Proteomics: Strategies for protein Quantitation

1
Proteomics Strategies for protein Quantitation

• Yao-Te Huang
• Oct 19, 2009

2
Supplementary material for the last-time lecture

• More on sequencing by MS

3
More on sequencing by MS
87.08 (Ser)
4
More on sequencing by MS
?S2
?S the sum of the residue mass of the amino
acids in a peptide fragment
5
More on sequencing by MS
An Example a hexapeptide NFESGK S1S2S3S4S5S6
The total mass (m) of such a parent peptide m
?S 18680.34 A peak in the mass spectrum for
the parent peptide ion (m/z value (m1)/1 for
the positive ionization mode) .Thus,
(680.341.008)/1 681.42
6
More on sequencing by MS
NFESGK K Y1 GK Y2
SGK Y3 ESGK Y4
FESGK Y5
An example Y4 (its m/z ratio)
(129.0487.0857.02128.0919)/1420.23
7
More on sequencing by MS
NFESGK N B1 (missing) NF
B2 NFE B3 NFES B4 NFESG B5
An example B3 (its m/z ratio)
(114.04147.07129.041)/1391.15
8
More on sequencing by MS
9
More on sequencing by MS
10
Outline

• Conventional ways to quantify the amount of
  individual proteins of interest.
• Methods to quantify the amount of the cellular
  proteins on the proteomic scale.

11
Part (A) the conventional ways

• To quantify the amount of our purified protein,
  we use
• (a) The Bradford assay
• (b) The direct spectrophotometric assay
• (c) ELISA (enzyme-linked immunosorbent assay)

12
Protein quantification The Bradford assay

• Principle (1)?max of Coomassie Brilliant Blue
  dye changes from 465 nm to 595nm upon binding to
  protein (2) measure A595
• Sensitivity High (5?g)
• Interferences detergents, Triton X-100, SDS
• Time rapid, 15 min

13
Protein quantification The direct
spectrophotometric assay

• The last assay (1) involves colored product
  formation (2) need to do standard curve using
  (e.g., BSA (Bovine Serum Albumin))
• The current assay
• Principle absorbance of 280 nm light by Trp
  Tyr residues in protein
• Time rapid, 5-10 min
• Sensitivity moderate sensitivity, 50-100 ?g
• Interferences nucleic acids, purines,
  pyrimidines

14
A UV-VIS spectrophotometer
capable of selecting light of a particular
wavelength (practically, most light emitting
from it is of a single wavelength but shorter
and longer wavelengths are present)
Light intensity/purity
Light source H or D lamp producing UV (200-320
nm) light W (tungsten) lamp
producing VIS (320-800 nm)
light
15
Cuvet (cuvette)
Cuvets are made of glass, quartz, or other
transparent material.
Glass cuvets absorb UV light, so they are used
for only above 320 nm For UV range (200 to 320
nm) Cuvets made of quartz or fused silica
Disposable (plastic) cuvets conveniently used
for cell culture suspensions growth rate
measurement at 600 nm One unit of A600 8?108
cells/ml
16
The Beer-Lambert Law

• Transmittance I/I0 (where I0 the intensity of
  light irradiating the sample I the intensity of
  light transmitted through the sample)
• Absorbance, A, (or Optical Density (O.D.)) is
  defined as
• -log(I/I0)
• A?lc (The Beer-Lambert Law)
• where
• ? the molar extinction coefficient (or the molar
  absorption coefficient) (M-1 ? cm-1)
• l path length of light through the sample (cm)
• c concentration of the absorbing material in the
  
• sample (M)

17
UV absorption spectroscopy of proteins aromatic
side chains
18
Calculation of the molar extinction coefficient
for any given protein
Once ? is calculated A is measured, the
concentration of protein can be determined
19
An example

• If protein X contains 3 Trp residues, 4 Tyr
  residues, and one disulfide bridge, its
  calculated molar extinction coefficient
• (?280nm) is
• 3 5500 41490 1125
• M-1cm-1

20
ELISA (enzyme-linked immunosorbent assay )

• Antibodies can be used as exquisitely specific
  analytic reagents to quantify the amount of a
  protein or other antigen. The technique is the
  enzyme-linked immunosorbent assay (ELISA). In
  this method, an enzyme, which reacts with a
  colorless substrate to produce a colored product,
  is covalently linked to a specific antibody that
  recognizes a target antigen. If the antigen is
  present, the antibody-enzyme complex will bind to
  it, and the enzyme component of the
  antibody-enzyme complex will catalyze the
  reaction generating the colored product. Thus,
  the presence of the colored product indicates the
  presence of the antigen. Such an enzyme-linked
  immunosorbent assay, which is rapid and
  convenient, can detect less than a nanogram (10-9
  g) of a protein. ELISA can be performed with
  either polyclonal or monoclonal antibodies, but
  the use of monoclonal antibodies yields more
  reliable results.

21
AP (Alkaline Phosphatase)
HRP (horseradish -peroxidase)
22
Indirect ELISA and Sandwich ELISA (A) In indirect
ELISA, the production of color indicates the
amount of an antibody to a specific antigen. (B)
In sandwich ELISA, the production of color
indicates the quantity of antigen.
23
Part (B) Methods to quantify the amount of the
cellular proteins on the proteomic scale.

• Quantitative proteomics with standard 2D gels.
• DIGE (Difference Gel Electrophoresis)
• Isotopically labeling methods for quantitative
  proteomics

24
Quantitative proteomics with standard 2D gels

• The abundance of different proteins on a 2D-gel
  is determined by the shape, size, and intensity
  of the corresponding spots.
• Hence, protein quantitation requires the
  conversion of an analogue gel image into digital
  data, resulting in a catalog of individual spots
  listed as x/y positions, shape parameters and
  quantitative values (integrated spot intensities).

25
Quantitative proteomics with standard 2D gels
(contd.)

• The first stage is image acquisition, and the
  method used depends on how the proteins were
  labeled or stained.
• (1) Radioactively labeled proteins are detected
  by X-ray film or phosphorimaging. The X-ray film
  may be scanned by a CCD (charge-coupled device)
  camera or a densitometer, whereas phosphorimeters
  come with their own scanning devices.

26
Quantitative proteomics with standard 2D gels
(contd.)

• (2) Coomassie-stained and silver-stained gels may
  also be scanned with a CCD camera or
  densitometer.
• (3) Gels stained with the fluorescent SYPRO
  agents or gels containing fluorescently labeled
  proteins may be scanned using a CCD camera or a
  fluorescence imager.

27
CCD (charge-coupled device)

• A CCD is simply a solid-state electrical
  component that is divided into a series of
  light-sensitive areas or photosites. Each
  photosite is composed of a material that emits
  electrons when struck by a photon of light.
• The image from a CCD camera is therefore
  generated by a microprocessor that counts the
  electrons at each photosite.

28
Densitometer

• A densitometer is a scanning device that works on
  similar principles. That is, light reflected from
  or transmitted through the surface of a film is
  detected by a photodiode, which therefore records
  the density of the light and dark areas on the
  image.

29
Quantitative proteomics with standard 2D gels
(contd.)

• The quality of the digital data depends on the
  resolution of the scanned image, which can be
  considered both in terms of spatial resolution
  (expressed as pixels per unit length or area) and
  densitometeric resolution (i.e., the range of
  gray values that can be interpreted).
• The densitometric resolution also depends on the
  labeling or staining method employed.

30
Quantitative proteomics with standard 2D gels
(contd.) silver staining

• (a) is 10-100 times more sensitive than Coomassie
  brilliant blue staining.
• (b) does not detect glycoproteins very
  efficiently.
• (c) may lead to chemical modification of Cys
  residues ( alkylating exposed amino groups),
  therefore interfering with downstream MS
  analysis.
• (d) has very narrow linear range of quantitation
  (about one order of magnitude, about 10-fold
  difference)

31
Quantitative proteomics with standard 2D gels
(contd.) SYPRO Ruby staining

• (a) is as sensitive as silver staining.
• (b) can detect glycoproteins efficiently.
• (c) does not cause any covalent modifications of
  the proteins.
• (d) has an extensive linear range (over three
  orders of magnitude) which means they can be used
  to compare protein abundances very efficiently.

32
Quantitative proteomics with standard 2D gels
(contd.) spot detection, and quantitation

• The first stage in automated spot detection is
  digital image enhancement, which helps to clear
  the background and improve the contrast of the
  image to make the spot boundaries easier to
  delineate.
• Once a processed image is available, a number of
  different algorithms can be detect and quantitate
  individual spots. These calculate the integrated
  spot intensities, which are essentially absolute
  values that represent protein abundances.

33
Watershed transformation method

• In which a grayscale image is converted into a
  topographical surface with darker sections
  representing peaks and lighter sections
  representing troughs.
• The idea is then to flood the image from the
  minima, which divides the image into catchment
  basins representing divisions.

34
Watershed transformation method contd.

• In practice, the indiscriminate flooding of gel
  images in this manner leads to over-segmentation
  due to background variation in pixel intensity.
• To avoid this outcome, flooding can be initiated
  from a previously defined set of markers, which
  avoids any over-segmentaion.

35
(No Transcript)
36
Quantitative proteomics with standard 2D gels
(contd.) spot comparison

• Gel-matching algorithm then apply image
  transformation procedure such as stretching,
  skewing, and rotating, at both local and global
  levels, to bring multiple gel images into
  register and make them comparable.

37
Quantitative proteomics with standard 2D gels
(contd.) spot comparison

• This can be thought of as a procedure in which
  several equivalent gels are stacked above each
  other and a pin is used to pierce the center of
  the first landmark spot through all the stacked
  gels.
• Further pins are inserted through other
  landmarks. When the gels are held in position by
  a number of pins, bendy wires can be inserted to
  link equivalent spots that are not perfectly in
  register.

38
(No Transcript)
39
Quantitative proteomics with standard 2D gels
(contd.) spot comparison

• The end result should be a table of spot values
  (x/y coordinates, shape parameters and integrated
  spot intensities) arranged as a N?M matrix where
  N represents all the different spots that have
  been identified and M represents all the gels.

40
(No Transcript)
41
Quantitative proteomics with standard 2D gels
(contd.) spot comparison

• The quantitative values must be normalized for
  any differences in the overall signal
  intensities on the gels (e.g., due to different
  exposure times) and then various statistical
  methods can be used to identify protein spots
  whose abundance varies over the experimental
  conditions.
• The above points are generally hard to achieve
  rigorously.

42
Multiplexed proteomics

• Multiplexed proteomics is the use of the
  fluorescent stains or probes with different
  excitation and emission spectra to detect
  different groups of proteins simultaneously on
  the same gel.
• An example DIGE (difference gel electrophoresis).

43
Ettans 2-D DIGE (difference gel electrophoresis)
system

• Different protein samples (e.g., healthy vs.
  disease) are labeled on Lys side chains with Cy3
  (green) and Cy5 (red), respectively.

3 main components (1)CyDye DIGE
fluors.(2)Typhoon Variable Mode Imager(3)
DeCyder 2-D Differential Analysis Software
44
Ettans 2-D DIGE (difference gel electrophoresis)
system

• The use of further labels, e.g., Cy2, can allow
  even more samples to be run concurrently, or used
  as for the purpose of internal control.
• Because the samples run together, all differences
  in gel preparation, running conditions and local
  gel structure are eliminated which considerably
  simplifies the downstream analysis.

45
(No Transcript)
46
Isotopically labeling methods for quantitative
proteomics

• ICAT
• Nonselective labeling of peptides after digestion
• Isotope tagging in vivo

47
The strategy of quantitative protein profiling
using ICAT (isotope-coded affinity tags)
Mass difference by 8 Da
48
The strategy of quantitative protein profiling
using ICAT (isotope-coded affinity tags) (contd.)
Using avidin affinity chromatography (
because biotin binds very strongly to avidin)
49
The strategy of quantitative protein profiling
using ICAT (isotope-coded affinity tags) (contd.)
50
Variants of ICAT reagents

• A cleavable ICAT reagent, which allows the biotin
  to be removed before mass spectrometry
• The use of heavy and light forms of acrylamide
  which also react with Cys side chains.

51
(No Transcript)
52
(No Transcript)
53
A disadvantage of ICAT reagents

• About 5-10 of all proteins do not have Cys
  residues, and therefore can not be used with
  ICAT.
• A solution to this problem ICPL (isotope-coded
  protein label) can be used, which will label
  amino groups of Lys residues of proteins. The
  light version of ICPL contains 6 C12 atoms
  whereas the heavy version of ICPL contains 6 C13
  atoms.

54
Nonselective labeling of peptides after tryptic
digestion

• When trypsin cleaves a protein and generates a
  peptide with a new C-terminus, it introduces an
  oxygen atom derived from a molecule of water into
  the carboyl group of the peptide.
• If H216O is used in one buffer and H218O is used
  in the other buffer, the abundance of the
  peptides in the two reaction buffers can then be
  compared because they will appear as doublets
  separated by two mass units.

55
(No Transcript)
56
(No Transcript)
57
Isotope tagging in vivo

• Cells treated under different conditions are
  grown in media containing either normal or heavy
  isotopes of nitrogen, carbon, or hydrogen.
• A useful approach is the use of labeled amino
  acids (stable-isotope labeling with amino acids
  in cell culture, SILAC).

58
(No Transcript)
59
(No Transcript)
60
A disadvantage of isotope tagging in vivo

• Isotope tagging in vivo can only be used for the
  analysis of live cells.
• It is not useful for tissue explants, biopsies,
  body fluids, or cells that are difficult to
  maintain in culture.

61
An additional method Mass-coded abundance tags
(MCATs)

• Proteins from one sample are labeled with
  O-methylisourea and those from the other sample
  are not labeled at all. This method is simple and
  inexpensive, but less accurate than those
  involving isotopes.