用等压标记、广泛的液相色谱法、质谱法和软件辅助定量技术进行深层蛋白质组分析
Summary
我们提出了一个协议, 以准确定量蛋白质与等压标签, 广泛的分馏, 生物信息学工具, 和质量控制步骤, 结合液相色谱与高分辨率质谱仪接口。
Abstract
在质谱 (ms) 的蛋白质组学中, 有许多特殊的进展, 特别是在液相色谱 (lc) 与串联质谱 (lc-ms) 和等压标记复用能力的技术进展。在这里, 我们引入了一个 deep-proteomics 的分析协议, 结合10丛串联质量标签 (TMT) 标签与广泛的 lc/lc/ms 平台, 和后 MS 计算干扰校正, 以准确定量整个蛋白质。该协议包括以下主要步骤: 蛋白质提取和消化, TMT 标签, 2 维 (2D) LC, 高分辨率质谱, 和计算数据处理。质量控制步骤包括在故障排除和评估实验变化。在哺乳动物样本中, 有超过1万的蛋白质可以自信地定量这个协议。这一协议也可以应用于量化的后平移修改与小的变化。这种多路复用、健壮的方法为蛋白质组分析提供了强有力的工具, 包括细胞培养、动物组织和人体临床标本。
Introduction
下一代测序技术的进步已经为研究生物系统和人类疾病带来了新的前景。这使得大量的基因组、转录、蛋白质、代谢和其他分子系统的测量变得有形。质谱 (MS) 是分析化学中最敏感的方法之一, 其在蛋白质组学中的应用在人类基因组测序之后迅速扩大。在蛋白质组学领域, 在过去的几年中取得了重大的技术进步 MS-based 定量分析, 包括等压标记和复用能力结合广泛的液相色谱法, 除了仪器仪表进展, 允许更快, 更准确的测量, 并要求较少的样品材料。定量蛋白质组学已经成为在高度复杂的生物样品中分析数以万计的蛋白质和后修饰的主流方法1,2,3,4,5,6。
多路等压标记方法, 如等压标签的相对和绝对定量 (即, iTRAQ) 和串联质量标签 (TMT) MS 大大提高了样本吞吐量, 并增加了样本数, 可以分析在一个单一的实验1,6,7,8。与其他 MS-based 定量方法, 例如无标签定量和稳定同位素标记与氨基酸在细胞培养 (即, SILAC), 这些技术的潜力在蛋白质组学领域是可观的9 ,10,11。例如, TMT 方法允许在1实验中使用10丛试剂对10蛋白质样品进行分析。这些结构相同的 TMT 标签具有相同的整体质量, 但重同位素是差异分布的碳或氮原子, 导致一个独特的记者离子在 ms/毫秒碎片的每个标签, 从而使相对定量在10样本之间TMT 策略通常用于研究生物通路、疾病进展和细胞过程12、13、14。
大量的技术改进有增强的液相色谱 (lc) –MS/ms 系统, 无论是在 LC 分离和 ms 参数, 以最大限度地提高蛋白质的鉴定, 而不牺牲定量的准确性。在这种类型的猎枪蛋白质组学方法中, 采用高正交分离技术对肽的一维分离是至关重要的, 从而达到最大结果20。高 pH 反相液相色谱 (色) 比传统的强阳离子交换色谱法20具有更好的性能。当 high-pH 色与 low-pH 色的第二维度相结合时, 分析的动态范围和蛋白质覆盖率都得到提高, 从而能够在执行全蛋白质组分析时识别表达的蛋白质的大部分15 ,16,17,18。其他技术进步包括小 C18 微粒 (1.9 µm) 和延长长的专栏 (~ 1 m)19。此外, 其他显著的改进包括具有快速扫描速率的质谱仪的新版本、改进的灵敏度和分辨率20以及用于 MS 数据挖掘的复杂生物信息学管道21。
在这里, 我们描述了一个详细的协议, 结合了最新的方法和修改, 以提高灵敏度和吞吐量, 同时侧重于质量控制机制的整个实验。该协议包括蛋白质提取和消化, TMT 10 丛标记, 碱性 ph 和酸 ph 色分馏, 高分辨率 ms 检测, 和 ms 数据处理 (图 1)。此外, 我们还实施了一些质量控制步骤, 用于诊断和评估实验变化。这一详细的协议旨在帮助研究人员在新的领域定期识别和准确定量数以千计的蛋白质从一个裂解或组织。
Protocol
警告: 请在使用前查阅所有相关的安全数据表 ( 即 、MSDS)。执行此协议时, 请使用所有适当的安全做法. 注意: 在本协议中, 一个 TMT 10 丛等压标签试剂集用于10样本的蛋白质组定量. 1. 细胞/组织的制备 注意: 在低温下收集样品以保持蛋白质的原始状态是至关重要的. 在 10 cm 板上两次用10毫升冰冷磷酸盐缓冲盐水 (PBS) …
Representative Results
我们使用先前描述的物种肽组合系统地分析了比压缩在3主要协议步骤中的影响, 包括前 ms 分馏、ms 设置和后 ms 校正23。采用碱性 ph 色和酸性 ph 色结合的方法对预 MS 分馏进行了评价和优化。对于后质谱分析, 只考虑 species-specific 肽。我们使用此干涉模型检查 lc/lc-ms/毫秒中的多个参数, 包括 MS2 隔离窗口、在线 lc 分辨率、在线酸性 pH 色的加载量以及脱机 hi…
Discussion
我们描述了一个高通量协议的蛋白质定量与10丛等压标记策略, 已经成功地实现了在几个出版物12,13,14,32.在本协议中, 我们可以在1实验中分析多达10种不同的生物蛋白样本。我们可以定期识别和定量超过1万的蛋白质, 并有很高的信心。虽然等压标记是一种有效的定量蛋白质的技术, 但它受比率压缩的限?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢所有其他实验室和设施成员进行有益的讨论。这项工作得到了 R01GM114260、R01AG047928、R01AG053987 和 ALSAC 的部分支持。MS 分析是在 St. 裘德儿童的研究医院蛋白质组学, 部分支持的 NIH 癌症中心支持补助金 P30CA021765。作者感谢莎 Badders 对编辑手稿的帮助。
Materials
| 1220 LC system | Agilent | G4288B | |
| 50% Hydroxylamine | Thermo Scientific | 90115 | |
| Acetonitrile | Burdick & Jackson | AH015-4 | |
| Bullet Blender | Next Advance | BB24-AU | |
| Butterfly Portfolio Heater | Phoenix S&T | PST-BPH-20 | |
| C18 tips | Harvard Apparatus | 74-4607 | |
| Dithiothreitol (DTT) | Sigma | D5545 | |
| DMSO | Sigma | 41648 | |
| Formic acid | Sigma | 94318 | |
| Fraction Collector | Gilson | FC203B | |
| Glass Beads | Next Advance | GB05 | |
| HEPES | Sigma | H3375 | |
| Iodoacetamide (IAA) | Sigma | I6125 | |
| Lys-C | Wako | 125-05061 | |
| Methanol | Burdick & Jackson | AH230-4 | |
| Pierce BCA Protein Assay kit | Thermo Scientific | 23225 | |
| Mass Spectrometer | Thermo Scientific | Q Exactive HF | |
| nanoflow UPLC | Thermo Scientific | Ultimate 3000 | |
| ReproSil-Pur C18 resin, 1.9um | Dr. Maisch GmbH | r119.aq.0003 | |
| Self Pck Columns | New Objective | PF360-75-15-N-5 | |
| Sodium deoxycholate | Sigma | 30970 | |
| Speedva | Thermo Scientific | SPD11V | |
| TMT 10plex Isobaric label reagent | Thermo Scientific | 90110 | |
| Trifluoroacetic acid (TFA) | Applied Biosystems | 400003 | |
| Trypsin | Promega | V511C | |
| Urea | Sigma | U5378 | |
| Xbridge Column C18 column | Waters | 186003943 | |
| Ziptips C18 | Millipore | ZTC18S096 | |
| SepPak 1cc 50mg | Waters | WAT054960 |
References
- Pagala, V. R., et al. Quantitative protein analysis by mass spectrometry. Methods Mol Biol. 1278, 281-305 (2015).
- Altelaar, A. F., Munoz, J., Heck, A. J. Next-generation proteomics: towards an integrative view of proteome dynamics. Nat Rev Genet. 14 (1), 35-48 (2013).
- Rauniyar, N., Yates, J. R. Isobaric labeling-based relative quantification in shotgun proteomics. J Proteome Res. 13 (12), 5293-5309 (2014).
- Ross, P. L., et al. Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Mol Cell Proteomics. 3 (12), 1154-1169 (2004).
- Aebersold, R., Mann, M. Mass-spectrometric exploration of proteome structure and function. Nature. 537 (7620), 347-355 (2016).
- Bai, B., et al. Deep Profiling of Proteome and Phosphoproteome by Isobaric Labeling. Extensive Liquid Chromatography, and Mass Spectrometry. Methods Enzymol. 585, 377-395 (2017).
- McAlister, G. C., et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal Chem. 84 (17), 7469-7478 (2012).
- Everley, R. A., Kunz, R. C., McAllister, F. E., Gygi, S. P. Increasing throughput in targeted proteomics assays: 54-plex quantitation in a single mass spectrometry run. Anal Chem. 85 (11), 5340-5346 (2013).
- Bai, B., et al. Integrated approaches for analyzing U1-70K cleavage in Alzheimer’s disease. J Proteome Res. 13 (11), 4526-4534 (2014).
- Bai, B., et al. U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease. Proc Natl Acad Sci U S A. 110 (41), 16562-16567 (2013).
- Thongboonkerd, V., LaBaer, J., Domont, G. B. Recent advances of proteomics applied to human diseases. J Proteome Res. 13 (11), 4493-4496 (2014).
- Churchman, M. L., et al. Efficacy of Retinoids in IKZF1-Mutated BCR-ABL1 Acute Lymphoblastic Leukemia. Cancer Cell. 28 (3), 343-356 (2015).
- Wang, X., et al. Joint mouse-human phenome-wide association to test gene function and disease risk. Nat Commun. 7, 10464 (2016).
- Mertz, J. L., et al. Sequential elution interactome analysis of the Mind bomb 1 ubiquitin ligase reveals a novel role in dendritic spine outgrowth. Mol Cell Proteomics. 14 (7), 1898-1910 (2015).
- Yang, F., Shen, Y., Camp, D. G., Smith, R. D. High-pH reversed-phase chromatography with fraction concatenation for 2D proteomic analysis. Expert Rev Proteomics. 9 (2), 129-134 (2012).
- Wang, H., et al. An off-line high pH reversed-phase fractionation and nano-liquid chromatography-mass spectrometry method for global proteomic profiling of cell lines. J Chromatogr B Analyt Technol Biomed Life Sci. 974, 90-95 (2015).
- Batth, T. S., Francavilla, C., Olsen, J. V. Off-line high-pH reversed-phase fractionation for in-depth phosphoproteomics. J Proteome Res. 13 (12), 6176-6186 (2014).
- Song, C., et al. Reversed-phase-reversed-phase liquid chromatography approach with high orthogonality for multidimensional separation of phosphopeptides. Anal Chem. 82 (1), 53-56 (2010).
- Wang, H., et al. Systematic optimization of long gradient chromatography mass spectrometry for deep analysis of brain proteome. J Proteome Res. 14 (2), 829-838 (2015).
- Hebert, A. S., et al. The one hour yeast proteome. Mol Cell Proteomics. 13 (1), 339-347 (2014).
- Wang, X., et al. JUMP: a tag-based database search tool for peptide identification with high sensitivity and accuracy. Mol Cell Proteomics. 13 (12), 3663-3673 (2014).
- Xu, P., Duong, D. M., Peng, J. Systematical optimization of reverse-phase chromatography for shotgun proteomics. J Proteome Res. 8 (8), 3944-3950 (2009).
- Niu, M., et al. Extensive Peptide Fractionation and y1 Ion-Based Interference Detection Method for Enabling Accurate Quantification by Isobaric Labeling and Mass Spectrometry. Anal Chem. 89 (5), 2956-2963 (2017).
- Ting, L., Rad, R., Gygi, S. P., Haas, W. MS3 eliminates ratio distortion in isobaric multiplexed quantitative proteomics. Nat Methods. 8 (11), 937-940 (2011).
- Savitski, M. M., et al. Delayed fragmentation and optimized isolation width settings for improvement of protein identification and accuracy of isobaric mass tag quantification on Orbitrap-type mass spectrometers. Anal Chem. 83 (23), 8959-8967 (2011).
- Wenger, C. D., et al. Gas-phase purification enables accurate, multiplexed proteome quantification with isobaric tagging. Nat Methods. 8 (11), 933-935 (2011).
- McAlister, G. C., et al. MultiNotch MS3 enables accurate, sensitive, and multiplexed detection of differential expression across cancer cell line proteomes. Anal Chem. 86 (14), 7150-7158 (2014).
- Zhou, F., et al. Genome-scale proteome quantification by DEEP SEQ mass spectrometry. Nat Commun. 4, 2171 (2013).
- Savitski, M. M., et al. Delayed fragmentation and optimized isolation width settings for improvement of protein identification and accuracy of isobaric mass tag quantification on Orbitrap-type mass spectrometers. Anal. Chem. 83 (23), 8959-8967 (2011).
- Ahrne, E., et al. Evaluation and Improvement of Quantification Accuracy in Isobaric Mass Tag-Based Protein Quantification Experiments. J Proteome Res. 15 (8), 2537-2547 (2016).
- Xu, P., Duong, D. M., Peng, J. M. Systematical Optimization of Reverse-Phase Chromatography for Shotgun Proteomics. J Proteome Res. 8 (8), 3944-3950 (2009).
- Tan, H., et al. Integrative Proteomics and Phosphoproteomics Profiling Reveals Dynamic Signaling Networks and Bioenergetics Pathways Underlying T Cell Activation. Immunity. 46 (3), 488-503 (2017).
- Wu, Z., Na, C. H., Tan, H., Peng, J. Global ubiquitination analysis by SILAC in mammalian cells. Methods Mol Biol. 1188, 149-160 (2014).
- Tan, H., et al. Refined phosphopeptide enrichment by phosphate additive and the analysis of human brain phosphoproteome. Proteomics. 15 (2-3), 500-507 (2015).
- Li, Y., et al. JUMPg: an Integrative Proteogenomics Pipeline Identifying Unannotated Proteins in Human Brain and Cancer Cells. J Proteome Res. 17 (7), 2309-2320 (2016).
- Yuan, Y., et al. Assessing the clinical utility of cancer genomic and proteomic data across tumor types. Nat Biotechnol. 32 (7), 644-652 (2014).
- Nesvizhskii, A. I. Proteogenomics: concepts, applications and computational strategies. Nat Methods. 11 (11), 1114-1125 (2014).
- Fagerberg, L., et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics. 13 (2), 397-406 (2014).
Cite This Article
High, A. A., Tan, H., Pagala, V. R., Niu, M., Cho, J., Wang, X., Bai, B., Peng, J. Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification. J. Vis. Exp. (129), e56474, doi:10.3791/56474 (2017).