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Current Analytical Chemistry
ISSN: 1573-4110
Current Analytical Chemistry
Volume 3, Number 4, October 2007
Contents
The Use of Atmospheric Pressure Chemical Ionization
Mass Spectrometry with High Performance Liquid Chromatography
and Other Separation Techniques for Identification of Triacylglycerols
Pp. 252-271
Tomáš Rezanka and Karel Sigler
[Abstract]
Purification of Transcription Factors and Identification
with Mass Spectrometry Pp. 272-282
Shoulei Jiang, Linda Nagore, Daifeng Jiang, Robert A.
Moxley and Harry W. Jarrett
[Abstract]
31P NMR Spectroscopy
of Phospholipids: From Micelles to Membranes Pp.
283-301
Jürgen Schiller, Matthias Müller, Beate Fuchs,
Klaus Arnold and Daniel Huster
[Abstract]
Pitfalls in High Throughput Screening for Drug Absorption
Optimization in Drug Discovery Pp. 302-309
Hiroshi Komura and Masahiro Iwaki
[Abstract]
Hollow-Fiber Flow Field-Flow Fractionation
Pp. 310-323
Andrea Zattoni, Sonia Casolari, Diana Cristina Rambaldi
and Pierluigi Reschiglian
[Abstract]
Abstracts
[Back to top]
The Use of Atmospheric Pressure Chemical Ionization Mass Spectrometry
with High Performance Liquid Chromatography and Other Separation
Techniques for Identification of Triacylglycerols
Tomáš Rezanka and Karel Sigler
Triacylglycerols (TAGs) are widely distributed, if not
universal, in edible plants and edible plant triacylglycerols
are nutrients forming an important part of the human diet.
TAGs of higher plants consist mainly of esters of glycerol
with fatty acids, many of which are essential as human nutrients
and are therefore in the center of increasing attention. Despite
their importance, the molecular species of each TAG class
have not yet been fully characterized. The use of state-of-the-art
techniques, especially in a combination such as that discussed
in this review, permits the identification of not only TAG
classes, but also their positional isomers and often also
individual molecular species.
Atmospheric pressure chemical ionization mass spectrometry
(APCI-MS) in combination with high-performance liquid chromatography
(HPLC) has proven to be a very valuable technique for analysis
of different lipids from a variety of sources. This paper
describes direct analyses of TAGs from natural oils, frequently
very unusual, using LC-MS/APCI.
[Back to top]
Purification of Transcription Factors and Identification
with Mass Spectrometry
Shoulei Jiang, Linda Nagore, Daifeng Jiang, Robert
A. Moxley and Harry W. Jarrett
Transcription factors are a large and diverse group of
proteins which bind to DNA response elements in the promoters
of genes either activating or repressing gene expression.
The purification and characterization of transcription factors
has been improving at a rapid pace. New purification techniques,
including oligonucleotide trapping, and improved methods of
detection, including chromatin immunoprecipitation have been
combined with improved characterization using mass spectrometry
methods. Here, we review and give examples of many of these
recent advances.
[Back to top]
31P NMR Spectroscopy
of Phospholipids: From Micelles to Membranes
Jürgen Schiller, Matthias Müller, Beate
Fuchs, Klaus Arnold and Daniel Huster
Phospholipids are the main constituents of biological
membranes. Their amphiphilic character is responsible for
the typical bilayer arrangement as the structural basis of
biological membranes. In addition to their structural role,
some phospholipids are of significant functional importance.
They act as intercellular messengers and are involved in the
pathogenesis of many diseases. Therefore, phospholipid research
(“lipidomics”) has significantly advanced in the
last decades, generating the need for fast, reliable, and
informative analytical techniques. The aim of this review
is to demonstrate the power of 31P
nuclear magnetic resonance (NMR) spectroscopy for structural
and analytical phospholipid research. High resolution 31P
NMR provides information on the composition of phospholipid
mixtures, whereas solid-state 31P
NMR gives structural information about the sample phase and
morphology. This review provides an introduction into the
field and a short overview of currently used analytical and
physicochemical methods to study these biomolecules. We will
provide a theoretical description of 31P
NMR spectroscopy and discuss methods for obtaining highly
resolved phospholipid spectra. Selected applications of 31P
NMR to aid phospholipid analysis and the investigation of
phospholipid structures, membrane-peptide interactions, and
enzyme activities are discussed. This review ends with an
overview on 31P NMR applications
to the analysis of body fluids, cells, and tissues.
[Back to top]
Pitfalls in High Throughput Screening for Drug
Absorption Optimization in Drug Discovery
Hiroshi Komura and Masahiro Iwaki
Implementation of combinatorial chemistry and high throughput
screening to biological targets has led to produce lipophilic
and poorly water soluble lead compounds. Since these compounds
show poor absorbability, high throughput methods for improving
solubility and membrane permeability have been developed.
Kinetic solubility of compounds after dilution of dimethyl
sulfoxide solution into aqueous buffer is determined using
high throughput methods with turbidimetry, nephelometry or
UV assays. The kinetic solubility tends to be overestimated
compared with the corresponding thermodynamic solubility obtained
using a traditional flask shaking method. A new solid-dissolution
method providing thermodynamic solubility similar to that
in traditional method has been developed using a 96-well plate
for equilibrium dialysis. In conventional Caco-2 assays, the
membrane permeability (Papp)
of certain lipophilic compounds was underestimated due to
insolubility in the apical compartment and adhesion to the
device, resulting in a poor relationship between the in
vivo absorption fraction and the Papp
values. The addition of a solubilizer into the apical compartment
and bovine serum albumin in the basolateral compartment improved
the relationship, enabling evaluation of widely diverse compounds.
The usefulness of these newly-developed methods for compounds
with poor physical properties will be discussed, with reviewing
currently available high throughput methods for optimizing
absorption.
[Back to top]
Hollow-Fiber Flow Field-Flow Fractionation
Andrea Zattoni, Sonia Casolari, Diana Cristina Rambaldi
and Pierluigi Reschiglian
Field-flow fractionation (FFF) is a family of separation
techniques able to fractionate a broad range of macromolecules,
nano- and micro-sized particles of different origin. Among
FFF, flow FFF (FlFFF) shows the highest separation versatility,
and today it is the only FFF technique with significant market
relevance. Commercial FlFFF employs macro-scale, flat-type
channels. The idea of hollow-fiber (HF) membranes as tubular,
micro-column channels for FlFFF (HF FlFFF) dates back 1974,
with fundamentals on HF FlFFF given only in the late 1980s,
and outstanding applications reported only over the last ten
years. Compared to standard FlFFF, the key advantage of HF
FlFFF lies in the instrumental simplicity and low-cost of
the fractionation device, and in the low volume able to favor
on-line coupling with orthogonal methods. Last-generation
HF FlFFF has been successfully applied to macromolecules and
particles such as proteins and whole cells, and shown particularly
suited to be coupled with time-of-flight (TOF) mass spectrometry
(MS) for intact protein profiling and characterization.
We review basics on HF FlFFF theory and instrumentation, the
early investigations, and the most recent technical and methodological
developments that have improved HF FlFFF to a performance
normally achieved by conventional FlFFF.
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