Quantitative proteomic analysis of the human nucleolus

Dalila Bensaddek, Armel Nicolas, Angus I. Lamond

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

5 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationThe nucleolus
Subtitle of host publicationmethods and protocols
EditorsAttila Németh
Place of PublicationNew York
PublisherSpringer
Pages249-262
Number of pages14
ISBN (Electronic)9781493937929
ISBN (Print)9781493937905
DOIs
Publication statusPublished - 2016

Publication series

NameMethods in molecular biology
PublisherSpringer
Volume1455
ISSN (Print)1064-3745

Keywords

  • Quantitative proteomics
  • Mass spectrometry
  • Nucleolus
  • Subcellular fractionation

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