PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.,Journal of Proteome Research

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PEPPI-MS: Polyacrylamide-Gel-Based Prefractionation for Analysis of Intact Proteoforms and Protein Complexes by Mass Spectrometry.
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2020-06-15 , DOI: 10.1021/acs.jproteome.0c00303
Ayako Takemori ₁ , David S Butcher ₂ , Victoria M Harman ₃ , Philip Brownridge ₃ , Keisuke Shima ₄ , Daisuke Higo ₅ , Jun Ishizaki ₆ , Hitoshi Hasegawa ₆ , Junpei Suzuki ₇ , Masakatsu Yamashita ₇ , Joseph A Loo _{8,

9} , Rachel R Ogorzalek Loo ₉ , Robert J Beynon ₃ , Lissa C Anderson ₂ , Nobuaki Takemori ₁

Affiliation

Division of Analytical Bio-Medicine, Advanced Research Support Center, Ehime University, Toon 791-0295, Ehime, Japan.
Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida 32310, United States.
Centre for Proteome Research, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool L69 7ZB, U.K.
Shimadzu Corporation, Nakagyo-ku, Kyoto 604-8511, Japan.
Thermo Fisher Scientific K.K., Yokohama 221-0022, Kanagawa, Japan.
Department of Hematology, Clinical Immunology and Infectious Diseases, Graduate School of Medicine, Ehime University, Toon 791-0295, Ehime, Japan.
Department of Immunology, Graduate School of Medicine, Ehime University, Toon 791-0295, Ehime, Japan.
Department of Chemistry and Biochemistry, University of California-Los Angeles, Los Angeles, California 90095, United States.
Department of Biological Chemistry, UCLA/DOE Institute for Genomics and Proteomics, and UCLA Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California 90095, United States.

Prefractionation of complex mixtures of proteins derived from biological samples is indispensable for proteome analysis via top-down mass spectrometry (MS). Polyacrylamide gel electrophoresis (PAGE), which enables high-resolution protein separation based on molecular size, is a widely used technique in biochemical experiments and has the potential to be useful in sample fractionation for top-down MS analysis. However, the lack of a means to efficiently recover the separated proteins in-gel has always been a barrier to its use in sample prefractionation. In this study, we present a novel experimental workflow, called Passively Eluting Proteins from Polyacrylamide gels as Intact species for MS (“PEPPI-MS”), which allows top-down MS of PAGE-separated proteins. The optimization of Coomassie brilliant blue staining followed by the passive extraction step in the PEPPI-MS workflow enabled the efficient recovery of proteins, separated on commercial precast gels, from a wide range of molecular weight regions in under 10 min. Two-dimensional separation combining offline PEPPI-MS with online reversed-phase liquid chromatographic separation resulted in identification of over 1000 proteoforms recovered from the target region of the gel (≤50 kDa). Given the widespread availability and relatively low cost of traditional sodium dodecyl sulfate (SDS)-PAGE equipment, the PEPPI-MS workflow will be a powerful prefractionation strategy for top-down proteomics.

中文翻译：

PEPPI-MS：聚丙烯酰胺凝胶预分馏，通过质谱分析完整的蛋白形式和蛋白质复合物。

通过自上而下的质谱（MS）进行蛋白质组分析时，必不可少的是将生物样品中蛋白质的复杂混合物进行预分离。聚丙烯酰胺凝胶电泳（PAGE）能够根据分子大小进行高分辨率的蛋白质分离，是生物化学实验中广泛使用的技术，并且有潜力用于自上而下的MS分析样品分离中。然而，缺乏有效地回收凝胶中分离的蛋白质的手段一直是其在样品预分离中的使用的障碍。在这项研究中，我们提出了一种新颖的实验流程，称为聚丙烯酰胺凝胶中的被动洗脱蛋白，作为MS的完整种类（“ PEPPI-MS”），它允许自上而下的PAGE分离出的MS。PEPPI-MS工作流程中考马斯亮蓝染色的优化以及随后的被动提取步骤使得能够在不到10分钟的时间内从广泛的分子量区域有效回收从商业预制凝胶分离出的蛋白质。离线分离PEPPI-MS与在线反相液相色谱分离相结合的二维分离可鉴定出从凝胶目标区域（≤50kDa）回收的1000多种蛋白形式。鉴于传统十二烷基硫酸钠（SDS）-PAGE设备的广泛可用性和相对较低的成本，PEPPI-MS工作流程将是自上而下蛋白质组学的强大预分级策略。在不到10分钟的时间内即可从各种分子量区域获得离线分离PEPPI-MS与在线反相液相色谱分离相结合的二维分离可鉴定出从凝胶目标区域（≤50kDa）回收的1000多种蛋白形式。鉴于传统十二烷基硫酸钠（SDS）-PAGE设备的广泛可用性和相对较低的成本，PEPPI-MS工作流程将是自上而下蛋白质组学的强大预分级策略。在不到10分钟的时间内即可从各种分子量区域获得将离线PEPPI-MS与在线反相液相色谱分离相结合的二维分离可鉴定出从凝胶目标区域（≤50kDa）回收的1000多种蛋白形式。鉴于传统十二烷基硫酸钠（SDS）-PAGE设备的广泛可用性和相对较低的成本，PEPPI-MS工作流程将是自上而下蛋白质组学的强大预分级策略。

更新日期：2020-06-15

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