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Printable Handouts
Navigable Slide Index
- Introduction
- Talk outline
- Proteomics is a component of systems biology
- What is proteomics?
- The dynamic proteome
- Proteome flux
- A distractive slice
- Current proteomic strategies
- Proteomics and scientific method
- Current approaches to proteomics
- Protein identification by mass spectrometry
- Intact proteins - identification
- Peptide mass fingerprinting
- Mass accuracy helps peptide mass fingerprinting
- Peptide sequence tags
- Example of MSMS experiment
- What about covalent modifications?
- Algorithms match sequence tag data
- Problem of complex mixtures
- Three aspects of the problem
- Shotgun proteomics
- Multidimensional protein identification technology
- Accurate mass tags
- Problem with 'shotgun' proteomics
- Intact proteins
- 2D-gel electrophoresis
- Blue native gel of membrane protein complexes
- 2D-chromatography
- Sub-fractionation
- Quantative proteomics and stable isotope labeling
- Quantitation is challenging
- Isotope-coded affinity tags (ICAT)
- ICAT experiment
- Net result of expereiment
- Mass spectrometric approaches to quantitation
- Subtle modification of isotope-ratio proteomics
- Isosolv algorithm
- Subtle modification of isotope ratio
- Intact mass measurement, 'top-down' proteomics
- Intact mass proteomics
- Intact mass tags from isolated thylakoids
- Identification of intact mass tags
- Sequence errors are predictable
- Intact protein mass spectrum defines gene product
- D1 herbicide receptor
- Integral membrane proteins
- Chromatography of membrane proteins
- Subunits of cytochrome b6f complex
- Intact mass proteomics of thylakoid membrane
- Crystal structure of cytochrom b6f complex
- Confirmatory data necessary
- Workflow for intact mass proteomics
- LC-MS+
- Determination of signal peptide cleavage sites
- Lhcb4 (CP29)
- Lhcb5 (CP26)
- PsbS
- PsbO isoforms
- 58 signal peptide cleavage sites
- PetM is nuclear encoded cleavage site
- Workflows for the integral membrane proteome (1)
- "Top-down" sequencing of intact proteins
- Top-down MSMS
- Top-down sequence of intact small subunits
- FT-ICR MS of integral membrane proteins
- SORI-CAD of bactriorhodopsin apoprotein
- 3 most abundant ions resulting from SORI-CAD
- Workflows for the integral membrane proteome (2)
- Reverse phase chromatogram
- Why are 'top-down' approaches important?
- Oxidative modification linked to phosphorylation
- Conclusions from thylakoids
- Summary
- Coworkers
Topics Covered
- What is proteomics and what does it measure?
- Protein identification by mass spectrometry
- The problem of complex mixtures and protein identification
- Quantitative proteomics and stable isotope labelling
- Intact protein mass measurements and top-down proteomics Update talk: What is proteomics and what does it measure
- Update talk: Top-down high-resolution FT-ICR mass spectrometry
- Update talk: Characterization of thylakoid membrane
- Update talk: Membrane proteins and lipids
- Update talk: Lipid-protein complexes in gels
- Update talk: PSI-ATP synthase supercomplex
Talk Citation
Whitelegge, J. (2021, October 31). The chloroplast proteome [Video file]. In The Biomedical & Life Sciences Collection, Henry Stewart Talks. Retrieved August 31, 2024, from https://doi.org/10.69645/VBPH9338.Export Citation (RIS)
Publication History
Financial Disclosures
- Dr. Julian Whitelegge has not informed HSTalks of any commercial/financial relationship that it is appropriate to disclose.
Update Available
The speaker addresses developments since the publication of the original talk. We recommend listening to the associated update as well as the lecture.
- Full lecture Duration: 42:32 min
- Update Duration: 9:28 min
A selection of talks on Cell Biology
Transcript
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0:00
The chloroplast proteome.
0:03
In this presentation,
I will review five subjects.
What is proteomics and
what does it measure?
Protein identification
by mass spectrometry,
including intact proteins, peptide
mass fingerprinting and sequence tags.
The problem of complex mixtures
and protein identification.
Quantitative proteomics and
stable isotope labeling,
intact protein mass measurement
and 'top-down' proteomics.
Let's start off by considering
what is proteomics and
what does it measure?
0:30
Proteomics is a component
of systems biology.
"We would like a computer model
that mimics life in silico
allowing accurate projections for
metabolic engineering experiments."
0:42
What exactly is proteomics?
Proteomics is the science
of describing the
complete set of proteins
expressed from an organism's
genetic material,
their interactions
with each other
and the influence
of development,
environment and disease
upon their expression.
Since understanding their
structure and function of
the gene products and their
interrelationships are the overall goals,
we can regard proteomics
alongside structural genomics
as a component of functional
genomics and systems biology.
1:09
The cellular proteome
is a dynamic beast
continuously expressed
from the genome
under the influence of the
environment and nutrient conditions.
The proteome impinges back upon
the genome and back upon itself
to accomplish short-term
acclimation over minutes,
medium-term acclimation
over hours and days,
often involving changes
in gene expression,
and ultimately accomplishing
development of growth,
reproduction and evolution.
1:35
We can consider proteome flux,
the flow of nutrients and
energy through transcription,
translation and
protein degradation
that is controlled
by the genetic code
and numerous factors that
influence expression.
Ideally, we would like to
monitor global gene expression,
protein post-translational
modification and turnover
with molecular resolution and
non-invasive technologies.