Proteomics-3.pdf protein science biology

Proteomic

1. Mass spectrometry is a technique to determine mass of
molecules
In mass spectrometry, a molecule is fragmented into different
ions whose masses are accurately measured
Ions generate a spectrum of peaks that is unique and therefore
determines identity of original molecule
Molecule 2-pentanol is used as an example in Figure
Here an electron beam aimed at sample fragments 2-pentanol
into different ions:
Mass Spectrometry for Protein Identification

2. oMolecular ion gains an electron
om-1 loses hydrogen from
alcohol group
om-15 loses a methyl group
om-17 loses alcohol group
om/e = 45 loses alkyl chain
Mass Spectrometry for Protein Identification
FIGURE – Basic Mass Spectrometry for 2-Pentanol
Every substance can be fragmented into multiple ions
This example shows molecular structure of all ions from 2-pentanol

3. FIGURE – Basic Mass Spectrometry for 2-Pentanol
oA mass spectrometer separates these ions by size and graphs results
oSpectrum is always same for each substance
oSo an unknown substance can be identified by comparing its spectrum
with a database of known substances

4. These ions are accelerated into a vacuum tube by an ion
accelerating array
Ions travel through tube at different speeds due to a magnetic
field that causes ions to follow a curved path within tube
Curves eliminate ions that are too small or too big
Ions that are too small gain so much momentum from magnetic
field that they collide with the wall
Those that are too big are not deflected by magnetic field and
also collide with the wall
Ions in right size range are deflected by magnetic field around
both curves to hit the collector, where they are recorded as
peaks in mass spectrum
Mass Spectrometry for Protein Identification

5. Base peak is the most intense, and other peaks are measured
relative to this
The time ions take from accelerator to collector correlates
directly to size of the ion
Each peak is plotted based on mass/charge ratio (m/z)
Losses of mass (such as m-17 or m-15) refer to loss of specific
groups from parent molecule
And are most informative to structure of sample molecule
because each such group has a characteristic mass
Mass Spectrometry for Protein Identification

6. Schematic Diagram of a Mass Spectrometry Tube
oSample travels through a narrow slit and then passes through a beam of electrons
that disrupts it into a mixture of ion fragments
oAccelerating array moves fragments into C-shaped tube
oThis is surrounded by a strong magnetic field that prevents ions that are too small
or too large from exiting tube
oA collector detects exiting fragments and measures time it took for them to travel
the tube
oComputer then converts time of travel into size and charge information and plots
this as a mass spectrum

7. Until recently, very large molecules such as proteins were
beyond range of mass spectrometry
Two different ionization techniques have been developed that
have made proteins manageable
1
st
technique embeds proteins in a solid matrix before
ionization, and is called MALDI , or matrix-assisted laser
desorption-ionization
Here proteins are embedded in a material such as 4-
methoxycinnamic acid that absorbs laser light
Matrix absorbs and transfers laser energy to proteins, causing
them to release different ions
Mass Spectrometry for Protein Identification

8. Ions are accelerated through a vacuum tube by a charged grid
At far end, time-of-flight (TOF) detector records intensity and
calculates mass
In between is a flight tube that is free of electric fields
Ions are accelerated with same kinetic energy, and when they
reach flight tube, lighter ions move faster than heavier ions
Time-of-flight is proportional to square root of mass to charge
ratio (m/z)
MALDI is able to handle ions up to 100,000 daltons
Mass Spectrometry for Protein Identification

10. oMass spectrometry can be used to determine molecular weight
of proteins
oProteins are crystallized in a solid matrix and exposed to a laser,
which releases ions from proteins
oThese travel along a vacuum tube, passing through a charged
grid, which helps separate ions by size and charge
oTime it takes for ions to reach detector is proportional to square
root of their mass to charge ratio (m/z)
oMolecular weight of protein can be determined from these data
Figure – Ions MALDI/TOF Mass Spectrometer

11. 2
nd
method for preparing ions from proteins is electrospray
ionization (ESI)
Here protein is dissolved in liquid and very small droplets are
released from a narrow capillary tube
Droplets enter electrostatic field, where a heated gas, such as
hydrogen, causes solvent to evaporate and droplets to break up
This causes protein to release ions into vacuum tube, where
they are accelerated by electric field
Detector at far end varies based on sample being studied
A TOF detector may be used, as described earlier
Mass Spectrometry for Protein Identification

12. Other detectors use quadrupole ion traps or Fourier transform
ion cyclotron resonance to determine mass of ions
Quadrupole ion traps capture ions in an electric field
Ions are then ejected into detector by a second electric field
Electric field controls what size ions can pass to detector, and
varying field allows different-sized ions to be detected
Combination detectors exist that use both TOF and quadrupole
ion traps
Advantage that ESI has over MALDI is that proteins isolated from
HPLC require no special preparation and can be used directly
Disadvantage of ESI is that masses of about 5000 are maximum
Mass Spectrometry for Protein Identification

14. Figure – Electrospray Ionization (ESI) Mass Spectrometer
ESI mass spectrometry uses a liquid sample of protein held in a
capillary tube
After exposure to a strong electrostatic field, small droplets are
released from end of capillary tube
A flow of heated gas within drift zone evaporates solvent and
releases small charged ions
Charged ions vary in size and charge and pattern of ions
produced is unique to each protein
Ions are separated by size using a charged grid to either impede
or promote flow toward detector

15. Tandem mass spectrometry is also known as MS/MS or MS
2
It involves multiple steps of mass spectrometry selection, with
some form of fragmentation occurring in between stages
Ions are formed in ion source and separated by mass-to-charge
ratio in 1
st
stage of mass spectrometry (MS1)
Ions of a particular mass-to-charge ratio (precursor ions) are
selected and fragment ions (product ions) are created
Resulting ions are then separated and detected in a second
stage of mass spectrometry (MS2)
A common use is for analysis of biomolecules such as proteins
and peptides
Tandem Mass Spectrometry

16. Schematic of tandem mass spectrometry

17. Determining peptide sequence of a short peptide is readily
achieved using current mass spectroscopy techniques
Determining entire sequence of a protein is too complex at this
point, but may someday be feasible
To determine sequence, a pure sample of protein must be
obtained either by cutting a spot from a 2D gel or by HPLC
purification
Protein is then digested into fragments using a protease such as
trypsin
Which cuts proteins on carboxy-terminal side of arginine and
lysine
Tandem Mass Spectrometry

18. Cutting a protein into peptides helps reduce undesirable
characteristics of entire protein
For example, membrane proteins are hydrophobic and stick
together, and digesting these into peptide fragments destroys
this characteristic
Solubility issues can also often be resolved by digesting a protein
into peptides
Tandem Mass Spectrometry

19. Mass Spectrometry of
Peptides
oBecause mass
spectrometry is so
sensitive, use of large
whole proteins is limited
oInstead, peptide
fragments are generated
by protease digestion
oPeptides are easily
separated with HPLC,
and then specific
peptides are subjected
to mass spectrometry

20. Determining sequence of these peptides will yield sequence of
original protein
The most common method of determining peptide sequences
begins by separating peptides using an HPLC column directly
attached to mass spectrometer
Column is a microscale capillary in order to keep sample volume
as small as possible
Mobile phase is an organic solvent that elutes peptides in order
of their hydrophobicity
From HPLC capillary tube, samples enter mass spectrometer
chamber
Each peptide is ionized into multiple fragments
Tandem Mass Spectrometry

21. For peptides, common ions include a doubly protonated form
(M + 2H)
2+
, where M is mass of peptide and H
+
is mass of a
proton
Ion peaks are plotted versus mass to charge ratio or m/z
For doubly protonated peptide ion, this would be mass of ion
divided by 2
For example
•If original peptide was 1232.55 daltons
•Double protonated ion would have a mass of:
o1232.55 daltons + (2 × 1.0073) for each added hydrogen
oTherefore, peak would appear at 617.2828
Tandem Mass Spectrometry

22. oNote: mass to charge ratio is where peak is plotted, that is,
mass for this ion is 1234.5646 and charge is +2
oPeak appears at m/z or 1234.5646/2
When mass spectrometer separates peptide ions, first step is to
determine charge state of ion
Usually a cluster of peaks occurs for each peptide ion
If peaks are 1 dalton apart, charge state of peptide is 1
If peaks are 0.5 dalton apart, charge state is 2
Tandem Mass Spectrometry

23. To determine peptide sequence, two rounds of mass
spectroscopy are used
This is called tandem mass spectroscopy because one ion is
produced in first round of mass spectroscopy
Then that ion is fragmented by collision with a gas such as
hydrogen, argon, or helium
As before, ion fragments are separated based on their mass to
charge ratio
Each peak usually varies by one amino acid, and size difference
between peaks determines amino acid sequence
Sometimes spectrum obtained for a peptide ion is unclear, so
databases of peptide ion spectra are used for comparison
Tandem Mass Spectrometry

26. Protein-detecting arrays may be divided into those that use antibodies
and those based on using tags
In ELISA assay, antibodies to specific proteins are attached to a solid
support, such as a microtiter plate or glass slide
Protein sample is then added and if target protein is present, it binds
its complementary antibody
Bound proteins are detected by adding a labeled second antibody
Another antibody-based protein-detecting array is antigen capture
immunoassay
Much like ELISA, this method uses antibodies to various proteins
bound to a solid surface
Protein Arrays

27. Experimental protein sample is isolated and labeled with a fluorescent
dye
If 2 conditions are being compared, proteins from sample 1 can be
labeled with Cy3, which fluoresces green, and proteins from sample 2
can be labeled with Cy5, which fluoresces red
Samples are added to antibody array, and complementary proteins
bind to their cognate antibodies
If both sample 1 and 2 have identical proteins that bind same
antibody, spot will fluoresce yellow
If sample 1 has a protein that is missing in sample 2, then spot will be
green
Conversely, if sample 2 has a protein missing from sample 1, spot will
be red
This method is good for comparing protein expression profiles for 2
different conditions
Protein Arrays

28. Ideal Results for Antigen Capture
Immunoassay
A variety of different antibodies
are fused to different regions of
a solid surface
Each spot has a different
antibody
If antibody recognizes only proteins labeled with Cy5, region will
fluoresce red (left)
If antibody recognizes only proteins labeled with Cy3, region will
fluoresce green (middle)
If antibody recognizes proteins in both conditions, spot will fluoresce
yellow (right)
Figure (Antigen Capture Immunoassay)

29. In 3
rd
method, direct immunoassay or reverse-phase array , proteins
of experimental sample are bound to solid support
Proteins are then probed with a specific labeled antibody
Both presence and amount of protein can be monitored
For example, proteins from different patients with prostate cancer can
be isolated and spotted onto glass slides
Each sample can be examined for specific protein markers or
presence of different cancer proteins
Levels of certain proteins may be related to stages of prostate cancer
This immunoassay helps researchers to decipher these correlations
Direct immunoassay binds protein samples to different regions on a
solid support
Protein Arrays

30. oEach spot has a different protein sample
oNext, an antibody labeled with a detection system is added
oAntibody binds only to its target protein
oIn this example, antibody recognizes only a protein in patient sample 1
and 2
Direct Immunoassay

31. Problems with immuno-based array
1.Main problem with immuno-based arrays is antibody
2.Many antibodies cross-react with other cellular proteins, which
generates false positives
3.In addition, binding proteins to solid supports may not be truly
representative of intracellular conditions
4.Proteins are not purified or separated; therefore, samples contain
very diverse proteins
5.Some proteins will bind faster and better than others
6.Also, proteins of low abundance may not compete for binding sites
7.Another problem is that many proteins are found in complexes, so
other proteins in complex may mask antibody binding site
Protein Arrays

32. Rather than using antibodies, protein interaction arrays use a fusion
tag to bind protein to a solid support
Use of protein arrays to determine protein interactions and protein
function is a natural extension of yeast two-hybrid assays and co-
immunoprecipitation
Protein arrays can assess thousands of proteins at one time, making
this a powerful technique for studying proteome
Protein arrays are often used in yeast because its proteome contains
only about 6000 proteins
Libraries have been constructed in which each protein is fused to a
His6 or GST tag
Proteins are then attached by tags to a solid support such as a glass
slide coated with nickel or glutathione
Protein Arrays

33. To build array, each protein is isolated individually and spotted onto
glass slide
Tagged proteins bind to slide and other cellular components are
washed away
Each spot has only one unique tagged protein
Once array is assembled, proteins can be assessed for a particular
function
To assemble a protein microarray, a library of His6-tagged proteins is
incubated with a nickel-coated glass slide
Proteins adhere to slide wherever nickel ions are present
In laboratory of Michael Snyder at Yale University, yeast proteome has
been screened for proteins that bind calmodulin (a small Ca
2+
binding
protein) or phospholipids

Protein Arrays

34. Both calmodulin and phospholipid were tagged with biotin and
incubated with a slide coated with each of yeast proteins bound to
slide via His6-nickel interactions
Biotin-labeled calmodulin or phospholipid was then visualized by
incubating slide with Cy3-labeled streptavidin (Streptavidin binds very
strongly to biotin)
Results identified 39 different calmodulin binding proteins (only six
had been identified previously), and 150 different phospholipid
binding proteins
Protein Arrays

35. Protein Interaction Microarray—Principle

36. Screening Protein Arrays Using Biotin/Streptavidin
Protein microarrays can be screened to find proteins that bind to
phospholipids
Protein microarray is incubated with phospholipid bound to biotin
Then bound phospholipid is visualized by adding streptavidin
conjugated to a fluorescent dye
Spots that fluoresce represent specific proteins that bind
phospholipids
Figure

37. Protein purification is generally a multi-step process on the basis
of wide range of biochemical and biophysical characteristics of
target protein
Such as its source, relative concentration, solubility, charge, and
hydrophobicity
Ideal purification attempts to obtain maximum recovery of
desired protein
With minimal loss of activity, combined with maximum removal
of other contaminating proteins
Proteins are fragile molecules that denature readily at extremes
of temperature and pH
Each protein offers its own unique set of physicochemical
characteristics
Protein Purification

38. Methods used for protein purification should be mild, to
preserve native conformation of molecule and its bioactivity
In most cases, having a reliable assay to be used as means of
following target protein is essential
There is no set procedure for isolating proteins
Purification schemes should be made to take advantage of
biochemical properties of target protein
As well as cellular properties of tissue that provides the most
abundant source of material
Whenever possible try to reduce complexity of sample
For example, isolate specific protein subsets or subcellular
organelles thereby enriching for low abundance target molecule
Protein Purification

39. When designing a purification protocol one should aim for the
following:
1.High recovery
2.Highly purified end product
3.Reproducibility, within lab, in other labs and also when
either scaled up or down
4.Economical use of reagents
5.Convenience with regard to time
Chemical structure and physical properties of protein are two
key parameters used to develop most purification protocols
Isoelectric point (pI), pH stability, and charge density are some of
properties of proteins that can be exploited during purification
Protein Purification

40. A number of separation techniques are capable of resolving
proteins on the basis of differences in net charge
These include isoelectric focusing, ion exchange
chromatography, and chromatofocusing
Chromatofocusing is a protein-separation technique that allows
resolution of single proteins and other ampholytes from a complex
mixture according to differences in their isoelectric points
Ampholytes are compounds that when dissolved in water (which is
itself an amphoteric compound) can act either as acid or as a base
In general, the most successful isolation procedures involve only
a few steps chosen to give the highest yields


Protein Purification

41. Rarely will a single technique fulfill requirements of any specific
separation
More frequently, 2, 3, or more steps are needed for purification
of desired protein
Before beginning to purify a protein it is important to have an
idea of degree of purity that is necessary for intended use of
target protein
Often, a 90–95% enrichment will be sufficient because
additional steps decrease yield and may not provide desired
purity of final product
Concern for purity will vary greatly depending on whether
protein is to be used for kinetic, sequence or crystallographic
studies, or is to be injected into humans
Protein Purification

42. Protein purification is routinely assessed by summarizing results
of each purification step as specific activities, total units, total
protein, and yields
Activity units are calculated based on assay system that is used
to track target protein
An example of expressing data is shown in Table using
purification of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA)
synthase from ox liver
If a step results in a major loss of activity, it should be changed
When undertaking any purification, always begin with enough
starting material so that a workable amount of final product can
be isolated
Protein Purification

44. Delays during a multistep protocol should be avoided
Stability of target protein will determine time necessary for
protocol
For example, in cases where stability is limited due to inherent
lability (changing continuously) of protein or copurifying proteolytic
activity, speed of operation can be much more important than
protein purity
Therefore, there will be instances when it will be beneficial to
rush one step after another, sacrificing purification, rather than
carrying out each step to perfection and taking a long time in
process
Protein Purification

45. Whenever possible, fractionation steps should be arranged to
follow one from the other without extensive manipulation
between steps
Some typical protocols that do not involve intermediate
treatments are listed below:
Salt precipitation  Gel filtration  Ion exchange
chromatography
Salt precipitation  Hydrophobic interaction chromatography 
Ion exchange chromatography  Reversed phase HPLC
Ion exchange chromatography  Hydrophobic interaction
chromatography  Affinity chromatography  Gel filtration
chromatography
Protein Purification

46. Organic solvent extraction  Affinity chromatography
Affinity chromatography  Salt precipitation  Gel filtration
chromatography
Positions of steps in above strategies are predicated on volume
and ionic strength of sample
In a typical purification protocol, 1
st
step after extracting target
protein in soluble form from starting material could be ion
exchange chromatography
Which has excellent resolving power that can concentrate target
protein from a dilute starting solution
Target protein is usually eluted with high concentrations of salt
Protein Purification

47. A temporary desalting step can be avoided if next step is
hydrophobic interaction chromatography
In which protein is loaded onto column in a high salt containing
solution
The eluate from this step, now free of lipids and other potential
problem-causing contaminants, can be put onto an affinity
column, if one is available
Using an immobilized ligand, target protein is specifically bound,
and contaminants are washed away
Protein of interest is then eluted
Often, this step is the most powerful in scheme, being good
enough to stand alone when a large quantity of semi-purified
protein is desirable
Protein Purification