|
Current
Analytical Chemistry
ISSN: 1573-4110
Current Analytical Chemistry
Volume 5, Number 2, April 2009
Contents
The Exciting Ionic Life of a Protein
in the Hands of a Mass Spectrometrist
Guest Editor: Andrea Armirotti
Editorial: Pp. 84
[Purchase
Article]
Protein Purification Pp. 85-105
William W. Ward and Gavin Swiatek
[Abstract] [Purchase
Article]
High Resolution Protein Display by Two-Dimensional
Electrophoresis Pp. 106-115
Gert Van den Bergh and Lutgarde
Arckens
[Abstract] [Purchase
Article]
Bottom-Up Proteomics Pp.
116-130
Andrea Armirotti
[Abstract] [Purchase
Article]
Mass Spectrometry Bioinformatics: Tools
for Navigating the Proteomics Landscape Pp.
131-143
Kevin Blackburn and Michael B.
Goshe
[Abstract] [Purchase
Article]
Analysis of Glycosylation and Other Post-Translational
Modifications by Mass Spectrometry Pp. 144-165
Willy Morelle
[Abstract] [Purchase
Article]
Advances in Quantitative Mass Spectrometry
Analysis: Weighing in on Isotope-Coding and Label-Free Approaches
for Expression and Functional Proteomics Pp.
166-185
Ko-yi Chien and Michael B. Goshe
[Abstract] [Purchase
Article]
Protein Folding and Protein-Ligand Interactions
Monitored by Electrospray Mass Spectrometry Pp.
186-204
Peter L. Ferguson, Mark C. Kuprowski, Brian
L. Boys, Derek J. Wilson, Jingxi Pan and Lars Konermann
[Abstract] [Purchase
Article]
Isotope Exchange and Covalent Modification
Strategies for Studying Protein Structure and Function
Pp. 205-212
Shugui Chen and John R. Engen
[Abstract] [Purchase
Article]
Abstracts
[Back to top]
[Purchase
Article]
Editorial: The Exciting Ionic Life
of a Protein in the Hands of a Mass Spectrometrist
Over the last twenty years, biomolecular scientists
learned to switch from the model to the result,
from a design based on a four letter alphabet to the final
twenty letters based outcome of the genetic project. From
genome to the proteome. This important step was taken because
the evolutive information is carried by a very rigid molecule,
the DNA, and becomes effective in much more flexible objects,
the proteins, whose chemical and physical freedom supports
every life process. “Proteomics” is a relatively
newborn science, whose aim is to study the entire complement
of proteins produced by an organism. No other science has
attracted such an impressive amount of human, technological
and economic resources among the large field of biochemical
research over the last decade, because proteins are involved
in every biological process. Scientific research against any
known disease has one or more protein as final target. If
any living being can be considered as an envelope protecting
its most important treasure, the genetic information, then
proteins must be thought as both results of the information
and tools for protecting, conserving and passing it on. In
this point of view, “proteomics” is not just that
somewhat mysterious core facility down the building, where
“upstair” scientists bring their samples just
to know “who’s in that 2D gel spot”. Proteomics
is putting every protein in place in its correct biochemical
contest, assessing its identity, its modifications map, its
degree of over or underexpression, its folding and its interactions
with other proteins. In this extremely ambitious project,
mass spectrometry plays a crucial role, because the development
of electrospray and MALDI ion sources (John B. Fenn and Koichi
Tanaka, Nobel laureates in 2002) opened a brand new world,
where even molecular “elephants” can be transferred
in the gas phase, opening unique and almost endless possibilities
for protein analysis.
The aim of this special issue, composed of eight reviews,
is to give the reader an introductory but exhaustive overview
of the analytical techniques used to purify, identify, quantify
and characterize proteins, keeping the main focus on mass
spectrometry. The first two articles have been written by
William Ward and Gert Van Den Berg, acknowledged experts on
protein purification and 2D gel electrophoresis, respectively.
These two steps are the foundation of the MS-based protein
research, because they allow a protein to be purified from
its biological environment, an extremely complex analytical
matrix. The third review describes how a protein can be identified,
through a “bottom-up” process and the extensive
use of bioinformatics tools, whose key role in proteomics
is to extract significant results from the enormous amount
of data generated by mass spectrometry. This last topic is
covered by a notable review of Michael Goshe. Post translational
modifications of proteins, such as phosphorylations and glycosilations,
play a crucial role in their biological activity and are one
of the most addressed issues in proteomics. In his review,
Willy Morelle exhaustively describes the most important techniques
for PTMs analysis. The fifth article, written by Michael Goshe
again, brightly describes an array of relatively new tools
for quantitative proteomics, based on both label and label-free
methods. The last two papers deal with an area of protein
science that is not usually included (unfortunately) in classical
MS-based “proteomics” reviews. These two articles
focus on a frontier area of mass spectrometry: the study of
protein structure, folding and function. Such an exciting
field of research is greatly discussed by Lars Konermann and
John Engen.
The Authors and the Editor sincerely hope that this special
issue will represent an outline of the state of the art of
several fields of proteomic sciences, possibly representing
an introductory approach for those analytical scientist not
(yet?) directly involved in protein research.
Finally, as Guest Editor of this special issue I would like
to express my gratitude to the Authors for their excellent
work and to the Publishers for giving me the opportunity to
work with such a brilliant group of scientists.
Dr. Andrea Armirotti
Department of Experimental Medicine
Center of Excellence for Biomedical Research
University of Genoa
Viale Benedetto XV, 7 Genova
Italy
[Back to top]
[Purchase
Article]
Protein Purification
William W. Ward and Gavin Swiatek
The science, art, and practice of protein purification
have been with us for more than a century, yet, in many respects,
the field is only now evolving past its adolescent roots.
New methods are replacing old methods at such a dizzying pace,
that even life-long experts in protein purification cannot
keep up. In this article, we present many state-of-the-art
protein purification techniques without totally ignoring the
past. Our goal is to enable those relatively new to the field
of protein purification to choose the best methods to solve
their own purification problems. Each method we describe has
been used and validated in our own research. We describe these
methods, pointing out advantages, disadvantages, and limitations
with practical examples rather than with complex, theoretical
equations. This paper covers methods of extraction, clarification,
batch purification, low pressure column chromatography, HPLC,
and electrophoresis as applied to both genetically engineered,
recombinant proteins and proteins isolated from natural sources.
The relatively new methods of three-phase partitioning, hydrophobic
charge induction chromatography, immobilized metal affinity
chromatography, and perfusion chromatography are featured.
[Back to top]
[Purchase
Article]
High Resolution Protein Display by Two-Dimensional Electrophoresis
Gert Van den Bergh and Lutgarde
Arckens
Two-dimensional electrophoresis has, for many years,
been the primary workhorse for performing functional proteomics,
the large-scale analysis of protein expression differences.
Despite its merits, limitations inherent to this technology
have been recognized for a long time, ranging from its gel-to-gel
variability to its inability to represent several classes
of proteins. Recently, however, technical advances in two-dimensional
electrophoresis have alleviated several of these drawbacks.
Fractionation approaches prior to two-dimensional electrophoresis,
e.g. by chromatography, organelle fractionation or Equalizer
Beads technology, have increased the number of visible proteins.
Fluorescent two-dimensional difference gel electrophoresis
has boosted the quantitative aspects of two-dimensional electrophoresis.
New protein stains have also enabled the analysis of post-translationally
modified proteins. As a result, two-dimensional electrophoresis
has been thoroughly modernized, enabling it to remain the
preferred method for protein expression analysis in a large
number of laboratories. In this review we will give an overview
of these technological advances.
[Back to top]
[Purchase
Article]
Bottom-Up Proteomics
Andrea Armirotti
In this work, the “bottom-up” protein identification
process is described, from sample digestion to the final database
search result. Both MALDI and LC-MS approaches to the identification
issue are illustrated. The state-of-the-art of these technique
is outlined along with several recent applications, such as
MSn data acquisition and chemical derivatization of peptides.
[Back to top]
[Purchase
Article]
Mass Spectrometry Bioinformatics: Tools for Navigating the
Proteomics Landscape
Kevin Blackburn and Michael B.
Goshe
Central to the successful implementation of a proteomics
pipeline are appropriate bioinformatics tools to provide a
level of automation and standardization to the qualitative
identification of proteins, quantitation of protein changes,
data capture and storage, and integration with other data
platforms. Many of these efforts are in various stages of
maturity; however, the identification, or recognition, of
peptides/proteins from mass spectrometry data, arguably the
most developed area, continues to remain challenging due to
the ever increasing size of proteomic datasets. Confident
peptide and protein identification, including assignment of
any post-translational modifications, is a necessary prerequisite
for any proteomic study aimed at elucidating biological and
physiological responses. This review includes discussions
of bioinformatic approaches for the qualitative identification
of peptides/proteins from mass spectrometry data as well as
the software tools and the analytical considerations required
for analysis. Issues related to placing a proteomics dataset
in a larger biological context which includes splice variants,
variations in databases, and neglected proteomes are also
discussed.
[Back to top]
[Purchase
Article]
Analysis of Glycosylation and Other Post-Translational Modifications
by Mass Spectrometry
Willy Morelle
The determination of post-translational modifications
is one of the main challenges in proteomics research. Mass
spectrometry is a powerful tool for the structural characterization
of proteins and different mass spectrometric techniques for
the analysis of post-translational modifications of individual
proteins or protein populations have been developed. This
review describes the most recent advances in mass spectrometry-based
approaches for the detection and determination of post-translational
modifications, with an emphasis on glycosylation and phosphorylation.
[Back to top]
[Purchase
Article]
Advances in Quantitative Mass Spectrometry Analysis: Weighing
in on Isotope-Coding and Label-Free Approaches for Expression
and Functional Proteomics
Ko-yi Chien and Michael B. Goshe
Mass spectrometry is an extremely versatile analytical
technique that is capable of characterizing proteins at various
levels of biochemical sophistication from recognition of protein
components and their modifications to their quantification
within a sample. With the development of electrospray ionization
and matrix-assisted laser desorption ionization, the last
decade of protein analysis using mass spectrometry has fully
established the field of proteomics within the life sciences
and a major player in the systems biology paradigm. The diversity
of proteins and their multi-facetted functions are indicative
of the numerous mass spectrometry methods that are used in
quantitative proteomic analysis. In this review, the various
techniques developed to quantify protein abundance by mass
spectrometry are presented in terms of those associated with
both stable isotope coding and label-free strategies. The
implementation of these methods to the quantitative mass spectrometry
analysis from “proof-of-concept” to those that
tackle investigations of protein expression and those of protein
function mediated by post-translation modifications are also
discussed.
[Back to top]
[Purchase
Article]
Protein Folding and Protein-Ligand Interactions Monitored
by Electrospray Mass Spectrometry
Peter L. Ferguson, Mark C. Kuprowski, Brian
L. Boys, Derek J. Wilson, Jingxi Pan and Lars Konermann
Electrospray ionization (ESI) mass spectrometry (MS)
has become an indispensable tool for studies on protein structure,
folding, dynamics, and interactions. The ESI process generates
intact and multiply protonated ions from proteins in solution.
The charge state distribution of these ions provides a highly
sensitive probe for the overall compactness of a protein in
solution. Unfolded conformers lead to the formation of higher
charge states than natively folded proteins. Due to its very
gentle nature, ESI allows the transfer of intact noncovalent
assemblies (protein-ligand and protein-protein complexes)
into the gas phase. Thus, ESI-MS is ideally suited for monitoring
coupled folding/binding events. The remarkable selectivity
of this technique facilitates the observation of co-existing
conformers and binding states. This review discusses mechanistic
aspects of the ionization process, as well as selected examples
that illustrate the use of ESI-MS for monitoring protein folding
and assembly reactions. The combination of ESI-MS with on-line
mixing techniques can provide mechanistic insights into processes
occurring on very rapid time scales. We also address the interesting
question whether biomolecular structures in the gas phase
resemble those in solution. Experimental approaches involving
hydrogen exchange and covalent labeling techniques are covered
in an accompanying article.
[Back to top]
[Purchase
Article]
Isotope Exchange and Covalent Modification Strategies for
Studying Protein Structure and Function
Shugui Chen and John R. Engen
Mass spectrometry can be used to obtain information about
all levels of protein structure. To gain access to the conformation
information found in the tertiary and quaternary structure,
various labeling methods have been developed. These methods
convert structural information into mass differences that
can be observed with high-resolution protein/peptide mass
spectrometry. Three methods are reviewed here: hydrogen/deuterium
exchange, covalent modification (also called chemical modification)
and hydroxyl radical footprinting. The general implementation
of these methods is described and comparisons are made between
the methods.
|
