Name: a mass spectrometry-based proteomics approach for global and high-confidence protein r-methylation analysis
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MM和EM是欧洲分子医学学院（SEMM）的博士生。EM是FIRC-AIRC助学金的3年期（项目代码：22506）的获得者。结核病组中R-甲基蛋白质组的全球分析得到了AIRC IG资助（项目代码：21834）的支持。

1. 细胞培养和蛋白质提取（时间：需要3-4周）
<ol><li>分别在用轻（L）或重（H）蛋氨酸提供的培养基中平行生长HeLa细胞（参见 表1 的培养基组成）。在至少八次细胞分裂后，从每个SILAC通道收集等分试样的细胞并进行掺入测试。 注意：为了检查掺入效率，请通过LC-MS/MS分析测试重质通道中重蛋氨酸（Met-4）的百分比尽可能接近100%。通过LC-MS/MS分析重标记细胞的等分试样（设置见表 <...

<ol>
	<li>Bulau, 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=Quantitative+assessment+of+arginine+methylation+in+free+versus+protein-incorporated+amino+acids+in+vitro+and+in+vivo+using+protein+hydrolysis+and+high-performance+liquid+chromatography.">Quantitative assessment of arginine methylation in free versus protein-incorporated amino acids in vitro and in vivo using protein hydrolysis and high-performance liquid chromatography.</a> Biotechniques. 40 (3), 305-310 (2006).</li><li>Blanc, R. S., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+methylation:+the+coming+of+age.">Arginine methylation: the coming of age.</a> Molecular Cell. 65 (1), 8-24 (2017).</li><li>Sylvestersen, K. B., Horn, H., Jungmichel, S., Jensen, L. J., Nielsen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+arginine+methylation+sites+in+human+cells+reveals+dynamic+regulation+during+transcriptional+arrest.">Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest.</a> Molecular &amp; Cellular Proteomics. 13 (8), 2072-2088 (2014).</li><li>Yang, Y., Bedford, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methyltransferases+and+cancer.">Protein arginine methyltransferases and cancer.</a> Nature Reviews Cancer. 13 (1), 37-50 (2013).</li><li>Bezzi, M., 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=Regulation+of+constitutive+and+alternative+splicing+by+PRMT5+reveals+a+role+for+Mdm4+pre-mRNA+in+sensing+defects+in+the+spliceosomal+machinery.">Regulation of constitutive and alternative splicing by PRMT5 reveals a role for Mdm4 pre-mRNA in sensing defects in the spliceosomal machinery.</a> Genes &amp; Development. 27 (17), 1903-1916 (2013).</li><li>Auclair, Y., Richard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+arginine+methylation+in+the+DNA+damage+response.">The role of arginine methylation in the DNA damage response.</a> DNA Repair. 12 (7), 459-465 (2013).</li><li>Spadotto, V., 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=PRMT1-mediated+methylation+of+the+microprocessor-associated+proteins+regulates+microRNA+biogenesis.">PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis.</a> Nucleic Acids Research. 48 (1), 96-115 (2020).</li><li>Musiani, D., Massignani, E., Cuomo, A., Yadav, A., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+and+computational+approaches+for+the+large-scale+analysis+of+protein+arginine+methylation+by+mass+spectrometry.">Biochemical and computational approaches for the large-scale analysis of protein arginine methylation by mass spectrometry.</a> Current Protein and Peptide Science. 21 (7), 725-739 (2020).</li><li>Ong, S. -. E., Mittler, G., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+and+quantifying+in+vivo+methylation+sites+by+heavy+methyl+SILAC.">Identifying and quantifying in vivo methylation sites by heavy methyl SILAC.</a> Nature Methods. 1 (2), 119-126 (2004).</li><li>Hart-Smith, G., Yagoub, D., Tay, A. P., Pickford, R., Wilkins, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+scale+mass+spectrometry-based+identifications+of+enzyme-mediated+protein+methylation+are+subject+to+high+false+discovery+rates.">Large scale mass spectrometry-based identifications of enzyme-mediated protein methylation are subject to high false discovery rates.</a> Molecular &amp; Cellular Proteomics. 15 (3), 989-1006 (2016).</li><li>Bedford, M. T., Clarke, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+arginine+methylation+in+mammals:+who,+what,+and+why.">Protein arginine methylation in mammals: who, what, and why.</a> Molecular Cell. 33 (1), 1-13 (2009).</li><li>Larsen, S. C., 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=Proteome-wide+analysis+of+arginine+monomethylation+reveals+widespread+occurrence+in+human+cells.">Proteome-wide analysis of arginine monomethylation reveals widespread occurrence in human cells.</a> Science Signaling. 9 (443), (2016).</li><li>Massignani, E., 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=hmSEEKER:+Identification+of+hmSILAC+doublets+in+MaxQuant+output+data.">hmSEEKER: Identification of hmSILAC doublets in MaxQuant output data.</a> Proteomics. 19 (5), 1800300 (2019).</li><li>Bradford, M. M. <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+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Analytical Biochemistry. 72, 248-254 (1976).</li><li>Smith, P. K., 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=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Analytical Biochemistry. 150 (1), 76-85 (1985).</li><li>Getz, E. B., Xiao, M., Chakrabarty, T., Cooke, R., Selvin, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+between+the+sulfhydryl+reductants+tris(2-carboxyethyl)phosphine+and+dithiothreitol+for+use+in+protein+biochemistry.">A comparison between the sulfhydryl reductants tris(2-carboxyethyl)phosphine and dithiothreitol for use in protein biochemistry.</a> Analytical Biochemistry. 273 (1), 73-80 (1999).</li><li>Nielsen, M. L., 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=Iodoacetamide-induced+artifact+mimics+ubiquitination+in+mass+spectrometry.">Iodoacetamide-induced artifact mimics ubiquitination in mass spectrometry.</a> Nature Methods. 5 (6), 459-460 (2008).</li><li>Huesgen, P. F., 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=LysargiNase+mirrors+trypsin+for+protein+C-terminal+and+methylation-site+identification.">LysargiNase mirrors trypsin for protein C-terminal and methylation-site identification.</a> Nature Methods. 12 (1), 55-58 (2015).</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 (2007).</li><li>Hartel, N. G., Chew, B., Qin, J., Xu, J., Graham, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+protein+methylation+profiling+by+combined+chemical+and+immunoaffinity+approaches+reveals+novel+PRMT1+targets.">Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets.</a> Molecular &amp; Cellular Proteomics. 18 (11), 2149-2164 (2019).</li><li>Bremang, M., 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=Mass+spectrometry-based+identification+and+characterisation+of+lysine+and+arginine+methylation+in+the+human+proteome.">Mass spectrometry-based identification and characterisation of lysine and arginine methylation in the human proteome.</a> Molecular bioSystems. 9 (9), 2231-2247 (2013).</li><li>Geoghegan, V., Guo, A., Trudgian, D., Thomas, B., Acuto, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+identification+of+arginine+methylation+in+primary+T+cells+reveals+regulatory+roles+in+cell+signalling.">Comprehensive identification of arginine methylation in primary T cells reveals regulatory roles in cell signalling.</a> Nature Communications. 6 (1), 1-8 (2015).</li><li>Tabb, D. L., Huang, Y., Wysocki, V. H., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+basic+residue+content+on+fragment+ion+peak+intensities+in+low-energy+collision-induced+dissociation+spectra+of+peptides.">Influence of basic residue content on fragment ion peak intensities in low-energy collision-induced dissociation spectra of peptides.</a> Analytical Chemistry. 76 (5), 1243-1248 (2004).</li><li>Guthals, A., Bandeira, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+identification+by+tandem+mass+spectrometry+with+alternate+fragmentation+modes.">Peptide identification by tandem mass spectrometry with alternate fragmentation modes.</a> Molecular &amp; Cellular Proteomics. 11 (9), 550-557 (2012).</li><li>Swaney, D. L., 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=Supplemental+activation+method+for+high-efficiency+electron-transfer+dissociation+of+doubly+protonated+peptide+precursors.">Supplemental activation method for high-efficiency electron-transfer dissociation of doubly protonated peptide precursors.</a> Analytical Chemistry. 79 (2), 477-485 (2007).</li><li>Kundinger, S. R., Bishof, I., Dammer, E. B., Duong, D. M., Seyfried, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle-down+proteomics+reveals+dense+sites+of+methylation+and+phosphorylation+in+arginine-rich+RNA-binding+proteins.">Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins.</a> Journal of Proteome Research. 19 (4), 1574-1591 (2020).</li><li>Batth, T. S., Francavilla, C., Olsen, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off-line+high-pH+reversed-phase+fractionation+for+in-depth+phosphoproteomics.">Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics.</a> Journal of Proteome Research. 13 (12), 6176-6186 (2014).</li><li>Yang, F., Shen, Y., Camp, D. G., Smith, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-pH+reversed-phase+chromatography+with+fraction+concatenation+for+2D+proteomic+analysis.">High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis.</a> Expert Review of Proteomics. 9 (2), 129-134 (2012).</li><li>Hartel, N. G., Chew, B., Qin, J., Xu, J., Graham, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+protein+methylation+profiling+by+combined+chemical+and+immunoaffinity+approaches+reveals+novel+PRMT1+targets.">Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets.</a> Molecular &amp; Cellular Proteomics. 18 (11), 2149-2164 (2019).</li><li>Uhlmann, T., 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+method+for+large-scale+identification+of+protein+arginine+methylation.">A method for large-scale identification of protein arginine methylation.</a> Molecular &amp; Cellular Proteomics. 11 (11), 1489-1499 (2012).</li><li>Wang, K., 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=Antibody-free+approach+for+the+global+analysis+of+protein+methylation.">Antibody-free approach for the global analysis of protein methylation.</a> Analytical chemistry. 88 (23), 11319-11327 (2016).</li><li>Moore, K. E., 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+general+molecular+affinity+strategy+for+global+detection+and+proteomic+analysis+of+lysine+methylation.">A general molecular affinity strategy for global detection and proteomic analysis of lysine methylation.</a> Molecular cell. 50 (3), 444-456 (2013).</li><li>Wu, Z., 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+chemical+proteomics+approach+for+global+analysis+of+lysine+monomethylome+profiling.">A chemical proteomics approach for global analysis of lysine monomethylome profiling.</a> Molecular &amp; Cellular Proteomics. 14 (2), 329-339 (2015).</li><li>Sylvestersen, K. B., Horn, H., Jungmichel, S., Jensen, L. J., Nielsen, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+arginine+methylation+sites+in+human+cells+reveals+dynamic+regulation+during+transcriptional+arrest.">Proteomic analysis of arginine methylation sites in human cells reveals dynamic regulation during transcriptional arrest.</a> Molecular &amp; Cellular Proteomics. 13 (8), 2072-2088 (2014).</li><li>Tay, A. P., Geoghegan, V., Yagoub, D., Wilkins, M. R., Hart-Smith, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MethylQuant:+A+Tool+for+Sensitive+Validation+of+Enzyme-Mediated+Protein+Methylation+Sites+from+Heavy-Methyl+SILAC+Data.">MethylQuant: A Tool for Sensitive Validation of Enzyme-Mediated Protein Methylation Sites from Heavy-Methyl SILAC Data.</a> Journal of Proteome Research. 17 (1), 359-373 (2018).</li></ol>

由于存在高FDR的风险，通过全球基于MS的蛋白质组学对 体内 蛋白质/肽甲基化进行高置信度鉴定具有挑战性，在样品制备过程中会发生几种氨基酸取代和甲酯化，这些氨基酸取代和甲酯化与甲基化同量异位，并且在没有正交MS验证策略的情况下可能导致错误的分配。该PTM的亚化学计量性质使全球甲基蛋白质组学的任务进一步复杂化，但可以通过选择性富集修饰肽10来克服。<...

精氨酸（R）-甲基化是一种翻译后修饰（PTM），可装饰约1%的哺乳动物蛋白质组1。蛋白精氨酸甲基转移酶（PRMTs）是通过一个或两个甲基以对称或不对称方式沉积到R侧链胍基的氮（N）原子上来催化R-甲基化反应的酶。在哺乳动物中，PRMT可分为三类 - I型，II型和III型 - 取决于它们沉积单甲基化（MMA）和不对称二甲基化（ADMA），MMA和对称二甲基化（SDMA）或仅MMA的能力，分别2，3。PRMT主要靶向位于富含甘氨酸和精氨酸的区域的R残基，称为GAR基序，但一些PRMTs，如PRMT5和CARM1，可以将脯氨酸-甘氨酸-富含蛋氨酸（PGM）基序4甲基化。R-甲基化已成为几种生物过程的蛋白质调节剂，例如RNA剪接5，DNA修复6，miRNA生物发生7和翻译2，促进了对该PTM的研究。
质谱 （MS） 被认为是以蛋白质、肽和位点分辨率系统研究全局 R-甲基化的最有效技术。但是，该PTM需要一些特殊的预防措施才能通过MS进行高置信度鉴定。首先，R-甲基化是亚化学计量的，肽的未修饰形式比修饰的肽丰富得多，因此在数据依赖性采集（DDA）模式下运行的质谱仪将比低强度甲基化对应物更频繁地破碎高强度未修饰肽8。此外，大多数用于R-甲基化位点鉴定的基于MS的工作流程在生物信息学分析水平上存在局限性。事实上，甲基肽的计算鉴定容易产生高错误发现率（FDR），因为这种PTM与各种氨基酸取代（例如，甘氨酸转化为丙氨酸）和化学修饰（例如天冬氨酸和谷氨酸的甲酯化）同量异位9。因此，基于甲基同位素标记的方法，例如细胞培养中氨基酸重甲基稳定同位素标记（hmSILAC），已被实施为正交策略，用于 体内甲基化的 可靠MS鉴定，显着降低了假阳性注释的发生率10。
最近，研究R-甲基化蛋白质的各种蛋白质组范围方案已经优化。基于抗体的R-甲基肽免疫亲和富集策略的开发导致了人类细胞中数百个R-甲基化位点的注释11，12。此外，许多研究3，13报告说，将基于抗体的富集与肽分离技术（如HpH-RP色谱分级分离）偶联可以增加鉴定的甲基肽总数。
本文介绍了一种基于各种生化和分析步骤的系统、高置信度鉴定人细胞中 R-甲基化位点的实验策略：从 hmSILAC 标记的细胞中提取蛋白质，使用胰蛋白酶和 LysargiNase 蛋白酶进行平行双酶消化，然后对消化肽进行 HpH-RP 色谱分级分离，再加上基于抗体的 MMA-、SDMA-、 和含ADMA的肽。然后，在DDA模式下通过高分辨率液相色谱（LC）-MS/MS对所有亲和富集肽进行分析，并使用MaxQuant算法处理原始MS数据以鉴定R-甲基肽。最后，使用hmSEEKER处理MaxQuant输出结果，hmSEEKER是一种内部开发的生物信息学工具，用于搜索重质和轻质甲基肽对。简而言之，hmSEEKER从msms文件中读取并过滤甲基肽鉴定，然后将每个甲基肽与其在allPeptides文件中相应的MS1峰相匹配，最后搜索重肽/轻肽对应物的峰。对于每个假定的重光对，计算Log2 H/L比（LogRatio）、保留时间差（dRT）和质量误差（ME）参数，并将位于用户定义的截止值内的双峰标记为真阳性。生化方案的工作流程如图 1所示。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Ammonium Bicarbonate (AMBIC)</td>
			<td>Sigma-Aldrich</td>
			<td>09830</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Sigma-Aldrich</td>
			<td>497363</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 Sep-Pak columns vacc 6cc (1g)</td>
			<td>Waters</td>
			<td>WAT036905</td>
			<td></td>
		</tr>
		<tr>
			<td>Colloidal Coomassie staining Instant</td>
			<td>Sigma-Aldrich</td>
			<td>ISB1L-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete Mini, EDTA-free</td>
			<td>Roche-Sigma Aldrich</td>
			<td>11836170001</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Dialyzed Fetal Bovine Serum (FBS)</td>
			<td>GIBCO ThermoFisher</td>
			<td>26400-044</td>
			<td></td>
		</tr>
		<tr>
			<td>DL-Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>3483-12-3</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM Medium</td>
			<td>GIBCO ThermoFisher</td>
			<td>requested</td>
			<td>&#160;with stabile glutamine and without methionine</td>
		</tr>
		<tr>
			<td>EASY-nano LC 1200 chromatography system</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EASY-Spray HPLC Columns</td>
			<td>ThermoFisher</td>
			<td>ES907</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerolo</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>ATCC CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide (IAA)</td>
			<td>Sigma-Aldrich</td>
			<td>144-48-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Jupiter C12-RP column</td>
			<td>Phenomenex</td>
			<td>00G-4396-E0</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M5308</td>
			<td>Light (L) Methionine</td>
		</tr>
		<tr>
			<td>L-Methionine-(methyl-13C,d3)</td>
			<td>Sigma-Aldrich</td>
			<td>299154</td>
			<td>Heavy (H) Methionine</td>
		</tr>
		<tr>
			<td>LysargiNase</td>
			<td>Merck Millipore</td>
			<td>EMS0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtip Cell Disruptor Sonifier 250</td>
			<td>Branson</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>GIBCO ThermoFisher</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP</td>
			<td>Roche-Sigma Aldrich</td>
			<td>4906837001</td>
			<td>Phosphatase Inhibitor</td>
		</tr>
		<tr>
			<td>Pierce C18 Tips</td>
			<td>ThermoFisher</td>
			<td>87782</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160; 0.1% Formic Acid (v/v) in Acetonitrile, LC-MS Grade</td>
			<td>ThermoFisher</td>
			<td>85175</td>
			<td>LC-MS Solvent B</td>
		</tr>
		<tr>
			<td>Pierce&#160; 0.1% Formic Acid (v/v) in Water, LC-MS Grade</td>
			<td>ThermoFisher</td>
			<td>85170</td>
			<td>LC-MS Solvent A</td>
		</tr>
		<tr>
			<td>Pierce&#160; Acetonitrile (ACN), LC-MS Grade</td>
			<td>ThermoFisher</td>
			<td>51101</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce&#160; Water, LC-MS Grade</td>
			<td>ThermoFisher</td>
			<td>51140</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>92560</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein&#160; All Blue Prestained Protein Standards</td>
			<td>Bio-Rad</td>
			<td>1610373</td>
			<td></td>
		</tr>
		<tr>
			<td>PTMScan antibodies &#945;-ADMA</td>
			<td>Cell Signaling Technology</td>
			<td>13474</td>
			<td></td>
		</tr>
		<tr>
			<td>PTMScan antibodies &#945;-MMA</td>
			<td>Cell Signaling Technology</td>
			<td>12235</td>
			<td></td>
		</tr>
		<tr>
			<td>PTMScan antibodies &#945;-SDMA</td>
			<td>Cell Signaling Technology</td>
			<td>13563</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequencing Grade Modified Trypsin</td>
			<td>Promega</td>
			<td>V5113</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultimate 3000 HPLC</td>
			<td>Dionex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Concentrator 5301</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Speed vac</td>
		</tr>
	</tbody>
</table>

a mass spectrometry-based proteomics approach for global and high-confidence protein r-methylation analysis

蛋白精氨酸（R）-甲基化是一种广泛的蛋白质翻译后修饰（PTM），参与多种细胞途径的调节，包括RNA加工，信号转导，DNA损伤反应，miRNA生物发生和翻译。
近年来，由于生化和分析的发展，基于质谱（MS）的蛋白质组学已成为以单位点分辨率表征细胞甲基蛋白质组的最有效策略。然而，通过MS鉴定和分析 体内 蛋白质R-甲基化仍然具有挑战性且容易出错，主要是由于这种修饰的亚化学计量性质以及存在各种氨基酸取代和酸性残基的化学甲酯化，这些残基与甲基化同量异位。因此，需要富集方法来增强R-甲基肽的鉴定和正交验证策略，以降低甲基蛋白质组学研究中的错误发现率（FDR）。
本文描述了一种专门为细胞样品中的高置信度R-甲基肽鉴定和定量而设计的方案，该方案将细胞的代谢标记与重同位素编码的蛋氨酸（hmSILAC）和双蛋白酶全细胞提取物的溶液内消化相结合，然后使用抗泛-R-甲基抗体对R-甲基肽进行离线高pH反相（HpH-RP）色谱分级分离和亲和富集。在高分辨率MS分析后，首先使用MaxQuant软件包处理原始数据，然后通过hmSEEKER分析结果，hmSEEKER是一款设计用于深入搜索MaxQuant输出文件中对应于轻质和重质甲基肽的MS峰对的软件。

本文介绍了一种高置信度鉴定全局蛋白R-甲基化的工作流程，该工作流程基于蛋白质提取物的酶消化与两种不同的蛋白酶并行结合，然后对蛋白水解肽进行HpH-RP液相色谱分级分离，以及使用抗泛-R-甲基抗体对R-甲基肽进行免疫亲和富集（图1）。
细胞在蛋氨酸存在下生长，蛋氨酸可以是天然的（Light，L，Met-0）或同位素标记的（重，H，Met-4）。在完全同位?...

Watch this Scientific Journal Video about A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis at JoVE.com

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

蛋白精氨酸（R）-甲基化是一种广泛的翻译后修饰，调节多种生物学途径。质谱法是全球分析R-甲基蛋白质组的最佳技术，当与修饰肽富集的生化方法相结合时。本文介绍了专为高置信度鉴定人细胞中全球R-甲基化而设计的工作流程。

基于质谱的蛋白质组学方法用于全局和高置信度的蛋白质R-甲基化分析

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

该协议非常重要，因为它代表了通过质谱分析蛋白质精氨酸甲基化的全球最先进的工作流程。这是以单一风格分辨率鉴定和分析R-甲基化蛋白质的唯一方法，这是其他生化技术无法实现的。当与PRMT抑制剂的使用相结合时，其中一些抑制剂正在临床试验和抗癌药物中，该协议允许深入分析其作用机制。首先，使用溶解在终浓度为4.5毫摩尔的超纯水中的DTT储备溶液还原蛋白质的硫醇基团，并在55摄氏度下反应30分钟。通过加入浓度为10毫摩尔的碘乙酰胺并在室温下在黑暗中孵育15分钟来进行蛋白质的硫醇基团的烷基化。为了验证蛋白水解效率，请保存等分试样的蛋白质提取物，以便在SDS-PAGE库马西染色凝胶上进行后续分析，并在消化时将其与相应量的样品进行比较。用四体积的 20 毫摩尔 HEPES 在 pH 8.0 下稀释剩余的蛋白质提取物，以达到 2 摩尔的最终尿素浓度。将样品分成两部分，然后将测序级修饰胰蛋白酶添加到一部分，将LysargiNase蛋白酶添加到另一部分。将样品在37摄氏度下以每分钟600转的速度在热混合器中过夜，以进行酶消化。对于每个色谱梯度，将所有级分收集到深 96 孔板中。将梯度开始前收集的分数合并为一个名为pre的单个分数。将高pH反相液相色谱梯度中的60个馏分连接起来，以不连续的方式将它们合并成14个最终馏分。为了获得非连续的串联，如文本手稿中所述，合并高pH反相部分。将梯度后收集的分数合并为一个名为 post 的唯一分数。分别对来自胰蛋白酶和赖氨酸酶消化的两个样品进行修饰肽的顺序免疫亲和富集。将10X浓缩免疫亲和纯化或IAP缓冲液稀释10倍。将冻干肽以2， 000 G离心五分钟，将肽旋转到管底部。每15毫升管用250微升稀释的IAP缓冲液重悬肽，并转移到1.5毫升低结合管中。使用石蕊纸检查pH值是否大于6。保留每个馏分的一小部分等分试样作为后续MS分析的输入。将每个部分分成两部分，以平行进行不对称二甲基化（ADMA）和对称二甲基化（SDMA）肽的免疫富集。每 10 毫克初始蛋白质提取物制备三瓶与蛋白 A 琼脂糖珠偶联的选定抗泛 R 甲基化抗体。通过将每个小瓶以2， 000 G离心30秒并从磁珠中除去缓冲液，制备与琼脂糖珠偶联的正确量的抗体。用一毫升1X PBS洗涤珠子三次，以2， 000 G离心30秒。最后一次洗涤后，将珠子重悬于每个小瓶的40微升1X PBS中，将它们合并，然后最终将它们平均分成16个部分。向每个试管中加入250微升1X IAP缓冲液并通过倒置混合。然后将试管在旋转轮上以四摄氏度孵育两个小时。孵育后，将含有肽的1.5毫升管和pan R-甲基抗体偶联珠以2， 000 G离心30秒以沉淀珠子并将每个级分的流过转移到干净的1.5毫升低结合管中。将磁珠加入与R-mono甲基化抗体偶联的流通液中，并重复重悬到PBS中，用IAP缓冲液孵育并离心。在用单甲基化或MMA珠孵育肽样品期间，用250微升IAP缓冲液洗涤先前用抗ADMA和SDMA免疫沉淀的级分两次，并在每次洗涤后弃去上清液。然后用LC-MS级水清洗三次。通过向每个试管中加入 50 微升 0.15 TFA，从琼脂糖珠中洗脱亲和富集的 SDMA 或 ADMA 肽。将此溶液在室温下放置10分钟，每两到三分钟倒置一次管。将第一次洗脱转移到干净的1.5毫升低结合管中，并用50微升0.15TFA重复洗脱。将两个洗脱级分汇集到一个管中。对与抗MMA抗体珠一起孵育的R-单甲基化肽重复此过程。混合轻标记细胞和重标记细胞后，提取蛋白质并通过胰蛋白酶和赖氨酸酶进行消化。使用SDS-PAGE库马斯染色凝胶验证肽中总蛋白的有效酶消化。评估了C18 Sep-Pak色谱柱纯化步骤的效率，确认了C18色谱柱的流出以及第一次和第二次洗涤中不存在肽，以及它们在淋洗液中的预期存在。评估了重通道中适当的MET4掺入以及正确的重或轻混合。此处显示了离线高pH反相液相色谱肽分馏和随后馏分的非连续级分串联的色谱图。通过250纳米紫外线检测肽，同时通过280纳米紫外线评估潜在未消化的蛋白质。获得了代表真阳性甲基肽注释的肽的完整MS谱图。在三个峰之间观察到的M在Z上的差异与酶甲基化残基的存在一致。获得了代表假阳性甲基肽注释的肽的完整MS谱图。在轻甲基化肽与其惩罚性重对应物之间观察到的M在Z上的差异偏离预期值0.0312。协议工作流程总体上是线性的，但有几个步骤需要特别注意。例如，IPH色谱分离与非连续馏分串联，以及IP设置。相同的方案可以与标准SILAC或无标记定量相结合，以分析精氨酸甲基化动力学，以响应各种扰动。该协议为表征几种细胞类型和模型系统中蛋白质甲基化的程度和动力学铺平了道路，支持专注于PRMT的基础和转化研究的各个方面。

Research

JoVE Journal

Biochemistry

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 ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

脂滴隔离定量质谱分析

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

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 ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

蛋白质消化、超滤和尺寸排除色谱法优选人血浆和血清外来体的分离

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Preparation of Chloroplast Sub-compartments from Arabidopsis for the Analysis of Protein Localization by Immunoblotting or Proteomics

拟南芥叶绿体子隔层的制备及免疫印迹蛋白质组化分析

Chloroplasts are major components of plant cells. Such plastids fulfill many crucial functions, such as assimilation of carbon, sulfur and nitrogen as ...

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 ...

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 ...