Current Proteomics
ISSN: 1570-1646
Current Proteomics
Volume 2, Number 4, December 2005
Contents
Current Progress in Sample Preparation for Two-Dimensional
Electrophoresis in Proteomics Pp.259-268
Z. Cai, J.-F. Chiu and Q.-Y. He
[Abstract]
Sample Preparation Techniques for Mass Spectrometry
in Proteomics Using Recently Developed Highly Selective Materials
Pp.269-285
C.W. Huck, R. Bakry and G.K. Bonn
[Abstract]
Recent Progress in Quantitative Proteomics Using Stable
Isotope Labeling, Multidimensional Liquid Chromatography and
Mass Spectrometry Pp.287-302
J.-L. Hsu and S.-H. Chen
[Abstract]
Proteomics of Human Pulmonary Surfactant Proteins
Pp.303-318
C. He
[Abstract]
Perspectives in Proteomics: Structural Folds of a
Predicted and an Experimentally Determined Cation Channel
Pp.319-324
P.P. Mager, A. Weber, H. Walter, K. Wirkner and P. Illes
[Abstract]
Rice Proteomics: A Step Toward Functional Analysis
of Stress Responses Pp.325-33
S. Komatsu
[Abstract]
Abstracts
[Back to top]
Current Progress in Sample Preparation for Two-Dimensional
Electrophoresis in Proteomics
Z. Cai, J.-F. Chiu and Q.-Y. He
Two dimensional polyacrylamide gel electrophoresis (2DE)
has been a core technology of current proteomics for its high
resolution and ability to detect proteins with post-translational
modifications. This technology has been widely applied in
numerous biomedical research projects including biomarker
discovery and drug development. The application of narrow-range
immobilized pH gradient strips (IPGs) and advanced detection
methodologies have increased the resolution of 2DE technology.
However, 2DE still suffers from its limitation in unambiguous
detection of proteins of low-abundance. To improve this, sample
preparation is the first and key step for successful 2DE analysis.
This article summarizes current progresses in sample handling
and prefractionation aiming to enrich proteins of interest
according to their special physical and chemical properties
so that the application of 2DE can be greatly extended.
[Back to top]
Sample Preparation Techniques for Mass Spectrometry
in Proteomics Using Recently Developed Highly Selective Materials
C.W. Huck, R. Bakry and G.K. Bonn
Efficient sample preparation in the micro and nano-litre
range, prior to analysis of proteins and peptides of biological
origin by liquid chromatography (LC), liquid chromatography
coupled to mass spectrometry (LC-MS), micro–liquid chromatography
(µ-LC) and matrix assisted laser desorption/ionization–time
of flight (MALDI-TOF) mass spectrometry, is the key to success
for different applications, such as biomarker discovery. Sample
preparation in proteomics mainly comprises purification and
preconcentration by solid-phase extraction (SPE) using different
polymers (polystyrene, poly acrylate, cellulose, etc.) as
a stationary phase following several cleaning-up procedures.
These polymers can be derivatized with several functional
groups, for example, C18, ion-exchanger or immobilized metal
affinity chroma-tography (IMAC) groups. Based on these “traditional”
techniques new methods enabling higher selectivity, sensitivity
and speed of separation are required. Capillary coatings,
for example, with latex particles has been used successfully.
Derivatization of these latex particles, for example, with
IMAC-functional groups enables a selective purification of
phosphorylated or His-tagged proteins. Alternatively, polymeric
disks possessing a thickness of approximately 2 mm can be
used even in the case of multidimensional separations. In
this review, we summarize currently available sample pre-treatment
techniques, provide an overview on the technical background
and discuss the advantages of the individual methods using
recently developed selective stationary phases such as silica
based materials, polymers, etc.
[Back to top]
Recent Progress in Quantitative Proteomics Using Stable
Isotope Labeling, Multidimensional Liquid Chromatography and
Mass Spectrometry
J.-L. Hsu and S.-H. Chen
Stable isotope labeling coupled with multidimensional liquid
chromatography/tandem mass spectrometry is an advanced platform
for global proteome-wide quantification. Compared to conventional
methods, such as two dimensional polyacrylamide gel or electroblot
based quantification methods, stable isotope labeling holds
greater promise for accurate and large-scale quantitative
analyses. Various labeling strategies have been developed
to analyze protein expression, post-translational modification,
and protein/protein interactions, as well as for absolute
quantification. There are advantages and disadvantages inherent
with each method, but their applicability is dependent on
many factors. In addition to the choice of which labeling
chemistry to be used, there are several bottleneck issues
associated with this approach that are of critical importance.
These include finding effective multidimensional separation
steps to resolve low abundant proteins present in relatively
complicated mixtures, validating the methods, and interpreting
the large amount of statistical data generated. This review
covers the current progress in solving these concerns and
summarizes the various labeling strategies and applications.
We believe that a judicious integration of each component
of the technique is crucial for the success of such a global
systems approach. Based on the current progress, it is clear
that the stable isotope labeling/mass spectrometry technique
will find tremendous use in many fields, such as drug discovery,
clinical diagnostics, disease prevention, basic biological
research, and biotechnology.
[Back to top]
Proteomics of Human Pulmonary Surfactant Proteins
C. He
Over many years, the protein components of pulmonary surfactant
have been the subject of a large number of analyses using
high resolution methods for protein analysis. In fact, identification
of protein biomarkers for lung disorder is extremely important
in order to gain insight into the mechanisms underlying lung
diseases. For separation of protein components of pulmonary
surfactant proteins, two-dimensional gel electrophoresis (2-DE)
coupled with Western blot analysis involving electrophoretic
transfer of the separated proteins onto a membrane followed
by immunodetection of proteins by enhanced chemiluminescence
has been used. This method allows very high resolution, sensitivity
and specificity for proteomic study of pulmonary surfactant-associated
proteins, particularly for water-soluble surfactant-associated
protein-A (SP-A). Analysis of SP-A has also been carried out
with 2-DE followed by either amino acid se-quence analysis
using Edman degradation or mass spectrometry. Other pulmonary
surfactant-associated proteins, SP-B, SP-C and SP-D were identified
using either gel electrophoresis based or non-gel-electrophoresis
based methods, such as high performance liquid chromatography.
This review summarizes the major achievements in proteomic
studies of pulmonary surfactant-associated proteins.
[Back to top]
Perspectives in Proteomics: Structural Folds of a
Predicted and an Experimentally Determined Cation Channel
P.P. Mager, A. Weber, H. Walter, K. Wirkner and P. Illes
A novel method of structure prediction for membrane-bound
proteins is reviewed. The approach is based on a sequence-function
analysis, secondary structure prediction and subsequent geometry
optimization. The prediction of the structure of ligand-gated
P2X4 receptor subunit, which is a membrane-embedded cation
channel-forming protein with extracellularly occuring ATP
binding sites, is shown as an example. The potential N-glycosylation
sites of the glycoprotein, location of the five disulfide
bridges, and phospholipid-dependent protein kinase C (PKC)
phosphorylation attach-ment sites were determined by sequence-function
analysis. Subsequently an attempt was made to predict its
conformation using homology-based comparative modeling and
threading; however, the modeling could not be accomplished.
Because of this, secondary structure prediction of the protein
was carried out. The input coordinates of the spatial structure
were obtained by a profile-based neural network prediction
method. The resulting secondary structure was converted into
a three-dimensional geometry. The secondary and tertiary structures
were optimized by the quantum chemistry RHF/3-21G minimal
basic set and all-atom molecular mechanics AMBER96 force field.
The predicted shape is similar to the shape of the experimentally
obtained monomeric structure of the classical ion channel,
the K+ion channel from Streptomyces lividans
(KcsA channel), and agrees with the P2X shape proposed by
biological experimenters. The geometry optimized structure
of the P2X4 receptor is freely available (Protein Data Bank
format) from the authors on e-mail request (magp@medizin.uni-leipzig.de).
[Back to top]
Rice Proteomics: A Step Toward Functional Analysis
of Stress Responses
S. Komatsu
Plants need to perceive and process information both from
the biotic and abiotic surroundings for their optimum growth
and development. Because plants are not motile, they have
to be especially responsive to environmental changes, including
stress conditions. Subjecting rice seedlings to environmental
stresses results in various biochemical changes, many of which
are poorly understood. Proteomics approaches to identifying
proteins that are regulated in response to different environmental
conditions are becoming common in the post-genomic era in
rice research. This review describes initial steps toward
determining the physiological significance of some proteins
identified from rice.
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