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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

Published: April 28, 2022
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Özet

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。

Abstract

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。

Introduction

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23。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1112。此外,许多研究313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。

Protocol

1. 细胞培养和蛋白质提取(时间:需要3-4周) 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 <…

Representative Results

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?…

Discussion

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。<…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

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Bu Makaleden Alıntı Yapın
Maniaci, M., Marini, F., Massignani, E., Bonaldi, T. A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis. J. Vis. Exp. (182), e62409, doi:10.3791/62409 (2022).

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