Sample Prep II
Transcript of Sample Prep II
Identification of Posttranslational ModificationsFor 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 post-translational 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)
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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
Statistics: Adducts in NIST12 MS/MS DB (80,000 spectra)Most common adducts for LC-MS ([M+H]+ [M+Na]+ [M+NH4]+ [M+acetate]+)
…around 290 different adducts
ExPASy – the proteomic server
Different types of PTMs & their modification sites
Phosphorylation
Glycosylation
Acylation
Alkylation
Hydroxylation
Ser, Thr, Tyr
Asn, Ser, Thr
Asn, Gln, Lys
Lys, Arg
Pro, Lys
Process of post-translational modification
Cytosol
Endoplasmic reticulum
(ER)
PP
GlcGlc
CH3CH3
Cleaved protein
Protein folding & PTMs
mRNARibosome
Protease
Removal of certain N- and
C-terminal residues
Translated Protein
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
Increased complexity of proteome due to PTMs
A C G G U G C C G U G C A C GA C A C U A C G C A C U
Gene sequence
AsnGly
Ala
ValHis
AlaLeu
Arg Thr
Expected protein structure
Actual protein structure
AsnGly
Ala
ValHis
AlaLeu
Arg Thr
PCH3
Glc
Phosphorylation reactions
Ser
R
CH2
CH
CH3
CH2
Thr
Tyr
ATP ADP
Kinase
Amino acid residue
Phosphorylated residue
OHC
NH3+
COO-
RH OC
NH3+
COO-
R PO43-H
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
Glycosylation reactions
Ser/Thr
Asn
Glycosyl transferase
N-linked Glycosylation
O-linked Glycosylation
Glycosyl transferase
Sugar residues
N-linked amino acid
O-linked amino acid
CONH2C
NH3+
COO-
CH2H CONC
NH3+
COO-
CH2H
OHC
NH3+
COO-
RHOC
NH3+
COO-
RH
Source: Modified from Biochemistry by A.L.Lehninger, 4th edition (ebook)
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 Two-dimensional 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
Completed 2-DE gel
Protein bands get fixed on gel and minimize diffusion.
Tubing connected & outlet opened
Dye stains the phosphorylated protein bands only.Excess dye removed
Tray with fixing solution (methanol + acetic acid)Pro-Q-diamond stainWashing solution (methanol + acetic
acid)
Gel scanning
Dec
reas
ing
mol
ecul
ar
wei
ght
Decreasing pH
Gel scanner
Emission maxima – 580 nm
Phosphoprotein image
Stained gel
Gel removed from scanner
Dual staining with SYPRO-Ruby Red
SYPRO-Ruby red staining solution
Tubing connected & outlet opened
Dye stains all protein bands.Excess dye removed
Washing solution (methanol + acetic
acid)
Gel scanning
Dec
reas
ing
mol
ecul
ar
wei
ght
Decreasing pH
Gel scanner
Emission maxima – 610 nm
Total protein image
Fluo
resc
ence
Phosphoprotein image
Fluo
resc
ence
Total protein image by SYPRO-Ruby Red
A comparative profile between total protein image and phosphoprotein image enables detection of phosphorylated proteins.
Phosphoprotein image
Stained gel
Immunoblotting
Direction of migration
Anode
Cathode-
+BufferAcrylamide
gel
Sample loading
Protein mixture
SDS-PAGE 2-D Electrophoresis
Proteins focused on IPG strip
Direction of migration
Completed stained gels
Immunoblotting (this one for phosphorylated tyrosines!)
Completed gels
Nitrocellulose sheet or PVDFBlotting
Specific phospho-tyrosine
antibodies added
Detection using labeled secondary
antibodies
Proteins phosphorylated at
Tyr residues
Proteins phosphorylated at
Tyr residues
PHOSPHORYLATION
Phospho – ProteomicsWestern 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/Threonine-phosphorylated 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 non-
specific binding by carboxylate groups. Ficarro et al., Nature Biotech. (2002), 20, 301.
Phosphoprotein and Sypro Ruby Stains with Laser Imaging
PeppermintStick phosphoprotein molecular weight standards (LifeTechnologies) separated on a 13% SDS polyacrylamide gel.
The gel was stained with Pro-Q Diamond phosphoprotein gel stain (blue) followed by SYPRO Ruby protein gel stain (red).
The digital images were pseudocolored
Phosphorylated
Beta-galactosidase
Bovine serum albumin (BSA)
Ovalbumin
Beta-casein
Avidin
lysozyme BAPTA
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
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)
Detection of glycoproteins and total protein on an SDS-polyacrylamide gel using the Pro-Q Fuchsia Glycoprotein Gel Stain Kit.
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 3-
chlorotyrosine. 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
• 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 Co-
immunoprecipitation, 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
Just ask!
PTM identification!
Mass Spec and Proteomics and Protein Expression and Purification Facility
Biomedical Research Tower Room 250460 West 12th StreetColumbus, OhioLab: 614-247-8789
Arpad Somogyi, PhD – [email protected] L. James, PhD – [email protected]
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