Transcript Proteomics and Mass Spectroscopy

Proteomics
and
Mass Spectroscopy
Proteomics
• The dream of having genomes completely sequenced is now
a reality. The complete sequence of many genomes
including the human one is known.
• However, the understanding of probably half a million
human proteins encoded by less than 30,000 genes is still a
long way away and the hard work to unravel the complexity
of biological systems is yet to come.
• A new fundamental concept called proteome (PROTEin
complement to a genOME) has recently emerged.
Proteomics
• should drastically help to unravel biochemical and
physiological mechanisms of complex multivariate diseases
at the functional molecular level.
• The discipline of proteomics has been initiated to
complement physical genomic research.
• The term “proteome” was coined in 1994 by an Australian
graduate student (Mark Wilkins), it has come to be used and
defined in a variety of different ways
Proteomics
• Definition-The identification, characterization and
quantification of all proteins involved in a particular
pathway, organelle, cell, tissue, organ or organism
that can be studied in concert to provide accurate
and comprehensive data about that system.
• Or - A complete description of proteins expressed in
any given cell at any given time
Proteomics
• A cellular proteome is the collection of proteins found in
a particular cell type under a particular set of
environmental conditions such as exposure to hormone
stimulation
• It can also be useful to consider an organism's complete
proteome, which can be conceptualized as the complete
set of proteins from all of the various cellular proteomes.
This is very roughly the protein equivalent of the genome.
• The term "proteome" has also been used to refer to the
collection of proteins in certain sub-cellular biological
systems. For example, all of the proteins in a virus can be
called a viral proteome.
Proteomics
• So where are we in our understanding of the
cell?
– 31-60 K total genes in the human genome with little
difference between the fruit fly and us!
– Where does the diversity come from?
Answer: It’s the proteins!!!!!!!!!!!!
Proteomics
• The proteome is larger than the genome, especially in
eukaryotes, in the sense that there are more proteins than
genes.
• This is due to:
• alternative splicing of genes
• post-translational modifications like glycosylation or
Phosphorylation.
•
alternative splicing of
genes
A given piece of pre-mRNA
which has been transcribed from
one gene can be chopped and
reconnected in different ways to
yield various new mRNAs which
then exit the nucleus to be
translated in the cytoplasm.
• When the pre-mRNA has been
transcribed from the DNA, it
includes several introns and
exons.
• The regulation and selection of
splice sites is done by
Serine/Arginine-residue proteins
alternative splicing of genes
• Four known modes
• A - Alternative selection of promoters:
this is the only method of splicing which
can produce an alternative N-terminus
domain in proteins. In this case, different
sets of promoters can be spliced with
certain sets of other exons.
• B - Alternative selection of
cleavage/polyadenylation sites:
• this is the only method of splicing which
can produce an alternative C-terminus
domain in proteins. In this case, different
sets of polyadenylation sites can be spliced
with the other exons.
alternative splicing of genes
• Four known modes
• C - Intron retaining mode: Instead of
splicing out an intron, the intron is retained
in the mRNA transcript.
– However, the intron must be properly
encoding for amino acids. The intron's
code must be properly expressible,
otherwise a stop codon or a shift in the
reading frame will cause the protein to
be non-functional.
• D -Exon cassette mode: Certain exons are
spliced out to alter the sequence of amino
acids in the expressed protein.
post-translational
modifications
• PTMs involving addition include:
• Acetylation - the addition of an acetyl group, usually at the
N-terminus of the protein
• Alkylation - the addition of an alkyl group (e.g. methyl,
ethyl)
• Methylation -the addition of a methyl group, usually at
lysine or arginine residues. (This is a type of alkylation.)
• Biotinylation - acylation of conserved lysine residues with a
biotin appendage
• Glutamylation - covalent linkage of glutamic acid residues
to tubulin and some other proteins.
post-translational
modifications
• PTMs involving addition include:
• Glycylation - covalent linkage of one to more than 40
glycine residues to the tubulin C-terminal tail
• Glycosylation - the addition of a glycosyl group to either
asparagine, hydroxylysine, serine, or threonine, resulting in
a glycoprotein
• Isoprenylation - the addition of an isoprenoid group (e.g.
farnesol and geranylgeraniol)
• Lipoylation - attachment of a lipoate functionality
post-translational
modifications
• PTMs involving addition include:
• Phosphopantetheinylation - the addition of a 4'phosphopantetheinyl moiety from coenzyme A, as in fatty
acid, polyketide, non-ribosomal peptide and leucine
biosynthesis
• Phosphorylation - the addition of a phosphate group,
usually to serine, tyrosine, threonine or histidine
post-translational modifications
• For instance, the peptide
hormone insulin
• Cut twice after disulfide
bonds are formed, and a
propeptide is removed from
the middle of the chain;
• The resulting protein
consists of two polypeptide
chains connected by
disulfide bonds.
Proteomics - Bridging the genome
to the functions of the cell
Areas of Proteomics
• Protein Analysis/Chemistry - Look at PT modifications,
structure and function, enzyme behavior
• Expression - what why and when - 2D gels, MS,
HPLC/protein chips,
• Cell Mapping - protein-protein interactions - affinity tags,
two hybrid, antibody pull down
Proteomics
• A surprising finding of the Human Genome Project is that
there are far fewer protein-coding genes in the human
genome than proteins in the human proteome
– 20,000 to 25,000 genes coding for proteins.
– about 1,000,000 proteins.
• The human body may contain more than 2 million proteins,
each having different functions.
• The discrepancy implies that protein diversity cannot be
fully characterized by gene expression analysis, thus
proteomics is useful for characterizing cells and tissues.
So how does it work?
• Most proteins function in collaboration with other proteins,
and one goal of proteomics is to identify which proteins
interact.
• This often gives important clues about the functions of
newly discovered proteins
So how does it work?
• Proteins are resolved, sometimes
on a massive scale. Protein
separation can be performed using
2-D gel electrophoresis,
– `usually separates proteins first by
isoelectric point and then by
molecular weight.
• Once proteins are separated and
quantified, they are identified
• Individual spots are cut out of the
gel and cleaved into peptides with
proteolytic enzymes
So how does it work?
• These peptides can then be
identified by mass spectrometry,
• Specifically: matrix-assisted laser
desorption-ionization time-offlight (MALDI-TOF) mass
spectrometry.
• In this procedure, a peptide is
placed on a matrix, which causes
the peptide to form crystals.
So how does it work?
• Then the peptide on the matrix is
ionized with a laser beam and an
increase in voltage at the matrix is
used to shoot the ions toward a
detector in which the time it takes
an ion to reach the detector
depends on its mass.
• The higher the mass, the longer
the time of flight of the ion.
So how does it work?
• In a MALDI-TOF mass
spectrometer, the ions can also be
deflected with an electrostatic
reflector that also focuses the ion
beam.
• Thus, the masses of the ions
reaching the second detector can
be determined with high precision
and these masses can reveal the
exact chemical compositions of
the peptides, and therefore their
identities!
So how does it work?
• Protein mixtures can also be analyzed without prior
separation.
• These procedures begin with proteolytic digestion of the
proteins in a complex mixture
• The resulting peptides are often injected onto a high
pressure liquid chromatography column (HPLC) that
separates peptides based on hydrophobicity.
• HPLC can be coupled directly to a time-of-flight mass
spectrometer using electrospray ionization
So how does it work?
• electrospray ionization:
A technique used in mass
spectrometry to produce
ions.
• It is especially useful in
producing
ions
from
macromolecules because it
overcomes the propensity
of these molecules to
fragment when ionized
So how does it work?
• Peptides eluting from the column
can be identified by tandem mass
spectrometry (MS/MS).
• The first stage of tandem MS/MS
isolates individual peptide ions,
and the second breaks the
peptides into fragments and uses
the fragmentation pattern to
determine their amino acid
sequences.
• Labeling with isotope tags can be
used to quantitatively compare
proteins concentration among two
or more protein samples.
So how does it work?
• Finally, use databases.
• Computer compares sequences to other sequences stored in an
internationally accessible database.
• Determines the identity of the isolated protein
• As the entire human genome is known, computers are able to
determine nearly every potential protein.
• New proteins are “discovered” when they match sequences predicted
by the computer that have not previously been found.
Medical Applications
 Alzheimer’s disease:
 Elevations in beta secretase creates amyloid/beta-protein,
which causes plaque to build up in the patient's brain, which
causes dementia.
 Targeting this enzyme decreases the amyloid/beta-protein
and so slows the progression of the disease.
 A procedure to test for the increase in amyloid/beta-protein
is immunohistochemical staining, in which antibodies bind
to specific antigens or biological tissue of amyloid/betaprotein.
Medical Applications
 Heart disease:
 Commonly assessed using several key protein based biomarkers.
 Standard protein biomarkers for CVD include interleukin-6,
interleukin-8, serum amyloid A protein, fibrinogen, and troponins.
 cTnI cardiac troponin I increases in concentration within 3 to 12 hours
of initial cardiac injury and can be found elevated days after an acute
myocardial infarction.
 A number of commercial antibody based assays as well as other
methods are used in hospitals as primary tests for acute myocardial
infarction.
Medical Applications
 Renal cell carcinoma:
 Proteomic analysis of kidney cells and cancerous kidney
cells is producing promising leads for biomarkers and
developing assays to test for this disease.
 In kidney-related diseases, urine is a potential source for
such biomarkers.
 Recently, it has been shown that the identification of urinary
polypeptides as biomarkers of kidney-related diseases
allows to diagnose the severity of the disease several
months before the appearance of the pathology.
Medical Applications
 . Phenylketonuria (PKU)
– Affects in in 5,000 newborns
– Most common nervous system disorder
• Allele is on chromosome 12
– Lack the enzyme needed for the metabolism of
the amino acid phenylalanine
– A build up of abnormal breakdown pathway
• Phenylketone
• Accumulates in urine. If diet is not checked,
can lead to severe mental retardation
Overview of
proteomics
• Nucleus
•
DNA – RNA
• Cytoplasm
– Protein expression and
modification
• Protein isolation
• Mass spectroscopy
• Protein sequence
• Identification
Overview of proteomics
• Proteomics research is
highly interdisciplinary,
bringing together:
•
•
•
•
•
biology
chemistry
instrumentation
Statistics
computer science
Overview of proteomics
Summary time!
• Spend 15- 20 mins going over this as a group
• MAKE NOTES!!!!!!!!!!!!
• http://www.childrenshospital.org/cfapps/researc
h/data_admin/Site602/mainpageS602P0.html
Mass Spectroscopy
Mass Spectroscopy
• An analytical technique used to measure the mass-to-charge
ratio of ions.
• It is most generally used to find the composition of a
physical sample by generating a mass spectrum representing
the masses of sample components.
• The technique has several applications, including:
• 1) identifying unknown compounds by the mass of the
compound molecules or their fragments
Mass Spectroscopy
• 2) determining the isotopic composition of elements in a
compound.
• 3) determining the structure of a compound by observing its
fragmentation
• 4) quantifying the amount of a compound in a sample using
carefully designed methods (mass spectrometry is not
inherently quantitative)
Mass Spectroscopy
• 5) studying the fundamentals of gas phase ion chemistry
(the chemistry of ions and neutrals in vacuum)
• 6) determining other physical, chemical, or even biological
properties of compounds with a variety of other approaches.
• A mass spectrometer is a device that measures the mass-tocharge ratio of ions.
• This is achieved by ionizing the sample and separating ions
of differing masses and recording their relative abundance
by measuring intensities of ion flux.
Mass Spectroscopy
• All Mass specs consist of:
• A high vacuum system
– 10-6 torr
• A sample inlet
– GC, HPLC, electron impact,
or direct chemical isolation
• An ion source
– Converts molecules to gasphase ions
– MALDI, fast atom
bombardment
Mass Spectroscopy
• All Mass specs consist of:
• A mass filter/ analyzer
– Time of flight, magnetic
sector, MALDI, or ion trap
• A detector
– Array detector, conversion
dynode, or electron
multiplyer
Ionization
• In mass spectrometry, a
substance is bombarded with an
electron beam having sufficient
energy to fragment the molecule
• The positive fragments which
are produced (cations and
radical cations) are accelerated
in a vacuum through a magnetic
field and are sorted on the basis
of mass-to-charge ratio.
Ionization
• Since the bulk of the ions
produced
in
the
mass
spectrometer carry a unit
positive charge, the value m/e is
equivalent to the molecular
weight of the fragment.
•
The
analysis
of
mass
spectroscopy
information
involves the re-assembling of
fragments, working backwards
to
generate
the
original
molecule.
Ionization
• Electron Impact ionization (EI):
• It comprises an electron gun, a time-of-flight mass spectrometer with
position-sensitive detector (PSD).
• Analyte must be in a vapor state, limiting biological material below
400Da
• Useful for metabolites, pollutants, and pharmaceutical compounds
Ionization
• Chemical ionization:
• Especially useful technique for organic chemists when no
molecular ion is observed in EI mass spectrum
• Ionization of sample (analyte) is achieved by interaction of
its molecules with reagents such as CH4 or NH3
• Very good for determining molecular mass
– as high intensity molecular ions are produced owing to less
fragmentation.
Ionization
• electrospray ionization:
A
technique used in mass spectrometry
to produce ions.
• It is especially useful in producing
ions from macromolecules because
it overcomes the propensity of these
molecules to fragment when ionized
• Is the primary ion source used in
liquid chromatography-mass spec
because it's a liquid-gas interface
that is capable of coupling liquid
chomatography with mass
spectrometry
Mass Analyzers
• Once ions are created and leave the ion source they pass
into a mass analyzer
• This separates the ions and measures their masses
– What is actually measured is the mass to charge ratio (m/z) of each
ion
• At any given time, ions of a particular mass pass through
and are counted by the detector
– In this way, the analyzer scans through a large range of masses
Mass Analyzers
• Quadrupole mass spectrometry:
• Essentially a mass filter that is
capable of transmitting only the ion
of choice. A mass spectrum is
obtained by scanning through the
mass range of interest over time.
• Two opposite rods have an opposite
applied potential and affect the
trajectory of ions traveling down the
flight path centered between the four
rods
Mass Analyzers
• Quadrupole mass spectrometry:
• The mass range of the oscillating
ions is scanned by changing the DC
voltage and the frequency.
• The resolution of the spectrometer
can be increased by either
employing eight poles or by
connecting two or three qaudupoles
in series
• Excel at applications where
particular ions of interest are being
studied because they can stay tuned
on a single ion for extended periods
Mass Analyzers
• Ion trap mass spectrometry:
• The ion trap consists of three
electrodes with hyperbolic
surfaces, the central ring
electrode, and two adjacent
endcap electrodes
•
The
device
is
radially
symmetrical the electrodes are
aligned and isolated using
ceramic spacers and posts
Mass Analyzers
•
•
•
•
Ion trap mass spectrometry:
Advantages:
(i) high sensitivity
(ii) compactness and mechanical
simplicity in a device which is
nevertheless capable of high
performance
• (iii) tandem mass spectrometry
experiments are available by
performing sequential mass analysis
measurements
• (iv) high resolution
Mass Analyzers
• Magnetic sector analyzer:
• Generated ions are accelerated
and are passed around a curved
track (the sector) leading to a
detector.
• By increasing the magnetic field
applied to the ions, heavier ions
with higher momentum can be
induced to follow the curved
track.
Mass Analyzers
• Magnetic sector analyzer:
• Only ions of mass-to-charge ratio
that have equal centripetal and
centrifugal forces pass through
the flight tube
• The ions that reach the detector
can be varied by changing either
the magnetic field or the applied
voltage of the ion optics.
• So the individual ion beams are
separated spatially and each has a
unique radius of curvature
according to its mass/charge ratio
Mass Analyzers
• Plasma desorption ionization:
• First available to analyze proteins and other large biomolecules
(well, less than 35,000 Mr).
• The technology is now relatively obsolete
• Used radioactive californium (252Cf)
• Required a time of flight (TOF) mass detector
Mass Analyzers
• matrix-assisted laser desorption-ionization time-of-flight
(MALDI-TOF) mass spectrometry.
• Relatively novel technique in which a co-precipitate of an
UV-light absorbing matrix and a biomolecule is irradiated
by a nanosecond laser pulse,which causes the peptide to
form crystals.
• Most of the laser energy is absorbed by the matrix, which
prevents unwanted fragmentation of the biomolecule.
• The ionized biomolecules are accelerated in an elctric field
and enter the flight tube
Mass Analyzers
• matrix-assisted laser desorption-ionization time-of-flight
(MALDI-TOF) mass spectrometry.
• During the flight in this tube, different molecules are
separated according to their mass to charge ratio and reach
the detector at different times.
• In this way each molecule yields a distinct signal.
• It is a very sensitive method, which allows the detection of
low (10-15 to 10-18 mole) quantities of sample with an
accuracy of 0.1 - 0.01 %.
Mass Analyzers - MALDI-TOF
• Protein identification by this
technique has the advantage of
short measuring time (few minutes)
and negligible sample consumption
(less than 1 pmol).
• Additional information on posttranslational modifications and
presence of by-products!
Mass Analyzers - MALDI-TOF
• Drawbacks of Maldi-tof
• The sample preparation for MALDI is important for the
result.
• Inorganic salts which are also part of protein extracts
interfere with the ionization process.
• The matrix protein mixture is not homogenous because the
polarity difference leads to a separation of the two
substances during crystallization
Mass Analyzers - MALDI-TOF
• The spot diameter of the target is much larger than that of
the laser, which makes it necessary to do several laser shots
at different places of the target, to get the statistical average
of the substance concentration within the target spot.
• Delay between laser pulses, delay time of the acceleration
power, laser wavelength, energy density of the laser and the
impact angle of the laser on the target are among others the
critical values for the quality and reproducibility of the
method.
Detector
• The electron multiplier is a
highly sensitive device to detect
individual energetic particles
such as electron, photons, or ions
•
Multipliers are based on two
principles:
• (1) the particle(s) to be detected
have to be converted to electrons
before the amplification can take
place (using a so called
conversion dynode)
Detector
• (2) the amplification is caused by
a cascade of acceleration
electrodes
(called
dynodes)
which accelerate the electrons to
speeds which allow them to
generate more than one new
electron when hitting the next
dynode.
Protein identification
• Peptide sequencing – a quick
example.
• A peptide and a protein digest of
it were studied by Mass Spec
• MALDI-TOF detected a peek at
3840.2
• Following HPLC-MS of the
3480.2 Da peak showed signals
at m/z = 176, 624, 1129, 1508.
• Looked at the 624 component
• The ions appeared at m/z = 521,
406, 293, 130, and 43
Protein identification
• Peptide sequencing – a
quick example.
• What is the molecular mass of
the peptide?
• 176+624+1229+1508 = 3538 Da
• But the peak was 3840.2!
• Why the difference?
What is the molecular mass of
the peptide?
• Why the difference?
• 3538 – 3480.2 = 57.8 (58 Da) why a difference of 58 Da
• There were four peaks following enzyme digest
–
–
–
–
–
Take into account enzymatic hydrolysis
Three cleavage points give four parts
Each requires the imput of one water molecule (H2O = 18)
18 X 3 =58
So peak is heavier than the actual peptide
What is the sequence of the
624 component?
The Mass (DA) of the amino acids
Symbol
Structure
Mass (Da)
Ala A
-NH.CH.(CH3).CO-
71.0
Arg R
-NH.CH.[(CH2)3.NH.C(NH).NH2].CO-
156.1
Asn N
-NH.CH.(CH2CONH2).CO-
114.0
Asp D
-NH.CH.(CH2COOH).CO-
115.0
Cys C
-NH.CH.(CH2SH).CO-
103.0
Gln Q
-NH.CH.(CH2CH2CONH2).CO-
128.1
Glu E
-NH.CH.(CH2CH2COOH).CO-
129.0
Gly G
-NH.CH2.CO-
57.0
His H
-NH.CH.(CH2C3H3N2).CO-
137.1
Ile I
-NH.CH.[CH.(CH3)CH2.CH3].CO-
113.1
Leu
-NH.CH.[CH2CH(CH3)2].CO-
113.1
Lys K
-NH.CH.[(CH2)4NH2].CO-
128.1
Met M
-NH.CH.[(CH2)2.SCH3].CO-
131.0
Phe F
-NH.CH.(CH2Ph).CO-
147.1
Pro P
-NH.(CH2)3.CH.CO-
97.1
Ser S
-NH.CH.(CH2OH).CO-
87.0
Thr T
-NH.CH.[CH(OH)CH3).CO-
101.0
Trp W
-NH.CH.[CH2.C8H6N].CO-
186.1
Tyr Y
-NH.CH.[(CH2).C6H4.OH].CO-
163.1
Val V
-NH.CH.[CH(CH ) ].CO-
99.1
Protein identification
• What is the sequence of the 624 component?
• m/z 624
521
406
293
• ▲
103
115
115
163
• aa
Cys
Asp
Asp
Tyr
130
43
87
Ser
• The difference in the m/z valve gives you the identity of the
corresponding amino acid
• So the peptide sequence for the 625 component is:
– Cys-Asp-Asp-Tyr-Ser
THE END!