Download Proteomics - University of Warwick

An Organism is typically an individual life form composed of interdependent parts (organs). The organs have specific functions and they are composed by cells. A cell is the smallest structural and functional unit of an
organ and is microscopic. Proteins do most of the work in cells and are required for the structure, function, and regulation. Proteins are made up of hundreds or thousands of smaller units called amino acids, which
are attached to one another in long chains. The proteome is the entire complement of proteins that is or can be expressed by a cell, an organ or an organism and the study of proteomes at each of these levels is
called Proteomics.
Peptide
Amino acid
Organ or Tissue
Cell
Protein
Organism
Proteome
The scientific method starts with a problem/question identification and study of relevant previous data. Then, a hypothesis is formulated from these observations and is empirically tested by a specific experiment.
At Warwick staff in the Proteomics Research Technology Platform are experts in mass spectrometry based proteomics which investigate the proteomes/proteins and their functions. We are here to help you with
proteomic experiments.
Mass spectrometry Bioinformatics
Test Hypothesis*
Application
Observation Hypothesis* Experimental design
Experiment
Protein digestion
Protein extract
Peptides
analysis
analysis
A standard proteomics experiment compares the protein levels of two or more biological conditions (e.g. Healthy vs Disease). The protein extracts are fractionated to reduce their complexity ( often using protein
electrophoresis, SDS-PAGE), before in gel digestion to create smaller fragments of proteins called peptides. These peptides enable protein identification and quantification by mass spectrometry. Other types of
experiments are possible, so please discuss your ideas with us before extracting proteins!
Experiment
SDS-PAGE
Protein extract
Reduction
Alkylation
S-S
SH
DTT
Control
Condition A
3 biological replicates
Protein digestion
Peptides
Mass spectrometry
StageTip C18
IAA
SH HS
SCH2-CONH2
50µg of total protein
Desalting and concentration
Trypsin cuts on arginine
and lysine
Mass spectrometric analysis usually starts with chromatography of the peptides by Reverse Phase C18 (RP-C18) coupled with a electrospray ionization source. RP-C18 reduces the complexity of the peptide mixture by
gradually eluting them off the column using a hydrophobic stationary phase composed by octadecyl carbon chain (C18)-bonded silica. Hydrophobic peptides bind to the column in 0.1% formic acid aqueous solvent
and eluted with increasing concentration in a linear gradient of acetonitrile (organic solvent). Eluted peptides are desolvated and ionised by Electrospray ionization (ESI). The positively charged peptides (now called
ions) fly down a voltage gradient into the mass spectrometer to strike a detector for analysis. The mass of the peptide and their fragments is calculated from the flight path.
1µg of peptides
% Organic solvent
Sample injection
Chromatography RP-C18
C18 Peptide
Elution
Binding
Full mass spectrum (MS)
Electrospray ionization (ESI)
+
Intensity
Technology
Workflow
Strategy Proteomics
Proteomics step by step
-
+
Fragmentation spectrum (MS/MS)
Peptide
+
+++++ +++ +
+
+++++ +++ +
+ +
+
+
++ +
+
+
+ ++
+++++
+++
RP-C18
x25
+
Mass spectrometer
Time (1-4 h)
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+
+
+
+
MS scan per second
The trick!
25 MS/MS scans per second
Data dependent analysis (DDA) first selects peptide precursors from a full mass spectrum scan (MS) and then acquires MS/MS scans centered on these chosen masses. The cycle of selection and fragmentation is
then repeated. The selected, individual peptides are accumulated in the ion trap and fragmented upon an energy pulse in the presence of an inert gas (collision gas). All fragments are measured in a new mass
spectrum (MS/MS). Finally, search engine software uses known protein sequences to match the peptide fragmentation patterns (MS/MS).
Peptide isolation and accumulation
Full mass spectrum (MS)
+
Helium
Peptide ion
H2N- M Y
H2N- M Y G A V D R -COOH
G
A
V
D R -COOH
M Y G A V D
M Y G A V
Y G A V D R
G A V D R
M Y G A
Precursor peptide selection
Fragmentation spectrum (MS/MS) and peptide identification
Peptide fragmentation
Ion trap
b ions
A V D R
y ions
Results
Protein and peptide identification software uses known protein sequences to match the calculated in silico protein digestion and peptide fragmentation with the real peptide fragmentation acquired in the mass
spectrometer. Each identified mass in the MS is assigned to a peptide and each mass in the MS/MS is assigned to a peptide fragment. All peptides that belong to the same protein will contribute to its identification
and relative quantification. Complex algorithms combine the peptide intensities from the same protein and calculate the protein relative abundance. Finally, data analysis involves relative quantitative comparison
and selection of regulated proteins as a candidates for further investigations.
Protein database in silico digestion
MS/MS peptide search
Peptide/protein identification and quantification
Re-building proteins from peptides
Data analysis
Control Condition A
Protein X1
Protein X1
Protein C8
Protein sequence coverage
Protein database
Protein F7
Proteomics RTP
University of Warwick
Contact us:
School of Life Sciences. Gibbet Hill Campus
University of Warwick
Coventry. CV4 7AL
Email: [email protected]
www2.warwick.ac.uk/fac/sci/lifesci/research/facilities/proteomics/
Our Team:
Director: Alex Jones
Manager: Juan Ramon Hernandez-Fernaud
Chief Technician: Cleidiane Zampronio
RTP Officer: Ian Hancox