| Chapter title |
Quantitative Proteomic Analysis of the Human Nucleolus
|
|---|---|
| Chapter number | 20 |
| Book title |
The Nucleolus
|
| Published in |
Methods in molecular biology, January 2016
|
| DOI | 10.1007/978-1-4939-3792-9_20 |
| Pubmed ID | |
| Book ISBNs |
978-1-4939-3790-5, 978-1-4939-3792-9
|
| Authors |
Dalila Bensaddek, Armel Nicolas, Angus I. Lamond, Bensaddek, Dalila, Nicolas, Armel, Lamond, Angus I |
| Editors |
Attila Németh |
| 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. |
X Demographics
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| Unknown | 1 | 100% |
Demographic breakdown
| Type | Count | As % |
|---|---|---|
| Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
| Country | Count | As % |
|---|---|---|
| United Kingdom | 1 | 6% |
| Unknown | 15 | 94% |
Demographic breakdown
| Readers by professional status | Count | As % |
|---|---|---|
| Researcher | 5 | 31% |
| Student > Ph. D. Student | 3 | 19% |
| Unspecified | 2 | 13% |
| Lecturer | 2 | 13% |
| Student > Bachelor | 1 | 6% |
| Other | 2 | 13% |
| Unknown | 1 | 6% |
| Readers by discipline | Count | As % |
|---|---|---|
| Biochemistry, Genetics and Molecular Biology | 7 | 44% |
| Unspecified | 2 | 13% |
| Medicine and Dentistry | 2 | 13% |
| Agricultural and Biological Sciences | 1 | 6% |
| Pharmacology, Toxicology and Pharmaceutical Science | 1 | 6% |
| Other | 2 | 13% |
| Unknown | 1 | 6% |