Transcript Sample Prep II

Identification of Posttranslational Modifications
For Sample Prep
Complexity of the Proteome
 Protein processing and modification comprise an important third
dimension of information, beyond those of DNA sequence and
protein sequence.
 The thousands of component proteins of a cell and their posttranslational modifications may change with the cell cycle,
environmental conditions, developmental stage, and metabolic state.
 Proteomic approaches that don’t just identify proteins but also find
their post-translational modifications are needed!
Post-translational Modification
• What purpose ?
- targeting (eg. some lipoproteins)
- stability (eg. secreted glycoproteins )
- function
(eg. surface glycoproteins)
- control of activity (eg. clotting factors, caspases)
• How can we study it ?
Definitions of the components:
1. Post-translational modification (PTM): The chemical modifications that take place at certain amino acid residues after the
protein is synthesized by translation are known as post-translational modifications. These are essential for normal functioning of the
protein. Some of the most commonly observed PTMs include:
a) Phosphorylation: The process by which a phosphate group is attached to certain amino acid side chains in the
protein, most commonly serine, threonine and tyrosine.
b) Glycosylation: The attachment of sugar moieties to nitrogen or oxygen atoms present in the side chains of amino
acids like aspargine, serine or threonine.
c) Acylation: The process by which an acyl group is linked to the side chain of amino acids like asparagine, glutamine or
lysine.
d) Alkylation: Addition of alkyl groups, most commonly a methyl group to amino acids such as lysine or arginine. Other
longer chain alkyl groups may also be attached in some cases.
e) Hydroxylation: This PTM is most often found on proline and lysine residues which make up the collagen tissue. It
enables crosslinking and therefore strengthening of the muscle fibres.
Definitions of the components
2. Protein translation: The process by which the mRNA template is read by ribosomes to synthesize the
corresponding protein molecule on the basis of the three letter codons, which code for specific amino acids.
3. Cytosol: A cellular compartment that serves as the site for protein synthesis.
4. Signal sequence: A sequence that helps in directing the newly synthesized polypeptide chain to its
appropriate intracellular organelle. This sequence is most often cleaved following protein folding and PTM.
5. Endoplasmic reticulum: A membrane-bound cellular organelle that acts as a site for post-translational
modification of the newly synthesized polypeptide chains.
6. Cleaved protein: The protein product obtained after removal of certain amino acid sequences such as N- or
C-terminal sequences, signal sequence etc.
Proteomic analysis of PTMs
Mann and
Jensen, Nature
Biotech. 21,
255 (2003)
Adduct formation – expect the unexpected
Adduct ion
Percent [%] Adduct ion
Percent [%] Adduct ion
Percent [%] Adduct ion
Percent [%] Adduct ion
Percent [%]
[M+H]+
62.55381
[M+H-C3H8O]+
0.02667
[M-CCl3]+
0.00381
[M(37Cl)]+.
0.00190
[M-2H+Na]-
0.00127
[M+2H]2+
11.44459
[M-H-H2O-CO2]-
0.02667
[M-H-CO2]-
0.00381
[M-CH3]+
0.00190
[M-H+Co]+
0.00127
[M+H-H2O]+
8.77598
[M-H-H2O-HCO2H]-
0.02667
[M+H-C5H7PO6]+
0.00381
[M+H-C4H11N]+
0.00190
[M+H-(CH3)2NH-C3H6]+
0.00127
[M-H]-
6.25214
[M+H-3H2O]+
0.02540
[M+H-HCl]+
0.00381
[M+H-NO2-CHO]+
0.00190
[M+H-C10H6(OH)N]+
0.00127
[M+Na]+
5.51055
[M+H-CHN]+
0.02540
[M+H-C12H12N2O3]+
0.00381
[M-H-HF]-
0.00190
[M-H+Ni]+
0.00127
[M+H-NH3]+
1.19494
[M+K-3H]2-
0.01905
[M+H-CH3CO2H]+
0.00381
[M(37Cl)+H]+
0.00190
[M-H-H2O-C4H7CO2H]-
0.00127
[M+NH4]+
0.73715
[M+H-(CH3)2NH]+
0.01524
[M+H-CH3]+.
0.00381
[M-H-C6H10O5]-
0.00190
[M+H-OH]+
0.00127
[M-H-H2O]-
0.34604
[M+H-CHNO]+
0.01333
[M+H-H2]+
0.00381
[M+H-H2O-C6H13N]+
0.00190
[M(81Br)+H]+...
0.00127
[M-H+2Na]+
0.32953
[M+H-C2H6O]+
0.01333
[M+H-C3H8NO6P]+
0.00317
[M+H-H2O-H3PO4]+
0.00190
[M-H-CH2O-CH2NH]-
0.00127
[M-H+H2O]-
0.24508
[M+H-CH4O]+
0.01270
[M+H-C5H14NO4P]+
0.00317
[M+H-C5H7PO6-NH3]+
0.00190
[M+H-CO-CONH]+
0.00127
[M+NH4-H2O]+
0.22984
[M+H-C7H13NO3]+
0.01143
[M+Li-(CH3)3N]+
0.00317
[M-H-C5H7PO6]-
0.00190
[M-H-CONH]-
0.00127
[M+H+H2O]+
0.19429
[M+Na-2H]-
0.00952
[M+Li-C5H14NO4P]+
0.00317
[M+H-H2S]+
0.00190
[M+H-C3H4O2]+
0.00127
[M+H+Na]2+
0.18286
[M-H-CH2O]-
0.00952
[M+Cl]-
0.00317
[M+H-H2O-C8H8]+
0.00190
[M+H-C3H6O4]+
0.00127
[M+H+K]2+
0.17524
[M+H-C11H12N2O3]+
0.00952
[M(35Cl)-H]-
0.00317
[M+H-H2O-NH3-C8H8]+
0.00190
[M+Na-H2S]+
0.00127
[M-2H]2-
0.13968
[M+H-C13H16N3O4]+
0.00952
[M(37Cl)-H]-
0.00317
[M+H-H2O-NH3-C8H8-CO]+
0.00190
[M-H+2Na-H2S]+
0.00127
[M+2Na]2+
0.13778
[M+H-C17H25N3O4]+
0.00952
[M-H-C5H7O6P]-
0.00317
[M+H-H2O-NH3]+
0.00190
[M-C5H5Cl]+
0.00127
[M+2H-NH3]2+
0.13714
[M+CH3CO2]-
0.00889
[M+H-C3H7O5P]+
0.00317
[M+H-C3H6]+
0.00190
[M+H-N2]+
0.00127
[M+K]+
0.13651
[M-H2O+Na]+
0.00825
[M-H-C6H6N8O]-
0.00317
[M+HCO2-320]-
0.00190
[M+H-H2O-CO]+
0.00127
[M+H-2H2O]+
0.11810
[M-H+NH3]-
0.00762
[M(81Br)+H]+
0.00317
[M+H-C3H7N]+
0.00190
[M-H-H3PO4]-
0.00127
[M+3H]3+
0.06667
[M+H-C9H9NO]+
0.00762
[M-C4H9]+
0.00317
[M-H-H2]-
0.00190
[M+H+CH3CN]+
0.00127
[M+2H-H2O]2+
0.06476
[M+H-C15H21N2O3]+
0.00762
[M-2H+3Li]+
0.00254
[M-H-C16H30O-H2O]-
0.00190
[M+H-C4H6]+
0.00127
[M]+.
0.05905
[M-2H+3Na]+
0.00698
[M-H-HCl]-
0.00254
[M-H-CH4O]-
0.00190
[M+H-CH3OH]+
0.00127
[M+2Na-H]+
0.05143
[M+HCO2]-
0.00635
[M+2Li-H]+
0.00254
[M+H-C10H8FN3]+
0.00127
[M+H-HCCl3]+
0.00127
[M-H+2K]+
0.05079
[M+H-NO2]+
0.00571
[M+H-C8H10O2]+
0.00254
[M+Li-C3H5NO2]+
0.00127
[M+H-C2H3N3]+
0.00127
[M+H-CO]+
0.04635
[M+H-C6H13NO2]+
0.00571
[M+H-C2Cl4]+
0.00254
[M+Li-H3PO4]+
0.00127
[M+H-C3H6O2]+
0.00127
[M+H-CO2]+
0.04318
[M-H-C3H5NO2]-
0.00508
[M-H-C7H5NO]-
0.00254
[M-2H+3Li-C15H31CO2H]+
0.00127
[M+H-CH2Cl2O]+
0.00127
[M+H-CH2O2]+
0.03810
[M(81Br)-H]-
0.00508
[M+H-C5H11N]+
0.00254
[M-2H+3Na-C3H5NO2]+
0.00127
[M(356)+H-HCl]+
0.00127
[M-H-NH3]-
0.03746
[M+H-HCO2H]+
0.00508
[M+Ba-H]+
0.00254
[M-2H+Na+Co]+
0.00127
[M-C4H4O4S]+
0.00127
[M.Cl]-
0.03556
[M-2H+Li]-
0.00444
[M+H-C14H25NO3]+
0.00254
[M-2H+Li-C3H5NO2]-
0.00127
[M+H-C8H14O3]+
0.00127
[M+Li]+
0.03111
[M+H-CH4]+
0.00444
[M+H-C6H5NO2S]+
0.00254
[M-2H+Li-C16H30O]-
0.00127
[M+H-C2H4]+
0.00127
…around 290 different adducts
Statistics: Adducts in NIST12 MS/MS DB (80,000 spectra)
Most common adducts for LC-MS ([M+H]+ [M+Na]+ [M+NH4]+ [M+acetate]+)
8
ExPASy – the proteomic server
Different types of PTMs & their modification sites
Ser, Thr, Tyr
Pro, Lys
Phosphorylation
Asn,Glycosylation
Ser, Thr
Lys, Arg
Acylation
Alkylation
Hydroxylation
Asn, Gln, Lys
Process of post-translational modification
mRNA
Ribosome
Cytosol
Translated
Protein
P
Endoplasmic
reticulum
(ER)
Removal of
certain N- and
Cleaved
C-terminal Proteaseprotein
residues
P
Protein folding
& PTMs
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
CH3
CH3
Glc
Glc
Increased complexity of proteome due to PTMs
A A C G G U G C C G U G C A C G C A C U A C G C A C U
Expected protein
structure
Gene sequence
Actual protein
structure
Glc
P
CH3
Phosphorylation reactions
COO-
COOKinase
C
H
NH3+
R
H
OH
ATP
Amino acid
residue
ADP
CH2
Ser
CH
Thr
R
CH3
CH2
Tyr
th
NH3+
C
R
O
PO43-
Phosphorylated
residue
Glycosylation reactions
N-linked Glycosylation
H
Sugar residues
COO-
COO-
Glycosyl transferase
C
CH2
H
C
CH2
CONH2
CON
NH3+
NH3+
Asn
N-linked amino acid
O-linked Glycosylation
COO-
COOGlycosyl transferase
H
C
R
C
H
R
O
OH
NH3+
NH3+
Ser/Thr
O-linked amino acid
th
Definitions of the components:
Gel-based detection techniques for PTMs
1. Pro-Q-diamond: This fluorescent dye detects modified proteins that have been
phosphorylated at serine, threonine or tyrosine residues. They are used with electrophoretic
techniques and offer sensitivity down to few ng levels, depending upon the format in which
they are used. This dye can also be combined with other staining procedures thereby allowing
more than one detection protocol on a single gel.
a) Gel staining: The process by which the protein bands on an electrophoresis gel are
stained by suitable dyes for visualization.
b) Gel scanning: The visualization of the stained protein bands on an electrophoresis gel by
exciting it at a suitable maximum wavelength such that the dye absorbs the light and emits its
own characteristic light at another emission wavelength.
2. Immunoblotting: This process, also known as Western blotting, is a commonly used
analytical technique for detection of specific proteins in a given mixture by means of specific
antibodies to the given target protein.
a) Electrophoresis: Electrophoresis is a gel-based analytical technique that is used for
separation and visualization of biomolecules like DNA, RNA and proteins based on their
fragment lengths or charge-to-mass ratios using an electric field. The protein mixture is first
separated by means of a suitable electrophoresis technique such as SDS-PAGE or Twodimensional Electrophoresis.
Definitions of the components:
Gel-based detection techniques for PTMs
b) Blotting: The process by which the proteins separated on the electrophoresis gel are
transferred on to another surface such as nitrocellulose by placing them in contact with each
other.
c) Nitrocellulose sheet: A membrane or sheet made of nitrocellulose onto which the protein
bands separated by electrophoresis are transferred for further probing and analysis.
d) Specific probe antibodies: Antibodies that are specific to a particular protein modification
can be used as probes to detect those proteins containing that particular PTM. Protein
phosphorylation is commonly detected using anti-phosphoserine, phosphothreonine or
phosphotyrosine antibodies. Recently, specific motif antibodies have also been developed
which detect a particular sequence of motif of the protein that contains a PTM.
e) Labeled secondary Abs: Antibodies labeled with a suitable fluorescent dye molecule are
used to detect the interaction between the modified protein and its antibody by binding to
another domain of the probe antibody.
Pro-Q-diamond staining
Dye
stains
the get
Protein
bands
Excess
dye
phosphorylated
fixed
on gel and
removed
protein
bands
minimize
diffusion.
only.
Completed 2-DE gel
Tubing connected &
outlet opened
Tray with fixing
solution (methanol +
acetic acid)
Pro-Q-diamond stain
Washing solution
(methanol + acetic
acid)
Gel scanning
Gel scanner
Stained gel
Decreasing
molecular weight
Emission maxima
– 580 nm
Gel removed
from scanner
Phosphoprotein
image
Decreasing pH
Dual staining with SYPRO-Ruby Red
Dye
stains
all
Excess
dye
protein
bands.
removed
Tubing connected &
outlet opened
SYPRO-Ruby red
staining solution
Washing solution
(methanol + acetic
acid)
Fluorescence
Gel scanning
Gel scanner
Phosphoprotein
image
Stained gel
Emission maxima
– 610 nm
Decreasing
molecular weight
Total protein
image
Decreasing pH
A comparative profile
between total protein image
and phosphoprotein image
enables detection of
phosphorylated proteins.
Fluorescence
Phosphoprotein
image
Total protein image by
SYPRO-Ruby Red
Proteins focused on
IPG strip
Immunoblotting
SDS-PAGE
-
Sample loading
2-D
Electrophoresis
Cathode
Protein
mixture
Acrylamide
gel
Direction of
migration
Direction of
migration
+ Anode
Buffer
Completed
stained gels
Immunoblotting (this one for phosphorylated tyrosines!)
Proteins
phosphorylated at
Tyr residues
Completed
gels
Nitrocellulose
Blotting sheet or PVDF
Specific phosphotyrosine
antibodies added
Detection using
labeled secondary
antibodies
Proteins
phosphorylated at
Tyr residues
PHOSPHORYLATION
Phospho – Proteomics
Western 2D gel , Ab specific to phospho-tyrosine
Phosphorylation and Mass Spec
 Analysis of the entire complement of phosphorylated proteins in cells: “phosphoproteome”
 Qualitative and quantitative information regarding protein phosphorylation important in
many cellular processes
 signal transduction, gene regulation, cell cycle, apoptosis
 Most common sites of phosphorylation: Ser, Thr, Tyr
 MS can be used to detect and map
locations for phosphorylation
 MW increase from addition of phosphate
group
 treatment with phosphatase allows
determination of number of phosphate
groups
 digestion and tandem MS allows for
determination of phosphorylation sites
Enrichment strategies to analyze phosphoproteins/peptides
 Chemical derivatization

Introduce affinity tag to enrich for phosphorylated molecules

e.g., biotin binding to immobilized avidin/streptavidin
Enrichment strategies to analyze phosphoproteins/peptides
 Oda et al., Nature Biotech. 2001, 19, 379 for analysis of pS and pT
 Remove Cys-reactivity by oxidation with performic acid
 Base hydrolysis induce ß-elimination of phosphate from pS/pT
 Addition of ethanedithiol allows coupling to biotin
 Avidin affinity chromatography to purify phosphoproteins
AND MORE~!
Enrichment strategies to analyze phosphoproteins/peptides
 Phosphospecific antibodies
 Anti-pY quite successful
 Anti-pS and anti-pT not as successful, but may be used
(M. Grønborg, T. Z. Kristiansen, A. Stensballe, J. S. Andersen, O. Ohara, M. Mann, O.
N. Jensen, and A. Pandey, “Approach for Identification of Serine/Threoninephosphorylated Proteins by Enrichment with Phospho-specific Antibodies.” Mol. Cell.
Proteomics 2002, 1:517–527.
 Immobilized metal affinity chromatography (IMAC)
 Negatively charged phosphate groups bind to postively charged
metal ions (e.g., Fe3+, Ga3+) immobilized to a chromatographic
support
 Limitation: non-specific binding to acidic side chains (D, E)
 Derivatize all peptides by methyl esterification to reduce nonspecific binding by carboxylate groups.

Ficarro et al., Nature Biotech. (2002), 20, 301.
Phosphoprotein and Sypro Ruby Stains with Laser Imaging
Beta-galactosidase
Bovine serum
albumin (BSA)
Phosphorylated
PeppermintStick phosphoprotein molecular
weight standards (LifeTechnologies)
separated on a 13% SDS polyacrylamide gel.
Ovalbumin
Beta-casein
The gel was stained with Pro-Q Diamond
phosphoprotein gel stain (blue) followed by
SYPRO Ruby protein gel stain (red).
Avidin
BAPTA
lysozyme
The digital images were pseudocolored
Phosphoprotein Stain
Visualization of total protein and
phosphoproteins in a 2-D gel
Proteins from a Jurkat T-cell lymphoma line cell
lysate separated by 2-D gel electrophoresis and
stained with Pro-Q Diamond phosphoprotein gel
stain (blue) followed by SYPRO Ruby protein gel
stain (red).
After each dye staining, the gel was imaged and
the resulting composite image was digitally
pseudocolored and overlaid.
T.H. Steinberg et al., Global quantitative phosphoprotein analysis using
Multiplexed Proteomics technology, Proteomics 2003, 3, 1128-1144
GLYCOSYLATION
Protein Glycosylation
• The
most important and complex form of PTM
• Approx.
1% mammalian genes
• Early
view about carbohydrates (non-specific, static
structures) has been challenged
Ann. Rev. Biochem. 72(2003)643
Glycoprotein Gel Stain
Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using
the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
CandyCane glycoprotein molecular weight standards (LifeTechnologies)
containing alternating glycosylated and nonglycosylated proteins
electrophoresed through a 13% polyacrylamide gel.
After separation, the gel was stained with SYPRO Ruby protein gel stain to
detect all eight marker proteins (left). Subsequently, the gel was stained by
the standard periodic acid–Schiff base (PAS) method in the Pro-Q Fuchsia
Glycoprotein Gel Stain Kit to detect the glycoproteins alpha2-macroglobulin,
glucose oxidase, alpha1-glycoprotein and avidin.
Pro-Q™ Glycoprotein Stain (DDAO phosphate)
Molecular Formula: C15H18Cl2N3O5P (MW 422.20)
Protein Glycosylation
Common in Eukaryotic Proteins
NITRATION
Nitro-Tyrosine Modification
 Oxidative modification of amino acid side chains:
 methionine oxidation to the corresponding sulfone
 S-nitrosation or S-nitrosoglutationylation of cysteine residues
 Tyrosine modification to yield o,o’-dityrosine, 3-nitrotyrosine and 3chlorotyrosine.

Tyrosine nitration is a well-established protein modification that occurs in disease states
associated with oxidative stress and increased nitric oxide synthase activity.
 The combination of 2D-PAGE, western blotting,
IMMUNOASSAY and mass spectrometry has been the more
typical strategy to identify 3-nitrotyrosine-modified proteins.
Nitro-Tyrosine Modification
“Proteomic method identifies proteins nitrated in vivo during inflammatory
challenge,” K. S. Aulak, M. Miyagi, L. Yan, K. A. West, D. Massillon, J. W.
Crabb, and D. J. Stuehr, Proc. Natl. Acad. Sci. USA 2001; 98: 12056-12061.
Anti-nitrotyrosine immunopositive proteins in lung of rats induced with LPS.
WHAT WE DO
AT OSU…
SERVICES at OSU Proteomics
Just ask! •
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•
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•
•
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PTM
identification!
•
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Protein Growth, Induction and Expression, Protein purification
Subcloning into recombinant cell lines, Plasmid design
DIGE
Develop novel protein protocols, individualized for experiment
Selective subfractionation, Salt fractionation, Enrichment, Solubility screening,
Inclusion body isolation
Western Blotting, Far Western Blotting, Immunoprecipitation and Coimmunoprecipitation, Protein-Protein interaction studies
Classic chromatography:
Affinity –Tag purification, ionic exchange, HIC reverse phase, SEC gel
chromatography 100,300, Immobilized metal affinity chromatography (IMAC),
Heparin affinity: Protein A/G affinity column, ENDOTOXIN removal
SDS-PAGE and DNA Electrophoresis, reduced and/or non-reduced
ProQ, LavaPurple, Sypro and other gel staining
Fluorescent and Bradford Protein Quantitation
Mass Spectrometry for protein identification
THANKS FOR LISTENING!
You can find us at…
Mass Spec and Proteomics and
Protein Expression and Purification
Facility
Biomedical Research Tower Room 250
460 West 12th Street
Columbus, Ohio
Lab: 614-247-8789
Arpad Somogyi, PhD – [email protected]
Cindy L. James, PhD – [email protected]