Current Proteomics
ISSN: 1570-1646
Current Proteomics
Volume 8 Number 1, April 2011
Contents
Hot Topic
Guest Editor: Sonja Hess
Editorial Pp. 1
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)
– an Introduction for Biologists Pp. 2-16
Robert L.J. Graham, Michael J. Sweredoski and
Sonja Hess
[Abstract]
[Full Text Article]
Methods in Quantitative Proteomics: Setting iTRAQ
on the Right Track Pp. 17-30
Josselin Noirel, Caroline Evans, Malinda Salim,
Joy Mukherjee, Saw Yen Ow, Jagroop Pandhal, Trong Khoa Pham,
Catherine A. Biggs and Phillip C. Wright
[Abstract]
[Purchase Article]
Quantitative Proteomics Using Isobaric Chemical Tagging and
HCD Pp. 31-38
Emily S. Boja
[Abstract] [Purchase
Article]
Recent Technological Developments in Proteolytic
18O
Labeling Pp. 39-46
Dagmar Hajkova, K. C. Sekhar Rao and
Masaru Miyagi
[Abstract]
[Purchase
Article]
A Quantitative Perspective on Hydrophobic Interactions
in the Gas-Phase Pp. 47-58
Michal Sharon and Carol V. Robinson
[Abstract] [Purchase
Article]
General Article
Exploring the Nucleolar Proteome: Novel Concepts for
Chaperone Trafficking and Function Pp. 59-82
Piotr Banski,
Mohamed Kodiha and Ursula Stochaj
[Abstract]
[Purchase Article]
Abstracts
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Editorial
This special edition on Quantitative Mass Spectrometry in
Proteomics focuses on some of the leading technologies that
are currently used in state-of-the-art laboratories. In recent
years, mass spectrometry-based proteomics has come a long
way and seen a tremendous advancement to the point where quantitative
measurements are no longer a distant goal, but have become
routine to some of the leading labs. However, this is not
a simple achievement, since it requires an interdisciplinary
effort, starting from experimental design, to adequate sample
preparation, to state-of-the-art technology, to bioinformatics
and statistical analyses.
The advancements and possibilities in quantitative proteomics
have, in turn, changed the way, biological research can be
done. Rather than focusing on a single protein, more ambitious
goals are pursued and entire networks of proteins or protein
complexes are interrogated in current proteomics experiments.
While there are other techniques available, this special edition
focuses on metabolically, chemically or enzymatically introduced
mass tags and the quantitative study of protein-ligand interactions.
The paper by Graham et al. introduces the commonly
used stable isotope labeling by amino acids in cell culture
(SILAC) technology to biologists and covers a broad range
of applications, bioinformatics tools and potential problems
that should be considered when using this technology.
Noirel et al. introduce the chemical labeling technique
iTRAQ. In a meta-analysis of the the current iTRAQ literature,
they give advice to researchers on experimental design, biological
and technical replicates and data analysis tools. This is
followed by a paper by Boja that specifically introduces the
iTRAQ technology with high collision dissociation that has
become available in present-day Orbitrap technologies.
The review by Hajkova et al. covers the recent developments
of the proteolytic 18O labeling technique to improve the reliablity
of the label, the use of computational tools to quantify peptide
and protein ratios, and a new strategy to compare a large
number of samples.
Finally, Sharon and Robinson describe how they use mass spectrometry
to determine the composition, stoichiometry, subunit interactions,
and architectural organization of non-covalent protein complexes.
This issue is aimed at both mass spectrometry practitioners
who want to familiarize themselves with the current state-of-the–art
as well as biologists who contemplate the pros and cons of
the current proteomics technologies. We certainly hope that
it will stimulate further research in the exciting area of
quantitative mass spectrometry.
Sonja Hess
(Guest Editor)
California Institute of Technology
BI 211, MC139-74, Pasadena
CA 91125
USA;
Tel: 001-626-395-2339
Fax: 001-626-449-4159;
E-mail: shess@caltech.edu
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Text Article]
Stable Isotope Labeling by Amino Acids in Cell Culture
(SILAC) – an Introduction for Biologists
Robert L.J. Graham, Michael J. Sweredoski and
Sonja Hess
Since its introduction in 2002 ‘stable isotope
labeling by amino acids in cell culture’ (SILAC) has
become a major tool for quantitative proteomics. Stable isotopes
are incorporated into proteins by introduction of labeled
amino acids in growth medium. This methodology’s compatibility
with virtually all cell culture conditions, ease of implementation
into existing workflows and the high quality quantitative
data that can be obtained have made it the quantitative method
of choice for many laboratories. Although originally used
for mammalian cell cultures, it has been adapted to a number
of organisms including yeast, bacteria, plants and higher
organisms. This review will look at the use of SILAC as a
key tool in quantitative biology. The purpose of this review
is to furnish the reader with a basic understanding of the
fundamental principles of SILAC including its implementation,
application, bioinformatic tools for its analyses and potential
problems that one should be mindful of.
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Article]
Methods in Quantitative Proteomics: Setting iTRAQ
on the Right Track
Josselin Noirel, Caroline Evans, Malinda Salim,
Joy Mukherjee, Saw Yen Ow, Jagroop Pandhal, Trong Khoa Pham,
Catherine A. Biggs and Phillip C. Wright
Isobaric tags for absolute and relative quantification
(iTRAQ) provide the quantitative proteomics community with
an easy-to-use tool to examine proteome changes. Five years
after the launch of the technique, has the community become
familiar with iTRAQ? In this review, we propose to answer
this question by looking at two of its facets. On the one
hand, we enquire whether iTRAQ is used ‘optimally’;
in other words, whether the community is keeping up with the
methods that have been devised and the requirements that have
become recognised, in quantitative proteomics. On the other
hand, there is the question of whether an iTRAQ paper is written
in such a way as to allow other researchers to challenge or
improve the results. To tackle these two problems, we have
reviewed the iTRAQ literature and gathered data about the
methods; in this study, we discuss the experimental designs,
the use of biological and technical replicates, the confirmation
through additional experiments, the strategies to identify
differential expression and supplementary information. Our
conclusions aim at giving recommendations in light of our
statistics.
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Article]
Quantitative Proteomics Using Isobaric Chemical
Tagging and HCD
Emily S. Boja
Multiplexed stable isotope reagents such as iTRAQ and
Tandem Mass Tag (TMT) designed for MS/MS-based quantitation
of peptides rely on accurate and robust detection of low mass
fragments for peptide precursor ions. In the past, such analyses
depended upon mass spectrometers capable of producing the
so-called “triple-quadrupole-like” fragmentation
including triple quadrupole mass spectrometers (TQMS) and
quadrupole time-of-flight (Q-TOF) instruments. The“one-third
rule”
on an ion trap (IT) instrument precluded the use of these
reagents on this widely available instrument platform until
the invention of Pulsed Q Dissociation (PQD), which in itself
manifests problems in generating sufficient reporter ion intensities
for accurate quantitation. The introduction of high energy
collisional-activated dissociation (HCD) on LTQ-Orbitrap XL
and LTQ-Orbitrap Velos platforms has opened up great opportunities
for accurate quantitative analysis of proteins and their post-translational
modifications (PTMs) using chemical tagging by generating
“triple–quadrupole
like”
fragmentation mainly in the low mass range in MS/MS mode.
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[Purchase
Article]
Recent Technological Developments in Proteolytic
18O
Labeling
Dagmar Hajkova, K. C. Sekhar Rao and Masaru Miyagi
The proteolytic 18O
labeling method determines the relative ratios of individual
proteins between two samples. This technique utilizes a protease
and water (H2
16O
and H2
18O)
to produce labeled peptides; peptides in one sample incorporate
16O
by labeling in H2 16O,
and the other sample incorporates 18O
by labeling in H2
18O.
Both samples are mixed in a 1:1 ratio and subjected to mass
spectrometric analysis to identify and quantify the proteins
from which the peptides originated. Technical issues in sample
preparation and data processing prevented this method from
becoming widely accepted in the field of quantitative proteomics,
however these problems have been resolved and the technique
is now rapidly gaining popularity. This review focuses on
the recent technological developments that have improved the
reliability and practicality of proteolytic 18O
labeling, including improved peptide labeling techniques,
techniques to prevent proteasecatalyzed 18O
to 16O
back exchange reaction, mass spectrometry platforms suitable
for this technique, computational tools for the calculation
of 16O/18O-peptide
ratios, and a new strategy that allows to compare a large
number of samples.
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Article]
A Quantitative Perspective on Hydrophobic Interactions
in the Gas–Phase
Michal Sharon, and Carol V. Robinson
Mass spectrometry has become a powerful tool for determining
the composition, stoichiometry, subunit interactions, and
architectural organization of non-covalent protein complexes.
The vast majority of assemblies studied so far by this approach
are those that contain a sufficient amount of electrostatic
interactions and hydrogen bonds that can survive the transition
from solution to the gas–phase
and maintain the structural features of the vaporized ions.
An intriguing question that naturally arises is whether mass
spectrometry can also be harnessed for the study of molecular
systems dominated by non-polar interactions. Here we address
this issue and discuss the fate of hydrophobic complexes in
the absence of bulk water molecules. We emphasize the progress
that has been accomplished in this field that is moving towards
the analysis of larger and more complex hydrophobic systems.
We attribute this advance to recent developments of mass spectrometry
and its application to non-covalent complexes in general,
and to the understanding of experimental and biochemical conditions
for the preservation of hydrophobic interactions in particular.
Furthermore, we discuss the ability of mass spectrometry to
serve as a quantitative tool for assessing the strength, binding
energies, and stoichiometries of hydrophobic interactions.
Overall, we aim to stimulate research in this area and to
establish mass spectrometry as a tool for analyzing hydrophobic
interactions within complex biological systems.
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Exploring the Nucleolar Proteome: Novel Concepts
for Chaperone Trafficking and Function
Piotr Banski,
Mohamed Kodiha and Ursula Stochaj
Organelles and compartments are distinguished by their
biological functions, which in turn are reflected by their
proteome profiles. Prerequisites for the organization of cellular
compartments are mechanisms that help to generate and maintain
their proper composition. The nucleolus is one of the compartments
that have been the focus of intense research during the past
years. It is now well-established that nucleoli are crucial
for a growing number of diverse cellular processes. Moreover,
it became clear that the functional organization of nucleoli
is important for the survival and growth of cells under normal,
stress and disease conditions. Recent research provided us
with information on the nucleolar proteome under different
physiological conditions. These studies demonstrated that
multiple chaperone families, cochaperones and other folding
factors are present in nucleoli. The exact functions chaperones
have in the nucleolus are currently not understood; however,
data provided by nucleolar proteomics put us in the position
to study novel aspects of chaperone biology. These include
the specific roles of chaperones in the nucleolus and the
mechanisms that regulate their transport in and out of nucleoli.
Our review summarizes the present knowledge of chaperones
and their co-factors in the nucleolus as it is emerging from
proteomics and other studies. Based on this information, we
speculate on the biological relevance of chaperones in the
nucleolus and the cellular pathways that promote their targeting
to this subnuclear compartment. We conclude by highlighting
the open questions that will have to be addressed in future
studies.
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