Transcript Proteomica - Uninsubria

Proteomics
• No correlation between mRNA and protein amounts (Gygi
et al., 1999)
• The proteome is an instant picture of the phenotype at
the biochemical level
• The proteome accounts for PTM
“the proteome is the complete set of
proteins of an organ or an organism at a
given time and under specific
physiological conditions” (Wilkins et al.,
1995).
Gygi et al., Mol. Cell. Biol. 19, 1720 (1999);
Wilkins et al., Biotechnol. Genet. Eng. Rev. 13, 19 (1995).
Proteomics
Structural
Large-scale analysis of proteins structure
(X-ray, NMR)
Differential
Large-scale analysis of proteins expression
(2-DE, MS)
Interactions
Large-scale analysis of proteins interactions
(pull-down, affinity purification, two-hybrid system,
network analysis...)
Proteomics challenges
1) Samples are complex
2) You can not amplify proteins
High specificity
=> MS
High sensitivity
=> From proteins to peptides
Proteomics approach
Proteomics approach
Proteomics technologies
Differential Proteomics
Alberio T, Fasano M. J Biotechnol. 2011 PMID: 21925549
Proteomics and Bioinformatics
1) Data analysis
2) Results
interpretation
Quantitative Proteomics
• Proteome = all the proteins expressed in a cell type/ tissue/
organism (at a given time)
• Proteomics = science to characterize the proteome
BUT
• The proteome is dynamic and the identification of a list of
proteins is not sufficient to describe a functional
statequantitative data are fundamental to describe
transitions among several states (environmental variations,
disease, drugs response…)
Gel-based vs. Gel-free
Webb-Robertson and Cannon, Brief Bioinform 2007;8:304-317.
Poor detection of acidic- basic- proteins
poor solubility of membrane proteins
limited loading capacity of gradient pH strips (crowding effect)
Low reproducibility of gels
relatively low throughput
2D gels perform robust separations
2D gels are well-suited for PTM analysis
Parallel, quantitative and label-free readout
Monteoliva and Albar, BRIEFINGS IN FUNCTIONAL GENOMICS AND
PROTEOMICS. VOL 3. NO 3. 220–239.
Proteins do the job, not peptides
Two-dimensional electrophoresis (2-DE)
Staining
pI 3-10 NL
170 kDa
95 kDa
72 kDa
170 kDa
95 kDa
72 kDa
55 kDa
43 kDa
55 kDa
43 kDa
34 kDa
34 kDa
26 kDa
26 kDa
17 kDa
17 kDa
Group B
Group A
pI 3-10 NL
Differential in-gel electrophoresis (DIGE)
 Matching not needed
o High cost
 Spatially accurate
o Weak signal
 Sensitive to small
quantitative changes
o Only binary
comparison
DIGE
Control [Cy5]
Pharmacological Treatment [Cy3]
Mass spectrometer
Source
Analyzer
Detector
To generate gas phase ions
•
Desorption
– MALDI matrix-assisted laser
desorption/ionization
•
Nebulization
– ESI electrospray ionization
m/z
MALDI
•Firing of a laser pulse into a surface on which
is coated a mixture of the sample of interest
(peptide) plus a UV light absorbing matrix
material (cinnamic acid).
•The matrix material acts a conduit for the
energy from the laser to reach the peptide
and generate an M+proton ion.
•The analyte ion is then accelerated out of the
region by the repulsion of the positive charge
on the surface of the sample plate.
•The ion flies down the flight tube
MALDI applications in proteomics
• MALDI imaging:
Direct profiling of tissue
sections. A powerful procedure
to study the spatial distribution
of large and small molecules
directly from a biological
matrix.
• MALDI profiling
ESI
Electrospray is a method used to
produce gaseous ionized molecules
from a liquid solution  from the
chromatographer
Vacuum Interface
charged
droplets form
High Voltage
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Sample
Flow
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Nebulizer Gas
Spray
Ions
released
To MS
Analyzers
• Time of Flight (TOF)
• Quadrupole
• Ion trap
time-of-flight (TOF)
Ion Source
Flight Tube
20-25 kV
Detector
Principle: If ions are accelerated with the same potential at a fixed
point and a fixed initial time and are allowed to drift, the ions will
separate according to their mass to charge ratios.
time-of-flight (TOF)
Ion Source
Flight Tube
Detector
The ions enter the flight tube with the lighter ions
travelling faster than the heavier ions to the detector
time-of-flight (TOF)
Ion Source
Flight Tube
Detector
The lighter ions strike the detector before the heavier ions.
This “time of flight” (TOF) can be converted to mass
MALDI is always combined with ToF
Quadrupole and Ion Trap
Uses a combination of Radio
Frequency (RF) and Direct Current
(DC) voltages to operate as a mass
filter.
• Has four parallel metal rods.
• Lets one mass pass through at
a time.
• Can scan through all masses
or sit at one fixed mass.
• Ions trapped and circulate until sequentially
ejected
• Compact, robust
Tandem MS: MS/MS,
Source
First
analyzer
Selection of a
parent ion with
a specific m/z
Collision
chamber
Fragmentation
1
MS ,
2
MS
Second
analyzer
Detector
Mesurement of
the m/z of
daugther ions
Applying two or more steps of mass analysis separated in space or time (in the same
instrument) to select an analyte (ion) of interest from a mixture and generate
fragments from it to give structural informationsequence
An example: Q-ToF
Gel-free: shotgun
Charged peptides
Issues:
•Ion intensity is not proportional to
quantity.
It depends on
peptides
physicochemical properties (charge,
hydrophobocity…)
•Spectra of the same ion in different
selected and
runs are influencedPeptides
by
external
fragmented
variables (e.g. chromatography)
which may casually alter ionization
efficiency
Reverse Phase Liquid
Chromatography
High pressure is used (High
Performance)
Peptides acelerated
by magnetic fields in
the analyzer and
separated by m/z
Strategies to have a quantitative MS
• Mix samples(e.g. control and treated
samples) to conduct only one analysis:
a priori
- Non-radioactive tracers (15N, 13C, 18O, 2H) to label different samples
identical proteomes (same carachteristics, same behaviour during
chromatography= ionization ) but ≠ mass
-Peptides from different samples generate a doublet of peaks. The ratio of
peak intensities is proportional to the protein abundance ratio
• Data analysis and elaboration from
different runs (label free): a posteriori
Quantitative Proteomics
•Labelling (ICAT, iTRAQ, SILAC, 18O enrichment, …)
•Label free (AQUA, SRM/MRM, …)
Isotope-coded affinity tagging (ICAT)
To purify with resins conjugated
with avidin
Reaction with Cys (-SH)
8 hydrogens or
deuteriums
PRO: simply spectra (only peptides with
Cys)
CONS:
•Quantification is based on one peptide
only (or few)
•Impossible to quantify proteins without
Cys
•In MS/MS the tag is fragmented 
noise in the MS/MS spectra
•light o heavy peptides do not perfectly
co-elute
•Only two samples
Isobaric Tagging for Relative and Absolute
Quantitation (iTRAQ)
PRO:
•Reaction with primary amines
•Multiplexing (till 8)
CONS:
•Variable efficiency of chemical labelling
•Tags added just before MS (differences
during samples preparation)
SILAC
(Stable Isotope Labeling of Amino acids in Cell culture)
•Stable isotopes are added in culture media.
•Isotopes are included in new-synthetized proteins.
Natural isotopes
in essential
aminoacids
(light medium)
Stable isotopes in
essential
aminoacids
(heavy medium)
•Relative quantification in MS1, on the basis of the intensities of the same
peptide
•PRO: early labelling : no technical variability accurate quantification
•CONS: only in proliferating models
SILAC in Silac-mouse
Label-free
Normally used for clinical proteomics (many samples, same tissue,
limited quantities)
1) Differential quantification of one or few peptides of the same protein
in different samples (“intensity-based”);
-Thanks to high resolution analyzers (ToF, Orbitrap..)
-High reproducibility of chromatografy and of the analyzer needed
-Algorithms to correct differences and allign chromatograms
2) Differential quantification based on spectral-counting
-Rationale: the number of peptides identified for one protein correlates with
its abundance
-BUT the number of peptides depends also on the protein length 
normalization index
-CONS: the proportion is not true for all the proteins
-Difficult to quantify little differences
Gel-free targeted
Selected/Multiple reaction monitoring (SRM/MRM)
•Only a defined number of peptides – corresponding to a protein of
interest – are monitored
Triple quadruple
Precursor/fragment
intensity is quantitative
Ritention time of
chromatography
•A lot of preliminary work: find proteotypic peptides
•Alternative to ELISA in clinical studies
Absolute Quantification
= to determine the
concentration of a protein
in a sample as ng/mL or
number of copies/cell
1) Quantification is possible thanks to isotope-labelled standards
(petides/proteins)
-Standard Peptides (Absolute QUAntification -AQUA-) or proteins (absolute SILAC, Protein
Standard for absolute Quantification - PSAQ-) are added to the sample at a known concentration
-A lot of preliminary work to choose and synthetize peptides with a known mass (incorporated
isotopes)
2) Quantification is obtained elaborating MS data and appliying algorithms
-Es. APEX algorithm based on machine-learning: thanks to experimental data is possible to
estimate the probability to detect a peptide
What next?
• You will call your preferred MS expert to ask
her/him to identify your spots
• You will get a list of protein names
• What tells you that list?