title: Quantitative Proteomic Analysis of the Human Nucleolus.
abstract: Recent years have witnessed spectacular progress in the field of mass spectrometry (MS)-based quantitative proteomics, including advances in instrumentation, chromatography, sample preparation methods, and experimental design for multidimensional analyses. It is now possible not only to identify most of the protein components of a cell proteome in a single experiment, but also to describe additional proteome dimensions, such as protein turnover rates, posttranslational modifications, and subcellular localization. Furthermore, by comparing the proteome at different time points, it is possible to create a "time-lapse" view of proteome dynamics. By combining high-throughput quantitative proteomics with detailed subcellular fractionation protocols and data analysis techniques it is also now possible to characterize in detail the proteomes of specific subcellular organelles, providing important insights into cell regulatory mechanisms and physiological responses. In this chapter we present a reliable workflow and protocol for MS-based analysis and quantitation of the proteome of nucleoli isolated from human cells. The protocol presented is based on a SILAC analysis of human MCF10A-Src-ER cells with analysis performed on a Q-Exactive Plus Orbitrap MS instrument (Thermo Fisher Scientific). The subsequent chapter describes how to process the resulting raw MS files from this experiment using MaxQuant software and data analysis procedures to evaluate the nucleolar proteome using customized R scripts.
authors: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD15EH, UK.
authors: Dalila
authors: D
authors: Bensaddek
authors: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD15EH, UK.
authors: Armel
authors: A
authors: Nicolas
authors: Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, DD15EH, UK. a.i.lamond@dundee.ac.uk.
authors: Angus I
authors: AI
authors: Lamond
mesh_terms: D002458:Cell Fractionation / Q000379:methods; D045744:Cell Line, Tumor; D002466:Cell Nucleolus / Q000378:metabolism*; D002853:Chromatography, Liquid; D019295:Computational Biology / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D020543:Proteome; D040901:Proteomics / Q000379:methods; D013347:Subcellular Fractions; D053719:Tandem Mass Spectrometry
chemical_list: D020543:Proteome
keywords: Mass spectrometry; Nucleolus; Quantitative proteomics; Subcellular fractionation