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![Page 1: Post-translational modifications Post-translational Modification of Proteins Expanding Nature’s Inventory (2006) by C.T. Walsh ISBN 0-9747077-3-2.](https://reader038.fdocuments.us/reader038/viewer/2022103022/56649d5d5503460f94a3cfe2/html5/thumbnails/1.jpg)
Post-translational modifications
Post-translational Modification of ProteinsExpanding Nature’s Inventory (2006)by C.T. Walsh
ISBN 0-9747077-3-2
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Genome↓
Transcription – mRNA↓
Translation – proteins + co-translational modifications
↓Post-translational modifications
Just for introduction
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Post-translational modifications I
• Enzymatic processing• N-, O-, C-linked glycosylation – Asn, Ser/Thr/Hyl/Hyp,
Trp• Phosphorylation – Tyr, Ser, Thr, His, Asp• Acylation
acetylation of the N-terminusfatty acid anchors on Cys
• Cross-linkage – Lys, Trp, Tyr, Met• Oxydation – Cys, Met, Trp, Tyr, His• Methylation – N-terminus, Arg, Lys• Ubiquitination - Lys
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Post-translational modifications II
• Cannot be predicted – though consensus sequences have been reported for some of them
• Organism-dependent• Can be tissue- or location-specific• Stable or dynamic – high and low level• Alters biological activity, and physical
properties• May alter the immune response
↓
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How to find PTMs?
Sometimes proteins give us a clue:• higher/lower/wider band on SDS-PAGEFor example, +~5 kDa on SDS-PAGE may indicate 1 occupied N-linked glycosylation site
• multiple spots with IEF or on 2D-gel• higher MW with gel-filtration or native gel-elfo• altered hydrophobicity• altered immunochemistry• altered biological activity• “blank” cycle in Edman sequencing (with proper
standard & altered chromatography the modification could be identified: Identification of glycosylation sites in mucin peptides by edman degradation. Zachara NE, Gooley AA. Methods Mol Biol. 2000;125:121-8. Review.
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How to find PTMs?
Sometimes we’re searching for something specific
• using a known donor and radioactive labeling
ATP for phosphorylation (Why cannot we use this approach for glycosylation?)
• using modification-specific derivatization and staining or enrichment
Periodate oxidation and labeling for glycosylation
• using modification-specific antibodiesAgainst phosphoTyr
• using modification-specific enzymes Phosphatase or glycosidase treatment
• Mass spectrometry Mass Spectrometry Mass Spectrometry!
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Sample preparation • Does the modification survive?• Losses due to enzymatic reactionsPhosphatase activity for phosphopeptides
• Losses due to chemical reactionsAcid-sensitive modifications, such as phosphoHis
• Losses due to different physical properties
Fatty-acylated or glycopeptides
• Do we introduced some modifications?* Biological artifacts* Chemical side reactions
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Some modifications that occur in vivo and can be introduced in vitro
• Carbamidomethylation of N-termini and Lys side-chains
• Formylation of Lys side-chains• Asn cyclization; -aspartame formation;
deamidation• Oxidation • Glu, Asp methyl ester formationAnd the list goes on…
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Finding the N-termini
• 80-90% of the eukaryotic proteins is acetylated
If 2nd aa: G, A, S, C, T, P, V - Met-1 is clipped of; Ac- is added to G,
A, S, TIf 2nd aa: E, D, Q, M, I, L, W, F – persisting Met gets acetylated
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Finding the N-termini
• N-terminal sequencing• Positive enrichment (Wells-group);
subtiligase-mediated selective N-terminal labeling with biotin-containing probe; enrichment; release with TEV protease; MS-analysis
“Global sequencing of proteolytic cleavage sites in apoptosis by specific labeling of protein N termini.” Mahrus S, Trinidad JC, Barkan DT, Sali A, Burlingame AL, Wells JA. Cell. 2008 Sep 5;134(5):866-76.
• Negative enrichment – next slide
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Finding the N-termini
• McDonald L, et al., Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides.Nat Methods. 2005 Dec;2(12):955-7. Epub 2005 Nov 18.
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Modified N-terminus
Modified side-chain
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Methylation (mono, di, tri - +14, +28, +42 Da)
• N-terminus, Lys, Arg, His• Trimethyl – acetyl = 36 mmuAccurate mass measurement helps;Fragmentation is different too
• Glu (Asp) may form Me-ester – upon CBB staining (MeOH + acid) + 14 Da
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7000
6000
5000
4000
3000
2000
1000
0
644.0643.8643.6643.4643.2643.0642.8642.6m/z
14
12
10
8
6
4
2
0
x103
795.0794.8794.6794.4794.2794.0793.8793.6m/z
642.77672 (10+)
b57 + 42 Da
Calculated masses 6418.6718 - with Ac 6418.7082 - with Me3
Experimental value 6418.6973 Me3b57 (-1.6 ppm)
Mass calculated: 7134.8569 Experimental: 7134.8463 (-1.4 ppm)
y64
793.65655 (9+)
FT ICR MS
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700
600
500
400
300
200
100
0
Ion
Cou
nts
400350300250200150100
m/z100
80
60
40
20
0
Ion
Cou
nts
800700600500400
m/z
72.08
87.05147.11 169.10
186.13
214.13
260.20
280.20(2+)
294.85(3+)308.86(3+)
329.74(2+)
373.26(2+)
745.50
623.36599.39
462.79(2+)
479.83(2+)
571.28
486.31
422.77(2+)
427.77(2+)
44
1.5
4(2
+)
45
8.2
9
471.29(2+)
314.5(3+)
320.2(3+)
354.73(2+)
141.1
V
N y1
SV-28-H2O
a2-NH3
a2
b2
y2
y42+
[MH3-NH3-NMe3]3+
[MH3-2NH3]3+
[MH3-NH3]3+
y52+
[a7-NH3-NMe3]2+ y6
2+
y72+
[MH2-CO2H-NMe3]2+
[MH2-NH3]2+
MH22+
SVK*ESVK*EI
SV
K*E
-28
[MH2-2xNH3]2+
y6
b6-NH3-NMe3
512.36
SVK*EI-28
SVK*EI-28-NMe3
Asn-Val-Ser-Val-Lys(Me3)-Glu-Ile-Lys
- 59 Da
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Disulfide-bridges
• in membrane and secreted proteins important 3D structure feature
• Some toxins and antibacterial peptides feature characteristic Cys network
• -S-S- prone to shuffling @ basic pH
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How to find & determine disulfide linkages?
• Alkylate the free SHs; digest the protein; fractionate the digest; reduce/or oxidize the fractions; look for altered chromatographic behavior; identify the components
* Optimal pH for alkylation is good for shuffling too* Digestion may be hindered by a compact structure* Multiple links could be present within one
cleavage product* If oxidation is used, Met, Trp also may be altered
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A “novel” methodology for assignment of disulfide bond
pairings in proteins.• Wu J, Watson JT. Protein Sci. 1997 Feb;6(2):391-8.
• Reduction by TCEP (works at low pH); cyanylation of nascent –SHs; fractionation of partially reduced forms; cleavage at the N-termini of the modified Cys-residues (with NH4OH)
* Fractionation of complex, partially reduced mixtures could be a problem…
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Digestion
@ low pH
Reduction/alkylation
Reduction/alkylation
I
m/z
I
m/z
I
m/z
I
m/z
I
m/z
MS/MS
Reduction/alkylation
Digestion
@ low pH
Reduction/alkylation
Assigning disulfide-bridges
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Synthetic Ac-TIMP-1(Ser175)126-184
ECTVFPCLSI PCKLQSGTHC LWTDQLLQGS EKGFQSRHLA CLPREPGLCS WQSLRSQIA
Where are the disulfide bridges?
* digestion with trypsin @ pH 6;
* with pepsin in acid
Bodi, N. et al., J. Pept. Sci. 9, 430-441 (2003).
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300028002600240022002000m/z
inte
nsity
12001000800600m/z
inte
nsity
1600140012001000800600m/z
[38-
44]
fre
e S
H
[33-
37]
[45-
55]
Fre
e S
H
[38-
55]
41-
49
[38-
44]-
[45-
55]
41-
49
Ac[
1-13
]-[4
5-55
]2-
7, 1
2-49
[14-
32]-
[38-
44]
20-
41
[33-
37]
[38-
44]
S*
[45-
55]
S*
Ac[
1-13
] 1
S-S
, 1 S
*
Ac[
1-13
] 3
S*
[45
-59]
1 S
*
30002800260024002200200018001600m/z
Ac[
1-13
] 1
free
SH
, 2 S
*
[38
-55]
2 S
* [
14-3
2] 1
S*
[38
-59]
2 S
*
[14
-37]
1 S
*
MALDI-TOF analysis of the tryptic digest
PSD
In-source reduction
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Disulfide bridges in TIMP-1 C-terminal domain: [38-44]-S-S-[45-55], PSD of MH+ at m/z 2082.1
1000
800
600
400
200
0
Inte
nsi
ty
200015001000500m/z
2X1
75
.6, y
1,
y 1*
25
1.6
, b2
25
5.2
, y2*
-NH
3
32
2.6
, b3
77
6.3 8
10
.08
41
.7 12
42
.1* 1
27
6.4*
13
08
.9*
11
0.1
, H
19
28
, b6+
H2O
4,
b 10*
+H
2O
His-Leu-Ala-NH-CH-CO-Leu-Pro-Arg
CH2
CH2
Glu-Pro-Gly-Leu-NH-CH-CO-Ser-Trp-Gln-Ser-Leu-Arg*
S
S
810
776
842.11242.4
1276.5
1308.5
38
45
MALDI-PSD/CID yields characteristic triplets
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4000
3000
2000
1000
0
Ion
Cou
nts
600500400300200100
m/z1200
800
400
0
Ion
Cou
nts
1000900800700600
m/z
400
300
200
100
0
Ion
Cou
nts
1600150014001300120011001000
m/z
Figure 9. Low energy CID of m/z 871.79 (most abundant ion in 9+ cluster), MW (monoisotopic) 7835.03 Da. This molecule was identifiedas disulfide linked peptides [443-496] and [519-531] of myosin heavy chain. Sequence and fragmentation are shown on the next page.
327.21
y3**y3**
y3**456.25
y4**
490.24y4 569.34
y5**
120.08F
201.12b2
637.32
y5 682.44y6**
763.10 (3+)b19
811.49y7**
796.13(3+)b20
872.18 (3+)
b22
706.41 (3+)b17
924.5y8**
1144.16 (2+)
1307.77 (2+)
1068.62
b9 b19b22
No characteristic triplets in ESI-CID
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Fragment ions used for the identification of disulfide linked myosin peptides. (see low energy CID spectrum in Figure 9.)
b-ions myosin heavy chain peptide [443-496] 763.10 (3+) 1144.16 (2+) 796.13 (3+) 872.18 (3+) 201.12 1068.62 706.41 (3+) 1307.77 (2+) 2 9 17 19 20 22 VTRINQQLDTKQPRQYFGVLDIAGFEIFDFNSLEQLCINFTNEKLQQFFNHHhS 5 4 637.32 490.24 y-ions
myosin heavy chain peptide [519-531] DLAACIELIEKPhS 8 7 6 5 4 3 924.5 811.49 327.21 682.44 456.25 569.34 **y-ions
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Disulfide BridgesThe ProteinProspector mass modification search can be used in conjunction with MS-Bridge to find peptides with disulfide bridges. For this example, mass shifts between 0-2000 Da were considered. (1023.3276+4).
KLSWADLIVFAGNCALESMGFK+4
VSFADLVVLGGCAAIEK
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Glycosylationhttp://glycores.ncifcrf.gov/
Reference: Essentials of Glycobiology by Varki et al.
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N-linked
AsnXxxSer/Thr/Cys
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Further processing
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N-linked glycosylation
• consensus sequence• GlcNAc2Man3 – coreoligomannose structure – just Man unitscomplex sugars– GlcNAc-Gal–SA
antennaehybrid structurescore fucosylationsulfate, phosphate modifications
• PNGase F removes all N-linked structures; Asn Asp
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N-linked glycosylation
• Incredible heterogeneity: a site may be only partially occupied and may display numerous different carbohydrates
• species-, tissue-, cell-type-specific modification, physiological changes, diseases may alter the sugars
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certain structures are immunogenic
Gal 1-3 capping, Fuc 1-3 on inner GlcNAc;blood group determinants
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Characterization of N-linked carbohydrate pool
• PNGase digestion; sugar/protein separation; carbohydrate analysis without derivatization
* High pH anion exchange chromatography with pulsed amperometric detection
* NMR* MS• After modifying the reducing terminus(Advantage of the derivatization: improved “visibility”)* Electrophoresis with a combination of endoglycosidase cocktails “Elucidation of N-linked oligosaccharide structures of recombinant human factor VIII using
fluorophore-assisted carbohydrate electrophoresis.” Kumar HP, Hague C, Haley T, Starr CM, Besman MJ, Lundblad RL, Baker D. Biotechnol Appl Biochem. 1996 Dec;24 ( Pt 3):207-16.
* Capillary electrophoresis
No information on the modification sites and site heterogeneity
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N-linked glycoproteins
• Periodate oxidation & color-reaction on gels
• Periodate oxidation & capture on hydrazide resin; release with PNGase F
• Western blot with protein- & sugar-specific antibody
Non-specific background; co-migrating proteins will interfere
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N-linked glycopeptides(analysis of purified proteins only)
• Identification from diagnostic fragments:* HexNAc m/z 204* HexHexNAc m/z 366 Precursor scan, or „ping-pong” acquisition• Identification from oligosaccharide
heterogeneity• enrichment by HILIC or lectin-
chromatography
K.F. Medzihradszky Meth. Enzymol. 405, 116-138 (2005).
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human lecithin:cholesterol acyltranferase and apolipoprotein D, tryptic digest, LC/MS analysis
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MHTVNGYVN*R
Recombinant Factor VIII, 50 kDa subunit
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AG(Man8GlcNAc2)NVSNIIPASATLNADVR
peptide+GlcNAc
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About the structures of N-linked glycopeptides
• from the measured mass, and the CID spectrum the modified peptide can be identified + the size and class of the sugar
Automated N-glycopeptide identification using a combination of single- and tandem-MS. Goldberg D, Bern M, Parry S, Sutton-Smith M, Panico M, Morris HR, Dell A. J Proteome Res. 2007 Oct;6(10):3995-4005.
• the identity of the sugar units and their linkage positions CANNOT be determined
• NMR, exo- and endoglycosidases are needed to complete the job
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[MHNa2]3+ of QV(Man10GlcNAc2)NIT and [MH2Na] 3+ of QV(Man9GlcNAc2)NITGK
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Scheme 1. Fragments observed in the low energy CID spectrum of [MH2Na]3+ of glycopeptide QV(Man9GlcNAc2)NITGK (Figure 6)
+Na 2159.81 1080.41(2+) +Na 671.18 +Na Gln Man1-2Man1 1997.77 999.39(2+)
36
Man1 Val Man1
Man1 36
Man1-4GlcNAc-GlcNAc-Asn 6 Man1-2Man1- 2Man1 Ile +Na +Na Thr 163.05 2483.87 1242.44(2+) 1684.46 2321.85 1161.43(2+) 962.49 Gly +Na 347.08 Lys +Na 1360.41 +Na Man1-2Man1 1036.30
36
Man1 Man1
Man1 36
Man1-4GlcNAc 6 Man1-2Man1 - 2Man1 204.08 +Na 1522.41 +Na 1198.36
One component from the previous slide
K.F. Medzihradszky Meth. Enzymol. 405, 116-138 (2005).
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O-linked sugars
• No consensus sequence• No common core structure• No universal enzyme-elimination works (NaOH)sugars have to be reduced upon release
Detection is problematic – because of heterogeneity & variable site occupancy
Site assignment is even harder
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O-linked sugars
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Other O-linked core structures
• FucHarris, R.J. & Spelmann, M.W. (1993) Glycobiology, 3, 219-224.
• GlcNishimura, H et al., (1989) J. Biol. Chem. 264, 20320-20325.• Man – in yeast____________________________________
• GlcNAc – single unit; INSIDE the cell
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Characterization of O-linked carbohydrate pool
• Release with NaOH or hydrazynolysis• Reducing end is usually derivatized i)
to prevent peeling ii) to increase “visibility”
• Methods just like for N-linked sugars• NMR; HPAE-PAD; capillary and gel-
elfo; glycosidase cocktails; MS
No information on site occupancy and heterogeneity
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O-linked glycopeptides
• Known protein - Elucidation of O-glycosylation structures of the
beta-amyloid precursor protein by liquid chromatography-mass spectrometry using electron transfer dissociation and collision induced dissociation. Perdivara I, Petrovich R, Allinquant B, Deterding LJ, Tomer KB, Przybylski M. J Proteome Res. 2009 Feb;8(2):631-42.
• Known sugar structure - Zsuzsanna Darula and Katalin
F. Medzihradszky “Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum” Mol. Cell. Proteomics, Nov 2009; 8: 2515 - 2526.
• Perseverance & luck - Crina I.A. Balog, Oleg M.
Mayboroda, Manfred Wuhrer, Cornelis H. Hokke, André M. Deelder, and Paul J. Hensbergen. “Mass spectrometric identification of aberrantly glycosylated human apolipoprotein C-III peptides in urine from Schistosoma mansoni-infected individuals.” MCP published January 13, 2010, 10.1074/mcp.M900537-MCP200
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500
400
300
200
100
0
Inte
nsi
ty
12001000800600400200m/z
274
292
SA
453
SA-Gal 657
SA-Gal-GalNAc
697.4 751.2
812.8
944.6
1045.4
1126.4(2+)
(2+)
(2+)
-SA
-SA-Gal
-SA-Gal-GalNAc
(3+)(3+)
1888.2 + GalNAcGalSA
Typical CID of an O-linked glycopeptide
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CID fragmentation of O-linked glycopeptides
J. Am. Soc. Mass Spectrom. 7, 1996, 319-328.
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CID vs ETD
NH2-CH(R1)-CO-NH-CH(R2)-CO-…-NH-CH(Rn)-COOH
The weakest bonds go first;glycosidic bond; phosphate …
radical ions fragment, side-chains usually survive
b
y
c
z.
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ETD of KTFMLQASQPAPT(GalNAcGalSA)HSSLDIK
Inter-alpha-trypsin inhibitor heavy chain H1 precursor
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O-glycosylation within the cell
• Regulatory modification of nuclear and cytoplasmic proteins
• Single GlcNAc, no extension• 1 glycosyl-transferase (at least 21 for
secreted O-linked GalNAc); 1 cytosolic N-Acetyl--D-glucosaminidase
• Poorly understood due to lack of effective methods for enrichment and detection.
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O-GlcNAc-detection/enrichment
1) Labeling with radioactive Gal and Gal-transferase
1/a) same enzyme + GalNAz (azido-derivative) + biotin-label + streptavidin - Enrichment and site mapping of O-linked N-acetylglucosamine by a combination of chemical/enzymatic tagging, photochemical cleavage, and electron transfer dissociation mass spectrometry. Wang Z, Udeshi ND, O'Malley M, Shabanowitz J, Hunt DF, Hart GW.Mol Cell Proteomics. 2010 Jan;9(1):153-60.2)
2) Immuno-precipitation with O-GlcNAc-specific antibodies
3) Lectin-affinity chromatography
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Lectin-affinity Chromatography
•WGA lectin has affinity for GlcNAc, but affinity to a single GlcNAc moiety is low: millimolar1.
WGA
Good recoveryof glycoprotein
Complex glycosylation
GlcNAcGlcNAcGlcNAc
protein
No recovery
WGAPSVPVSerGSAPGR
O-GlcNAcO-GlcNAcylation
Load Wash Elute
Load Wash Elute
Peptide Mixture
Non-enriched
O-GlcNAc modified enriched
UV
abs
orba
nce
214
Time (minutes isocratic HPLC)
Solution?Isocratic chromatography on a long!!! column
Vosseller, K. et al. Mol Cell Proteomics (2006) 5 5: p.923-934
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CID Analysis of O-GlcNAc-Modified Peptides
Chalkley, R. J. and Burlingame, A. L. J. Am. Soc. Mass Spectrom. (2001) 12 p.1106-1113
•O-glycosidic link is significantly more labile under CID conditions than peptide backbone.
•Modification site identification using CID often not possible.
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ECD MS Spectrum of GlcNAc-modifiedPeptide from Spectrin
Vosseller, K et al. Mol Cell Proteomics (2006) 5 5: p.923-924
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One of the most important regulatory events:
turns proteins on and off
induces or prevents other post translational
modifications in the same protein
signaling pathways : phosphorylation cascades
PhosphorylationBiological significance
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Difficulties
a) Dynamic process : kinase vs. phosphatase
– both must be blocked during isolation
b) Phosphorylation often @ low level (<5%)
c) Lower ionization efficiency – signal of
phosphopeptides suppressed
Enrichment is a must at protein level
at peptide level
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Confirming the presence of phosphorylation
Western blot
pTyr large enough for sequence independent
recognition - works well
pSer, pThr – not reliable
Dyes – questionable reliability
Phosphatase treatment + isoelectric focusing – pI
shift
In vitro/in vivo assay with radioactive phosphate
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Determining site of phosphorylation
Mikrosequencing
Mutation studies
Mass spectrometry
MS spectrum : + 80 Da shift
MS/MS fragmentation :
pSer, pThr - H3PO4 loss : –98 Da
pTyr – always retains the modification
immonium ion at m/z 216 Da
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Enrichment methods
Ion exchange on SCX
IMAC : Fe(3+), Ga(3+)…
binding at low pH
methyl-esterification prior to IMAC
TiO2, ZrO2
Immunoprecipitation (only for pTyr)
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Reproducible isolation of distinct, overlapping segments of the phosphoproteome
Overlap between phosphopeptide isolation methods on the level of identified phosphorylation sites.
B. Bodenmiller et al, Nat. Methods 2007
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Large scale vs. 1 protein
large scale phosphorylation studies: large amounts of a complex mixture is
analyzed compensating for losses during sample preparation
leads to more PTM identification but low end results are incidental
single protein samples:sample amount is usually limitedmore challengingcombination of techniques may be necessary
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MALDI-TOF MS without enrichment
1200 1700 2200 m /z
0
5000
10000
15000
20000
25000
a.i.
/O = /bruker/2006/06novem ber/ke061110_02/2S R ef/pdata/1 A dm inistrator F ri N ov 10 11:56:05 2006
P P
PP
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Enrichment with TiO2
1400 1900 2400 m /z
0
10000
20000
30000
40000
50000
60000
70000
a.i.
/O = /bruker/2006/06novem ber/ke061109_01/2S R ef/pdata/1 A dm inistrator F ri N ov 10 10:15:26 2006
P
P
P
P
P P
P
P
P
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CID of CDSSPDpSAEDVR140x10
3
120
100
80
60
40
20
0
Inte
nsity
800600400200m/z
40x103
30
20
10
0
Inte
nsity
140012001000800600400200m/z
456
.7 M
-98
3+
518
.3 y
4
389
.3
b3
324
.1 b
2
274
.3 y
2
213
.1
PD
175
.1 y
1 435
.9 y
8-9
82+
597
.2 P
Dp
SA
ED
-98
695
.2 P
Dp
SA
ED
498
.2 b
4
871
.4 y
7
477
.3 M
## 3
+
109
4.4
b
10-9
8
870
.5 y
7
968
.4 y
8
119
2.4
b
10
389
.2 y
3
324
.1 b
2
274
.2 y
2
462
.2 b
42#
715
.4 M
## 2
+
136
0.6
M
-py
684
.4 M
-98
2+
126
2.5
M
-py-
98
756
.3 y
6
658
.4 y
6-9
8m/z 733.7 (2+)
m/z 489.6 (3+)
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Some new resultsSci Signal. 2010 Jan 12;3(104):ra3. “Quantitative phosphoproteomics reveals widespread full
phosphorylation site occupancy during mitosis.”Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, Brunak S, Mann M.
“… quantified 6027 proteins and 20,443 unique phosphorylation sites and their dynamics.”
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Some other tricks to study phosphorylation
• Shokat-method: utilizing analog-specific kinases
“Design and use of analog-sensitive protein kinases.” Blethrow J, Zhang C, Shokat KM, Weiss EL. Curr Protoc Mol Biol. 2004 May;Chapter 18:Unit 18.11.
• ERLIC-chromatography – single step enrichment & fractionation of acidic/hydrophilic modifications
“Simultaneous characterization of glyco- and phospho-proteomes of mouse brain membrane proteome with electrostatic repulsion hydrophilic interaction chromatography (ERLIC).” Zhang H, Guo T, Li X, Datta A, Park JE, Yang J, Lim SK, Tam JP, Sze SK. Mol Cell Proteomics. 2010 Jan 4. [Epub ahead of print]