Biological
Mass Spectrometry and Proteomics
We
are using modern mass spectrometry based on both MALDI or electrospray
ionization to characterize a wide range of biomolecules as well as their
complexes. In the context of proteomics we are using these techniques
to discover the function of new proteins and their modifications in
various biological systems. These studies will provide fundamental knowledge
on how cells work and, in the biomedical context, how diseases evolve.
These latter studies could lead to new diagnostics and to new drug targets.
We collaborate with numerous scientists from UWO, the Robarts Research
Institute, the Child Health Research Institute and from elsewhere in
Canada. In parallel, we are developing alternative approaches for protein
isolation and purification. In addition, in collaboration with colleagues
from the Department of Computer Science, we are developing new software
tools to handle the large number of complex data generated by mass spectrometry.
Currently, we are working on the following projects.
1. Proteomic analysis of undifferentiated human embryonic stem cell growth
Human
embryonic stem cells (hESC), derived from the inner cell mass of pre-implanted
embryos, are stable, pluripotent cell lines that can proliferate indefinitely
in culture. These
cell types and their derivatives have shown huge clinical potential
in cell replacement therapies and regenerative medicine.
Unfortunately, little is known with respect to molecular
mechanisms controlling in vitro
proliferation and differentiation.
Furthermore, most of what is known about hESC has proven to
be in contrary to previous discoveries made using their mouse counterparts.
Knowledge of these fundamental mechanisms will be essential
in order to effectively proliferate and differentiate hESC into various
enriched cell populations for future therapeutic use.
My project involves the proteomic characterization of undifferentiated
hESC growth. It is a
two pronged approach investigating both essential protein factors
in hESC growth media as well as key phosphorylation events induced
by basic fibroblast growth factor stimulation.
This work is being carried out in conjunction with Mick Bhatia
of the Krembil Centre for Stem Cell Biology at Robarts Research Institute.
Preeclampsia is a disease affecting
5-8% of pregnancies worldwide and is characterized by hypertension,
proteinuria and edema during pregnancy.
Despite current research efforts, very little is known about
what causes this condition and the underlying factors involved in
its development. By sampling
preeclamptic placental tissues and comparing them to gestationally
age matched controls some insight will be gained as to what the causative
factors are in this disease.
My
project is the proteomics based comparison of placental tissues from
preeclamptic pregnancies and gestationally age matched controls.
Access to clinical samples is facilitated through collaboration
between our lab and Dr. Victor Han at the Child Health Research Institute. Through the development and application of new and existing
proteomic differential display strategies, we hope to elucidate the
underlying factors of this disease.
3.
Proteomics of Intrauterine Growth Restriction
(Dr. Suya Liu)
Intrauterine Growth Restriction (IUGR) is among the major causes of
neonatal morbidity and mortality in western countries. The pathogenic
reason of IUGR is not well understood and the early diagnosis is hindered
due to lack of a biochemical test. This project is performed in collaboration
with Dr Victor Han and Dr Madhulika Gupta from the Child Health Research
Institute and includes two parts: one is to find biomarkers for IUGR
by using mass spectrometry for a global proteomics analysis of the
IUGR patient plasma; another is to study the phosphorylation of the
insulin-like growth factors binding protein 1 (IGFBP1) and to answer
the question whether the abnormal phosphorylation of IGFBP1 is responsible
for the development of IUGR, as IGFBP1 is a key factor in regulation
of fetal growth by binding the IGF.
4. Proteomic Analysis of Lung Surfactant Proteins
(Dr.
Suya Liu)
Pulmonary
surfactant consists of surfactant proteins and phospholipids. One
of its essential roles is to reduce the surface tension at air-liquid
interface of the alveolar lining layer so maintain a normal lung function.
Surfactant oxidation and dysfunction could contribute to several lung
related diseases. It
is very important to study how the oxidation affects the chemical
structure of surfactant proteins and phospholipids and what is the
consequence of those changes on the function of surfactant. We use
mass spectrometry to analyze the surfactant proteins and phospholipids
for identification of chemical modifications(s) upon the oxidation.
5. Functional proteomics by MS of
non-covalent complexes of protein-ligand
(Dr. Suya Liu)
Nuclear receptors are transcription factors which regulate the expression
of target genes and have important biological functions in control
of development and metabolism of eukaryotic cells. The activities
of these proteins are controlled by small hydrophobic ligands. However,
for many of these nuclear receptors their nature ligand(s) have not
been identified yet. The aim of this project is to identify these
unknown ligands for selected nuclear receptors using electrospray
mass spectrometry. Electrospray
is the softest ionization technique for protein analysis so that the
binding of a ligand to a nuclear receptor would be maintained and
analyzed by mass spectrometry.
The observation of the binding of an endogenous small molecule
to the protein will provide a good starting point for identification
of a nature ligand for the nuclear receptor. Identification of a nature
ligand will allow the investigators to study the mechanism of actions
in new and novel ways and will be very important for the design of
new drugs target these receptors. This work is done in collaboration
with Professor Aled Edwards from the University of Toronto.
6.
Software Development
for Proteomics
(Cunjie Zhang,
Amanda Doherty-Kirby, Ryan McLeod)
Despite major advances in mass spectrometry instrumentation, the interpretation
of the data is often the slow step in analyzing samples.
Our group has been working with Dr Bin Ma and Dr Kaizhong
Zhang and their students Baozhen
Shan, Hailing
Zhang, Yonghua Han, Li Wang and Weiwu Chen from the UWO Computer Science
Department to develop various softwares for data analysis. Current projects include the development of software for de
novo sequencing by MS/MS (PEAKS); a software for deconvolution of
raw MS data of complex protein mixtures (FOOTPRINT); a software to deduce
the structure of glycans of from MS/MS spectra of glycopeptides (GlycoMaster)
and a software to predict post- translational of proteins (PredictPTM)
and their corresponding mass.
Inhibitors
Proteins of Serine Proteases
Many
physiological processes are controlled by inhibitory proteins. An imbalance
in the protease-antiprotease can result in severe disease states. The
study the binding region of the inhibitory proteins is important to
understand these interactions and could lead to novel therapeutic agents.
We are studying a small inibitory protein eglin c, isolated from the
leech Hirudo medicinalis. The goal is to develop highly selective
and non-immunogenic proteins as inhibitors of physiologically relevant
proteases. With the appropriate mutation in the binding domain of eglin
c, we have obtained extremely potent inhibitors (Ki ~10-14 M) which
are ~10,000 times more potent for human leukocyte elastase (HLE) than
for chymotrypsin and cathepsin G. These enzymes have been implicated
in the development of emphysema and rheumatoid arthritis.
Antimicrobial
Peptides
Histatins
are a class of antimicrobial peptides found in the saliva of man and
of other primates. They have a broad range of antimicrobial activity
and are believed to be an important in the non-immune response to invading
organisms. We have synthesized many members of the histatins family
and analogs. We are studying their conformational properties by various
biophysical techniques such as CD and NMR in different environments
in order to gain insight in their mode of action. Based on NMR studies,
we have designed analogs that are 100 times more potent than the natural
sequence as antifungal agent.
Amino Acids Chemistry
We
have recently discovered a general method for the synthesis of optically
active alpha-amino acids. The method is based on the utilization of
an ortho ester as a blocking group on the carboxylic acid function of
serine. The hydroxyl function can then be oxidized to the corresponding
aldehyde or substituted without racemization. This approach allows for
the synthesis of several uncommon amino acids that are found in many
natural products. We have used this approach to prepare amino acid analogs
as selective receptor agonists for the glutamate receptors or as enzyme
inhibitors for important enzymes such as nitric oxide synthase(NOS)
(in collaboration with Prof. Guy Guillemette, University of Waterloo).
