tips, 1000 &#956;L</td>
			<td>Eppendorf</td>
			<td>22491555</td>
			<td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td>
		</tr>
		<tr>
			<td>Pipette tips, 200 &#956;L</td>
			<td>Eppendorf</td>
			<td>22491539</td>
			<td>CRITICAL: Other tips may leach polymer into samples, contaminating the analysis.</td>
		</tr>
		<tr>
			<td>plastic syringe, 10 mL</td>
			<td>BD</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease inhibitor cocktail III</td>
			<td>Research Products International</td>
			<td>P50700-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer, Oribtrap Fusion, Orbitrap Fusion Lumos, or Orbitrap Eclipse Tribrid Mass Spectrometer&#160;</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Refrigerated benchtop centrifuge</td>
			<td>Eppendorf</td>
			<td>model no. 5424 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotator (Labquake Shaker Rotisserie)</td>
			<td>Thermo Scientific</td>
			<td>13-687-12Q</td>
			<td>8 rpm rotation</td>
		</tr>
		<tr>
			<td>Sample collection plate, 96- well, 1 mL</td>
			<td>Waters</td>
			<td>WAT058957</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Fisher Scientific</td>
			<td>BP1311-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing grade modified trypsin</td>
			<td>Promega</td>
			<td>V511C</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>EMD Chemicals</td>
			<td>SX0299-1</td>
			<td>CAUTION: Sodium azide is explosive and toxic; wear gloves, work in a chemical fume hood and avoid contact with metals.</td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Chemical</td>
			<td>S27110</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator (Branson digital sonifier)</td>
			<td></td>
			<td>model no. SFX 250</td>
			<td></td>
		</tr>
		<tr>
			<td>SPE C18 desalting plate</td>
			<td>Waters</td>
			<td>186001828BA</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedBeads magnetic carboxylate modified particles (SP3 beads)</td>
			<td>Cytiva</td>
			<td>6.51521E+13</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer</td>
			<td>Eppendorf</td>
			<td>model no. 5350</td>
			<td></td>
		</tr>
		<tr>
			<td>TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent, 3 &#215; 0.8 mg per tag</td>
			<td>ThermoFisher</td>
			<td>A37725</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Honeywell</td>
			<td>T6508-25ML</td>
			<td>CAUTION: TFA is corrosive and will irritate skin on contact. Wear gloves and eye protection, and work in a chemical fume hood.</td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Research Products International</td>
			<td>T60040</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum centrifuge and vapor trap</td>
			<td>Thermo Scientific</td>
			<td>model nos. SpeedVac SPD120 and RVT5105</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer (Vortex-Genie 2)</td>
			<td>Scientific Industries</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water LC-MS</td>
			<td>Honeywell</td>
			<td>LC365-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

a mass spectrometry-based approach to identify phosphoprotein phosphatases and their interactors

大多数细胞过程受动态蛋白质磷酸化调节。超过四分之三的蛋白质被磷酸化，磷酸蛋白磷酸酶（PPP）协调超过90%的细胞丝氨酸/苏氨酸脱磷酸化。蛋白质磷酸化的失调与各种疾病的病理生理学有关，包括癌症和神经变性。尽管其活性广泛，但控制PPP和PPP控制的分子机制的分子机制特征不佳。在这里，描述了一种称为磷酸酶抑制剂磁珠和质谱（PIB-MS）的蛋白质组学方法，以使用任何细胞系或组织在短短12小时内鉴定和量化PPP，其翻译后修饰及其相互作用者。PIB-MS利用非选择性PPP抑制剂微囊藻毒素LR（MCLR），固定在琼脂糖珠上以捕获和丰富内源性PPP及其相关蛋白质（称为PPPome）。该方法不需要标记版本的PPP的外源性表达或使用特异性抗体。PIB-MS提供了一种创新方法来研究进化保守的PPP，并扩展了我们目前对去磷酸化信号传导的理解。

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63805/63805fig02.jpg"> 图2：鉴定特定的PIB粘合剂（A）可以通过PIB-MS分析各种组织类型或细胞。生物一式三份的HeLa细胞用DMSO或PPP抑制剂MCLR处理，与PIB一起孵育，并通过LC-MS / MS进行分析。PPP催化亚基被标记为蓝色并显示为蓝色。新的候选PPP亚基或?...

Watch this Scientific Journal Video about A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors at JoVE.com

A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors

在这里，我们提出了一种方案，用于富集来自细胞和组织的内源性磷酸蛋白磷酸酶及其相互作用的蛋白质，并通过基于质谱的蛋白质组学鉴定和定量它们。

一种基于质谱的磷酸化蛋白磷酸酶及其相互作用物鉴定方法

Most cellular processes are regulated by dynamic protein phosphorylation. More than three-quarters of proteins are phosphorylated, and phosphoprotein ...

PIB-MS是一种新技术，用于富集内源性磷酸蛋白磷酸酶，并且它们与来自细胞和组织的蛋白质相互作用。它涉及使用基于质谱的蛋白质组学对这些蛋白质进行鉴定和定量。该技术不需要标记版本的磷蛋白磷酸酶的外源性表达，这些版本可以改变蛋白质活性或定位。此外，它不使用可能非特异性或无法区分特定亚型的抗体。这种研究PPPM的方法可以扩展用于从细胞到临床样品的所有方面。首先，通过在室温下以277次g离心两分钟来收集细胞沉淀。取出培养基并用5毫升PBS洗涤细胞。按照文本协议中所述准备裂解缓冲液并保持在冰上。向样品中加入一毫升冷冻裂解缓冲液，然后通过超声处理使样品均质化，并将细胞保持在脉冲之间的冰上。将裂解物转移到新鲜的1.5毫升管中。将超声处理的样品以21，130倍g在4摄氏度下离心15分钟。使用裂解物的等分试样使用双新角蛋白酸测定法量化总蛋白浓度。此步骤对于确保每个样品中的蛋白质浓度相等至关重要。对于磷酸蛋白磷酸酶抑制剂控制，为每个样品制备两个等同一蛋白质浓度的等分试样。裂解缓冲液可用于稀释样品，使每个试管具有相等的蛋白质浓度。向一个等分试样中加入一微摩尔游离微囊藻素-LR，向另一个等分试样加入等体积的DMSO。涡旋样品并将其在冰上孵育15分钟。磷酸酶抑制剂磁珠的制备应提前或在样品制备部分中描述的孵育步骤中进行。使用10微升固体磷酸酶抑制剂珠或PIB树脂，用于一毫克蛋白质，并将它们加入1.5毫升管中。用0.5毫升裂解缓冲液涡旋洗涤PIB三次，然后在室温下以376倍g离心30秒。向洗涤的PIB中加入适量的裂解缓冲液，以制成50%体积的PIB缓冲液。通过轻轻移液混合并旋转移液器尖端和浆料以重悬PIB。将20微升浆液转移到含有0.5毫升裂解缓冲液的1.5毫升管中。以376次g旋转管30秒。每个管子中含有等体积的PIB。丢弃上清液，同时在每个管中仅留下固体树脂和最多50微升的裂解缓冲液。将裂解物转移到含有PIB的适当标记的管中。将裂解物与PIB一起在4摄氏度下孵育一小时，以8 rpm的速度旋转。与裂解物孵育后，在4摄氏度下以376倍g离心PIB30秒并除去上清液。通过加入0.5毫升裂解缓冲液并倒置管来洗涤磁珠通过离心收集磁珠并除去上清液。重复此步骤，总共洗涤三次。最终洗涤后，尽可能多地去除裂解缓冲液，而无需移取PIB。制备含有2%SDS的洗脱缓冲液，并加入洗脱缓冲液4至5倍于PIB体积。在65摄氏度下孵育一小时，以从磁珠中洗脱磷酸蛋白磷酸酶。在室温下以376次g离心管30秒。将洗脱液收集到单独的管中，并进行质谱分析或蛋白质印迹。要再生PIB，请将它们在2%SDS溶液中孵育，在室温下以8rpm旋转一小时。在25毫摩尔Tris-HCl pH 7.5下洗涤珠子三到五次，每次洗涤旋转30分钟。将PIB储存在25毫摩尔三元HCL pH 7.5和叠氮化钠中。对于质谱分析，数据过滤和分析的方法各不相同，可以由研究人员或核心设施执行。针对特定物种的蛋白质组数据库搜索原始数据后，筛选搜索结果并将其导出为 Microsoft Excel 工作表。一式三份分析数据。对于无标记定量，请使用 MS1 峰面积测量来量化数据。为了从头鉴定PPP亚基及其相互作用者，比较微囊藻毒素-LR抑制和DMSO处理的样品的生物一式三份。过滤数据，以便仅存在总肽计数超过三个DMSO对照处理样品中至少两分之一的蛋白质。为了过滤掉与树脂非特异性结合的蛋白质，请去除微囊藻毒素-LR处理条件下总肽计数高于任何PPP催化亚基的蛋白质。从分析中排除常见的污染物，如角蛋白、胶原蛋白、核糖体蛋白和异质核糖核蛋白。CSV文件对于将数据导入英仙座至关重要。通过单击“加载”部分中的“通用矩阵上传”，将筛选的数据导入英仙座。通过转到“基本转换”，选择数据并将转换函数指定为 x 的对数基数 2 来转换数据。通过转到插补、替换正态分布中的缺失值、选择数据并指定计算的宽度和下移，从正态分布中插补缺失值。通过转到归一化、分位数规范化来执行分位数规范化。通过转到“注释行”、“分类注释行”来批注数据。通过转到测试，双样本测试，选择组，要执行的测试以及用于截断的多个假设测试校正方法来执行学生的T检验。此函数还计算对数 2 比率。使用PIB下拉实验在HeLa细胞中鉴定磷酸化蛋白磷酸盐及其相互作用者，然后对DMSO处理和微囊藻毒素- LR处理的样品中的蛋白质丰度进行无标记定量。质谱分析的火山图鉴定出红色显示的特定PIB结合剂，蓝色显示催化亚基，白色显示新的候选PPP亚基或相互作用蛋白。非特异性粘合剂以灰色显示。DMSO处理的HeLa细胞裂解物log2面积散点图显示，其丰度高度相关，表明数据的可重复性。对所有磷蛋白磷酸酶亚基和相互作用蛋白的网络分析表明，这些蛋白质是PIB下拉中的特异性相互作用物。可以进行全球蛋白质组学分析，并将结果与PIB分析进行比较，以确定PPPome组成是由PPP丰度驱动的还是由翻译后机制调节的。PIB-MS是一种广泛适用的工具，使我们能够识别各种条件下PPP表达模式的差异，例如相间与有丝分裂样品或癌细胞系之间的差异。

Research

Education

JoVE Core

JoVE Journal

Analytical Chemistry

Biochemistry

Unit 1

Principles of Mass Spectrometry

SCIENTISTS IN ACTION

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

科研

生物化学

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

解决由蓝变性PAGE和蛋白质相关剖析亲和纯化的蛋白复合物

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

A Tandem Liquid ChromatographyÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œMass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

RNA 结合区质量谱鉴定的样品制备

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

A Rat Methyl-Seq Platform to Identify Epigenetic Changes Associated with Stress Exposure

大鼠甲基 Seq 平台识别与应力接触相关的表观变化

As genomes of a wider variety of animals become available, there is an increasing need for tools that can capture dynamic epigenetic changes in these ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

通过肽浓缩和质谱检测蛋白质泛化位点检测

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Studying RNA Interactors of Protein Kinase RNA-Activated during the Mammalian Cell Cycle

哺乳动物细胞周期内蛋白质激酶 rna 激活的 rna 相互作用研究

Protein kinase RNA-activated (PKR) is a member of the innate immune response proteins and recognizes the double-stranded secondary structure of viral ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

肽聚糖的液相色谱质谱和生物信息学半定量分析

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...