The
multidisciplinary science of chemical proteomics studies how small molecules of
synthetic or natural origin bind to proteins and modulate their function.
Scientists in the field have different backgrounds including molecular and cell
biology, biochemistry, pharmacology, organic chemistry, and physics. Chemical
Proteomics: Methods and Applications is directed at molecular biologists
and biochemists with either an interest in small molecules themselves, e.g., in
drug discovery projects, or in using small-molecule probes as research tools to
study protein function. The book may also be useful for organic chemists with
an interest in biology and for specialists in protein mass spectrometry.
In the
introductory chapters, we discuss analytical strategies for chemical proteomics
projects, with a focus on the current state-of-the-art in protein mass
spectrometry, and describe several examples how chemical proteomics can impact
the field of drug discovery. The consecutive chapters provide detailed
experimental protocols. Most chemical proteomics projects consist of three
parts. In the first part, the chemical probe is selected or designed, and then
synthesized. In the second part, the probe compound is exposed to the protein sample
or cell extract. In the final part, proteins binding to the probe compound are
identified and often quantified. In simple applications, this is often achieved
by antibody-based detection, but if the aim is the discovery of targets in an
unbiased fashion, mass spectrometry is the method of choice. The following
chapters cover all of these aspects.
The first set of
chapters describes how probes are generated from commercially available reagents
without elaborate chemical synthesis procedures, and how the proteins binding
to the probes can be analyzed by immunodetection or by mass spectrometry. Rix
et al. And Saxena provide protocols for direct noncovalent affinity capture
using protein kinase inhibitors as an example, which serves to profile the
targets of these compounds and provides probes for kinase expression and
activity. Ge and Sem have developed a target class-specific probe for the
labeling of dehydrogenase enzymes. Kawamura and Mihai, and Codreanu et al.
describe the use of biotin-conjugated probes which form covalent adducts with
defined subproteomes, here consisting of adenine-binding proteins and targets
of lipid electrophiles.
Lenz et al.
combine features of noncovalent and covalent capturing strategies in their bifunctional
ligands designed to target methyl transferases, an emerging class of drug
targets. The second set of chapters is concerned with techniques for the
discovery of small molecule targets and the probing of target function. Ong et
al. describe the use of stable isotope labeling of amino acids in cell culture
(SILAC) in identifying proteins that bind small-molecule probes in cell
extracts. Hopf et al. perform affinity enrichment of target proteins on a probe
matrix in the presence of competing free test compound in solution, thus enabling
determination of binding potencies of the free test compound to
affinity-captured proteins from cell extracts. The method employs quantitative
mass spectrometry with isobaric mass tags to determine the potencies for a
large number of targets in a single analysis.
Ge and Sem have
developed a protocol for the detection and purification of dehydrogenase
enzymes, which may represent targets, but also unwanted off-targets, for
certain types of drugs. Kovanich et al. describe a combination of cAMP-based
affinity chromatography with quantitative mass spectrometry to investigate
protein kinase A complexes in extracts of cells and tissues. De Jong et al. use
activity-based chemical probes to profile the activity of the proteasome, which
has recently emerged as an important cancer target, in cells and tissues. The
next three chapters provide innovative protocols for the study of potential
drug targets by chemical cross-linking and mass spectrometry. Mueller et al.
provide a method to study protein–drug interactions, and Gasilova et al. employ
cross-linking and MALDI-mass spectrometry to study ligand modulation of
protein–protein interactions. Jeon et al. provide a protocol for in vivo
cross-linking via time-controlled transcardiac perfusion, which in principle
enables the direct analysis of protein targets in animal models. The final set
of chapters is concerned with small-molecule ligand and drug discovery. Casalena
et al. describe the discovery of probe compounds by utilizing compound
libraries immobilized on microarrays. Wolf et al. delineate general guidelines
for working with small molecules, including aspects like storage, the
preparation of solutions, and the determination of solubility. The chapter by
de Matos et al. provides guidelines for the use of the ChEBI database, which
should be very helpful for researchers tasked with the selection of a
particular probe or with building a small molecule collection to purpose. They
describe the Chemical Entities of Biological Interest (ChEBI) database which
helps to find probe molecules with the desired structural or biological
features. Finally, many researchers will consider whether their research tool
compound might have the potential to be developed into a drug. Zhang delivers a
lucid analysis of the features that distinguish drugs from probe molecules, and
lays out a set of rules for “drug likeness.” Affinity- and activity-based
chemical probes, combined with quantitative immunodetection and mass
spectrometry techniques, are increasingly gaining appreciation as powerful
strategies for the molecular analysis of complex biological systems in
homeostasis and disease. We hope that the methodologies described in this volume
will contribute to a wider application of chemical proteomics methods in
biochemical and cell biological laboratories.
0 comments:
Post a Comment