Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)

387 Pages·2014·7.71 MB·English

Methods in
Molecular Biology 1188
Bettina Warscheid Editor
Stable Isotope
Labeling by Amino
Acids in Cell Culture
(SILAC)
Methods and Protocols
M M B
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hat fi eld, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

Stable Isotope Labeling
by Amino Acids
in Cell Culture (SILAC)
Methods and Protocols
Edited by
Bettina Warscheid
Institute of Biology II, University of Freiburg, Freiburg, Germany
Editor
Bettina Warscheid
Institute of Biology II
University of Freiburg
Freiburg, Germany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-4939-1141-7 ISBN 978-1-4939-1142-4 (eBook)
DOI 10.1007/978-1-4939-1142-4
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Prefa ce
Since its introduction about a decade ago, SILAC—stable isotope labeling by amino acids
in cell culture—combined with mass spectrometry has not merely been integrated into the
spectrum of quantitative methods applied in proteomics and protein research laboratories.
In fact, it has become a prime tool of functional proteomics research, which allows us to
address important questions in the fi elds of biology, biotechnology, medicine, and beyond.
SILAC could obtain this status by virtue of its versatility. This book tries to provide a
synopsis of the large array of different SILAC methods by presenting a set of protocols that
have been established by renowned scientists and their working groups. These protocols
describe basic applications such as the labeling of various model organisms but also highly
advanced strategies relying on SILAC, e.g., for the analysis of protein interactions, the map-
ping of posttranslational modifi cations, or the characterization of subcellular proteomes.
The book aims at applicability, and so all chapters entail step-by-step instructions that
are easy to follow.
Chapter 1 provides a historical overview in which Matthias Mann outlines, from a very
personal perspective, important steps in the development and implementation of SILAC as
well as further signifi cant innovations in the use of this unique method over the last 12 years.
The main feature of the SILAC technology is the potential to label the entire proteome with
defi ned combinations of stable isotopes during an organism’s growth. At fi rst applied to
mammalian cell lines, its applicability to various other organisms has since been demon-
strated as well. Chapter 2 is about a protocol for the effective labeling of both Gram positive
and negative bacteria using only lysine in its “light” and “heavy” version. Chapter 3 contin-
ues with the description of an experimental procedure for complete SILAC labeling of the
yeast Saccharomyces cerevisiae , a widely used model system for higher eukaryotes, including
details on the generation of auxotrophic strains and the SILAC-based analysis of membrane
protein complexes. SILAC applied to the protozoan T rypanosoma brucei , a further unicel-
lular eukaryotic organism, enables new studies on the parasite’s unique biology. A straight-
forward protocol for the metabolic labeling of both the procyclic and the bloodstream form
of T. brucei with SILAC amino acids in a cell culture system can be found in Chapter 4 .
Since the complete incorporation of SILAC amino acids into proteomes requires
approximately fi ve cell doublings, issues of partial labeling arise when working with nondi-
viding cells. Chapter 5 addresses this problem by introducing an elegant multiplex SILAC
labeling approach. It allows for the quantifi cation of partially labeled proteins from nondi-
viding cell types as exemplifi ed here by primary neurons.
The following four chapters deal with the applicability of SILAC to the metabolic label-
ing of multicellular organisms including the higher plant model system Arabidopsis thali-
ana as well as D rosophila melanogaster , Mus musculus, and C aenorhabditis elegans . In
Chapter 6 , limitations of the SILAC technology in plant cells are discussed and an alterna-
tive protocol for labeling whole A . thaliana plants using 1 5 N salts is provided. SILAC label-
ing of D . melanogaster , as described in Chapter 7 , relies on feeding “heavy” lysine-labeled
yeast to fl ies, which can easily be implemented for quantitative analyses. Chapter 8 informs
about the details how to generate SILAC mice and how to utilize them as spike-in standard
v
vi Preface
for quantitative proteomics studies of organs. And Chapter 9 presents an innovative proto-
col for SILAC-based quantitative phosphoproteome analyses in C. elegans along with
RNAi-mediated gene knockdown.
The high potential SILAC offers for the global study of signaling networks is high-
lighted in Chapter 10 providing the reader with state-of-the-art knowledge and practical
information about how to conduct large-scale quantitative and time-resolved phosphopro-
teomics studies using SILAC. Detailed experimental procedures for the global analysis of
dynamic changes in protein ubiquitination and methylation are described in Chapters 11
and 1 2 , respectively.
A further important fi eld of application for SILAC is the study of protein interactomes,
in which SILAC-based protein quantifi cation provides an effective measure to reliably dis-
tinguish between specifi c interaction partners and co-purifi ed background binders in
affi nity- based protein purifi cation or coimmunoprecipitation experiments. Complementary
to Chapter 3 , the potential and versatility of SILAC approaches for the study of protein–
protein interactions are discussed in Chapters 1 3 – 16 , i.e., the comparative analysis of
human protein complexes (Chapter 13 ) , the identifi cation of stable and dynamic interac-
tion partners exemplifi ed by the human 26S proteasome complex (Chapter 1 4 ) , the char-
acterization of nuclear protein–protein interactions in mammalian cells (Chapter 15 ) , and
the delineation of dynamic processes involved in the assembly of the human splicosome
(Chapter 1 6 ) .
Chapter 1 7 turns to a protocol for protein interaction studies in autotrophic organisms,
in which 14 N/1 5 N labeling combined with coimmunoprecipitation and antigen competition
is used.
At fi rst introduced as a label-free approach, the original protein correlation profi ling
method has been refi ned using SILAC. Dynamic aspects of protein interactomes and organ-
ellar proteomes can be analyzed by such spike-in SILAC standard-enhanced variations, for
which protocols are presented in Chapters 1 8 and 1 9 , respectively.
An important innovation of the SILAC technology has been its implementation in the
study of cancer tissues, which is based on the generation of SILAC-labeled reference pro-
teomes used as spike-in standards for relative protein quantifi cation. In Chapter 20 , the
reader fi nds detailed information about how to properly design and successfully conduct
such super-SILAC experiments for quantitative proteome analyses of tissue samples.
Chapter 21 provides an optimized protocol for SILAC labeling of D . melanogaster , both in
cell culture and in tissues, which allows tackling various questions in genetics and develop-
mental biology. As outlined in Chapter 22 , SILAC was also applied to the global study of
secreted proteins, a new promising approach to identify protein biomarker candidates for
human diseases. Furthermore, employed in pulse experiments, SILAC is a powerful tech-
nology for the large-scale analysis of protein turnover rates. Chapter 2 3 is about a protocol
for pulsed SILAC that enables the identifi cation of microRNA-mediated changes in the
synthesis rates of proteins. The protocol is complemented by the description of computa-
tional and experimental approaches for the analysis of potential microRNA targets identi-
fi ed in a pulsed SILAC study.
The SILAC technology is intimately linked to high-resolution mass spectrometry facili-
tating the generation of a wealth of data about both the identity and the abundance of
proteins in biological samples. Effi cient processing and computational analysis of large
SILAC-encoded mass spectrometry datasets is therefore a key step in quantitative p roteomics
studies. Chapter 24 presents an easy-to-follow protocol for the quantitative analysis of
SILAC-based proteomics data using the freely available software MaxQuant.
Preface vii
In sum, the methods and experimental strategies described in this volume hopefully
give an impression of the amazing diversity of SILAC applications. Smartly combined with
different molecular and cell biology or biochemical techniques, SILAC is and will be a most
valuable and multifunctional tool for many research studies aiming at a better understand-
ing of cellular organization, protein interaction, and signaling networks as well as protein
translation, turnover, and expression changes in health and disease.
The chapters also provide important information about practical aspects of sample frac-
tionation, enrichment, and/or further processing steps as well as—to different extent—
details on how to conduct LC/MS and downstream data analyses. In doing so, the book
hopefully will serve students and experienced scientists alike as a valuable reference of how
to make use of the SILAC technology for their own research.
I would like to thank the editors of the book series for their initial suggestion and their
ongoing support in all editorial matters and also members of my group, especially Silke
Oeljeklaus and Ida Suppanz, for providing much needed and welcomed assistance.
Finally and most importantly, I would like to cordially thank all contributors for sharing
their knowledge!
Freiburg, Germany Bettina Warscheid

Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x i
1 Fifteen Years of Stable Isotope Labeling by Amino Acids
in Cell Culture (SILAC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Matthias Mann
2 S table Isotope Labeling by Amino Acids Applied
to Bacterial Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Boumediene S oufi and Boris M acek
3 S ILAC Labeling of Yeast for the Study of Membrane Protein Complexes . . . . 2 3
Silke O eljeklaus, Andreas S chummer, I da Suppanz,
and Bettina Warscheid
4 Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei
Using Stable Isotope Labeling by Amino Acids in Cell Culture
and Mass Spectrometry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Olivera C irovic and Torsten O chsenreiter
5 S table Isotope Labeling by Amino Acids in Cultured Primary Neurons . . . . . . 5 7
Guoan Zhang, Katrin D einhardt, and Thomas A . N eubert
6 S ILAC and Alternatives in Studying Cellular Proteomes of Plants . . . . . . . . . . 6 5
Annemarie M atthes, Karin Köhl, and Waltraud X. Schulze
7 I n Vivo Stable Isotope Labeling by Amino Acids in Drosophila melanogaster . . 85
Matthias D. Sury, Jia-Xuan C hen, and Matthias S elbach
8 S table Isotope Labeling for Proteomic Analysis of Tissues in Mouse . . . . . . . . 9 5
Soraya H ölper, A aron R uhs, and Marcus Krüger
9 I dentification of Novel Protein Functions and Signaling
Mechanisms by Genetics and Quantitative Phosphoproteomics
in Caenorhabditis elegans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Julius F redens, Kasper Engholm-Keller, J akob Møller-Jensen,
Martin R øssel Larsen, and Nils J . F ærgeman
1 0 SILAC-Based Temporal Phosphoproteomics. . . . . . . . . . . . . . . . . . . . . . . . . . 1 25
Chiara F rancavilla, O mid H ekmat, Blagoy Blagoev,
and Jesper V. O lsen
11 Global Ubiquitination Analysis by SILAC in Mammalian Cells . . . . . . . . . . . . 1 49
Zhiping Wu, Chan Hyun Na, H aiyan T an, and Junmin P eng
12 Quantifying In Vivo, Site-Specific Changes in Protein
Methylation with SILAC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
Ho-Tak L au, Karen A . Lewis, and Shao-En O ng
ix