Özet

使用胺还原甲基化进行样品制备和相对定量,用于肽学研究

Published: November 04, 2021
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Özet

本文介绍了一种基于热灭活的样品制备方法,以保存内源性肽,避免死后降解,然后使用同位素标记和LC-MS进行相对定量。

Abstract

肽学可以定义为生物样品中肽的定性和定量分析。其主要应用包括鉴定疾病或环境应激的肽生物标志物,鉴定神经肽,激素和生物活性细胞内肽,从蛋白质水解物中发现抗菌和营养保健肽,并可用于研究以了解蛋白水解过程。最近在样品制备,分离方法,质谱技术和与蛋白质测序相关的计算工具方面的进步有助于增加已鉴定的肽数量和所表征的肽。肽研究经常分析细胞中自然产生的肽。这里描述了一种基于热灭活的样品制备方案,其消除了蛋白酶活性,并且在温和的条件下提取,因此没有肽键裂解。此外,还显示了通过胺的还原甲基化使用稳定同位素标记的肽的相对定量。这种标记方法具有一些优点,因为试剂是市售的,与其他试剂相比价格低廉,化学稳定,并且允许在单次LC-MS运行中分析多达五个样品。

Introduction

“组学”科学的特点是对分子集进行深入分析,例如DNA,RNA,蛋白质,肽,代谢物等。这些生成的大规模数据集(基因组学、转录组学、蛋白质组学、肽学、代谢组学等)彻底改变了生物学,并导致了对生物过程的高级理解1。肽学一词在20世纪初 开始引入,一些作者将其称为蛋白质组学的一个分支2。然而,肽学具有独特的特殊性,其主要兴趣是研究细胞过程中自然产生的肽含量,以及这些分子的生物活性的表征34

最初,生物活性肽研究仅限于通过Edman降解和放射免疫测定的神经肽和激素肽。然而,这些技术不允许进行全局分析,这取决于高浓度中每种肽的分离,抗体产生的时间,以及交叉反应性的可能性5

只有在液相色谱耦合质谱(LC-MS)和基因组项目取得多项进展后,肽学分析才成为可能,这些项目为蛋白质组学/肽学研究提供了全面的数据池67。此外,需要建立针对肽酶的特定肽提取方案,因为第一批分析大脑样本中全球神经肽的研究表明,检测受到蛋白质大规模降解的影响,蛋白质在死后1分钟后主要发生在该组织中。这些肽片段的存在掩盖了神经肽信号,并不代表 体内的肽组。这个问题主要通过使用微波照射对蛋白酶进行快速加热灭活来解决,这大大减少了这些伪影片段的存在,不仅允许鉴定神经肽片段,而且揭示了来自胞质,线粒体和核蛋白的一组肽的存在,与降解689不同。

这些方法论程序允许肽组扩展到众所周知的神经肽之外,其中主要由蛋白酶体作用产生的数百种细胞内肽已在酵母10,斑马鱼11,啮齿动物组织12和人类细胞中鉴定出来13。数十种这些细胞内肽已被广泛证明具有生物学和药理活性1415。此外,这些肽可用作疾病生物标志物,并可能具有临床意义,如颅内囊性动脉瘤患者的脑脊液16所示。

目前,除了肽序列的鉴定外,还可以通过质谱法获得绝对和相对定量的数据。在绝对定量中,将生物样品中的肽水平与合成标准品进行比较,而在相对定量中,比较两个或多个样品中的肽水平17。可以使用以下方法进行相对定量:1)”无标签”18;2) 体内 代谢标记或3)化学标记。最后两个是基于使用掺入肽中的稳定同位素形式1920。在无标记分析中,通过考虑LC-MS18期间的信号强度(光谱计数)来估计肽水平。然而,同位素标记可以获得更准确的肽相对水平。

许多肽研究使用丁酸三甲基铵(TMAB)标记试剂作为化学标记,最近,胺的还原甲基化(RMA)与氘化和非氘代形式的甲醛和氰基硼氢化钠试剂已被使用112122。然而,TMAB标签不是市售的,合成过程非常费力。另一方面,在RMA中,试剂是市售的,与其他标记相比便宜,程序易于执行,并且标记的肽稳定2324

RMA的使用涉及通过允许肽与甲醛反应来形成希夫碱,然后通过氰基硼氢化物进行还原反应。该反应引起N端和赖氨酸侧链和单甲基化N端脯氨酸上的游离氨基的二甲基化。脯氨酸残基在N端通常很少见,几乎所有在N端具有游离胺的肽都标有两个甲基232425。

Protocol

以下肽提取和还原甲基化程序改编自先前发表的程序24,25,26,27。该协议遵循国家动物实验控制委员会(CONCEA)的指导方针,并得到圣保罗州立大学生物科学研究所动物使用伦理委员会(CEUA)的批准。协议步骤如图 1 所示。 注意:在超纯水中准备所有水?…

Representative Results

从质谱仪上进行的运行中获得的结果存储在原始数据文件中,可以在质谱仪软件中打开。在MS光谱中,可以根据所使用的标记方案观察代表标记肽的峰组,范围从2-5个标记。例如,在 图2中,在色谱时间内检测到的峰对在实验中表示,其中在同一次运行中,在两个不同的样品中仅使用了两个同位素标记。 图3 显示了在每次LC-MS运行中使用3个和4个不同标?…

Discussion

在大多数肽学研究中,毫无疑问,关键步骤之一是应仔细进行的样品制备,以避免在死后几分钟后存在蛋白酶产生的肽片段。对从非微波样品制备的脑提取物的初步研究表明,10 kDa微滤液中存在大量的蛋白质片段。已经描述了避免蛋白质降解肽光谱的不同方法:聚焦微波照射动物牺牲68,冷冻器解剖,然后煮沸提取缓冲液31,</sup…

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

本文所述技术的开发和使用得到了巴西国家研究委员会420811/2018-4(LMC)赠款的支持;圣保罗国家保护基金会(www.fapesp.br)赠款2019/16023-6(LMC),2019/17433-3(LOF)和21/01286-1(MEME)。资助者在研究设计,数据收集和分析,发表决定或准备文章方面没有任何作用。

Materials

10 kDa cut-off filters Merck Millipore UFC801024 Amicon Ultra-4, PLGC Ultracel-PL Membrane, 10 kDa
Acetone Sigma-Aldrich 179124
Acetonitrile Sigma-Aldrich 1000291000
Ammonium bicarbonate Sigma-Aldrich 11213
analytical column (EASY-Column) EASY-Column (SC200)  10 cm, ID75 µm, 3 µm, C18-A2
Ethyl 3-aminobenzoate methanesulfonate Sigma-Aldrich E10521 MS-222
Fluorescamine Sigma-Aldrich F9015
Formaldehyde solution Sigma-Aldrich 252549
Formaldehyde-13C, d2, solution Sigma-Aldrich 596388
Formaldehyde-d2 solution Sigma-Aldrich 492620
Formic acid Sigma-Aldrich 33015
Fume hood Quimis Q216
Hydrochloric acid – HCl Sigma-Aldrich 258148
LoBind-Protein retention tubes Eppendorf EP0030108116-100EA
LTQ-Orbitrap Velos Thermo Fisher Scientific LTQ Velos
Microwave oven Panasonic NN-ST67HSRU
n Easy-nLC II nanoHPLC Thermo Fisher Scientific LC140
PEAKS Studio Bioinformatics Solutions Inc. VERSION 8.5
Phosphate-buffered saline Invitrogen 3002 tablets
precolumn (EASY-Column) Thermo Fisher Scientific (SC001) 2 cm, ID100 µm, 5 µm, C18-A1
Refrigerated centrifuge Hermle Z326K for conical tubes
Refrigerated centrifuge Vision VS15000CFNII for microtubes
Reversed-phase cleanup columns   (Oasis HLB 1 cc Cartridge) Waters 186000383 Oasis HLB 1 cc Cartridge
Sodium cyanoborodeuteride – NaBD3CN Sigma-Aldrich 190020
Sodium cyanoborohydride – NaBH3CN Sigma-Aldrich 156159
Sodium phosphate dibasic Sigma-Aldrich S9763 NOTE: 0.2 M PB= 0.1 M phosphate buffer pH 6.8 (26.85 mL of Na2HPO3 1M) plus 0.1 M phosphate buffer pH 6.8 (23.15 mL of NaH2PO3 1M) to 250 ml of water
Sodium phosphate monobasic Sigma-Aldrich S3139
Sonicator Qsonica Q55-110
Standard peptide Proteimax amino acid sequence: LTLRTKL
Triethylammonium buffer – TEAB 1 M Sigma-Aldrich T7408
Trifluoroacetic acid – TFA Sigma-Aldrich T6508
Ultra purified water Milli-Q Direct-Q 3UV
Vacuum centrifuge GeneVac MiVac DNA concentrator
Water bath Cientec 266
Xcalibur Software ThermoFisher Scientific OPTON-30965

Referanslar

  1. Kandpal, R., Saviola, B., Felton, J. The era of ‘omics unlimited. Biotechniques. 46 (5), 354-355 (2009).
  2. Farrokhi, N., Whitelegge, J. P., Brusslan, J. A. Plant peptides and peptidomics. Plant Biotechnology Journal. 6 (2), 105-134 (2008).
  3. Schulz-Knappe, P., Schrader, M., Zucht, H. D. The peptidomics concept. Combinatorial Chemistry & High Throughput Screening. 8 (8), 697-704 (2005).
  4. Dallas, D. C., et al. Current peptidomics: applications, purification, identification, quantification, and functional analysis. Proteomics. 15 (5-6), 1026-1038 (2015).
  5. Chard, T. An introduction to radioimmunoassay and related techniques (3rd Ed). FEBS Letters. 238 (1), 223 (1988).
  6. Svensson, M., Sköld, K., Svenningsson, P., Andren, P. E. Peptidomics-based discovery of novel neuropeptides. Journal of Proteome Research. 2 (2), 213-219 (2003).
  7. Baggerman, G., et al. Peptidomics. Journal of Chromatography B. 803, 3-16 (2004).
  8. Theodorsson, E., Stenfors, C., Mathe, A. A. Microwave irradiation increases recovery of neuropeptides from brain tissues. Peptides. 11, 1191-1197 (1990).
  9. Che, F. Y., Lim, J., Pan, H., Biswas, R., Fricker, L. D. Quantitative neuropeptidomics of microwave-irradiated mouse brain and pituitary. Molecular & Cellular Proteomics. 4, 1391-1405 (2005).
  10. Dasgupta, S., et al. Analysis of the yeast peptidome and comparison with the human peptidome. PLoS One. 11 (9), 0163312 (2016).
  11. Teixeira, C. M. M., Correa, C. N., Iwai, L. K., Ferro, E. S., Castro, L. M. Characterization of Intracellular Peptides from Zebrafish (Danio rerio) Brain. Zebrafish. 16 (3), 240-251 (2019).
  12. Fricker, L. D. Analysis of mouse brain peptides using mass spectrometry-based peptidomics: implications for novel functions ranging from non-classical neuropeptides to microproteins. Molecular BioSystems. 6 (8), 1355-1365 (2010).
  13. Gelman, J. S., Sironi, J., Castro, L. M., Ferro, E. S., Fricker, L. D. Peptidomic analysis of human cell lines. Journal of Proteome Research. 10 (4), 1583-1592 (2011).
  14. De Araujo, C. B., et al. Intracellular peptides in cell biology and pharmacology. Biomolecules. 9, 150 (2019).
  15. Gewehr, M. C. F., Silverio, R., Rosa-Neto, J. C., Lira, F. S., Reckziegel, P., Ferro, E. S. Peptides from natural or rationally designed sources can be used in overweight, obesity, and type 2 diabetes therapies. Molecules. 25 (5), 1093 (2020).
  16. Sakaya, G. R., et al. Peptidomic profiling of cerebrospinal fluid from patients with intracranial saccular aneurysms. Journal of Proteomics. 240 (3), 104188 (2021).
  17. Fricker, L. Quantitative peptidomics: General considerations. Methods in Molecular Biology. 1719, 121-140 (2018).
  18. Southey, B. R., et al. Comparing label-free quantitative peptidomics approaches to characterize diurnal variation of peptides in the rat suprachiasmatic nucleus. Analytical Chemistry. 86 (1), 443-452 (2014).
  19. Chen, X., Wei, S., Ji, Y., Guo, X., Yang, F. Quantitative proteomics using SILAC: Principles, applications, and developments. Proteomics. 15 (18), 3175-3192 (2015).
  20. Boonen, K., et al. Quantitative peptidomics with isotopic and isobaric tags. Methods in Molecular Biology. 1719, 141-159 (2018).
  21. Gewehr, M. C. F., et al. The relevance of thimet oligopeptidase in the regulation of energy metabolism and diet-induced obesity. Biomolecules. 10 (2), 321 (2020).
  22. Fiametti, L. O., Correa, C. N., Castro, L. M. Peptide profile of zebrafish brain in a 6-OHDA-induced Parkinson model. Zebrafish. 18 (1), 55-65 (2021).
  23. Boersema, P. J., Raijmakers, R., Lemeer, S., Mohammed, S., Heck, A. J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nature Protocols. 4 (4), 484-494 (2009).
  24. Dasgupta, S., Castro, L. M., Tashima, A. K., Fricker, L. Quantitative peptidomics using reductive methylation of amines. Methods in Molecular Biology. 1719, 161-174 (2018).
  25. Tashima, A. K., Fricker, L. D. Quantitative peptidomics with five-plex reductive methylation labels. Journal of the American Society for Mass Spectrometry. 29 (5), 866-878 (2018).
  26. Che, F. Y., et al. Optimization of neuropeptide extraction from the mouse hypothalamus. Journal of Proteome Research. 6 (12), 4667-4676 (2007).
  27. Lyons, P. J., Fricker, L. D. Peptidomic approaches to study proteolytic activity. Current Protocols in Protein Science. , 13 (2011).
  28. Udenfriend, S., et al. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 178 (4063), 871-872 (1972).
  29. Ma, B., et al. PEAKS: powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Communications in Mass Spectrometry. 17 (20), 2337-2342 (2003).
  30. Zhang, J., et al. PEAKS DB: de novo sequencing assisted database search for sensitive and accurate peptide identification. Molecular & Cellular Proteomics. 11 (4), 1-8 (2012).
  31. Sturm, R. M., Dowell, J. A., Li, L. Rat brain neuropeptidomics: tissue collection, protease inhibition, neuropeptide extraction, and mass spectrometric analysis. Methods in Molecular Biology. 615, 217-226 (2010).
  32. Fricker, L. D. Limitations of mass spectrometry-based peptidomic approaches. Journal of the American Society for Mass Spectrometry. 26 (12), 1981-1991 (2015).
  33. Ross, , et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Molecular & Cellular Proteomics. 3, 1154-1169 (2004).
  34. Thompson, A., et al. Tandem mass tags: a novel quantification strategy for comparative analysis of complex protein mixtures by MS/MS. Analytical Chemistry. 75, 1895-1904 (2003).

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Bu Makaleden Alıntı Yapın
Correa, C. N., Fiametti, L. O., Mazzi Esquinca, M. E., Castro, L. M. d. Sample Preparation and Relative Quantitation using Reductive Methylation of Amines for Peptidomics Studies. J. Vis. Exp. (177), e62971, doi:10.3791/62971 (2021).

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