SLIDE 1
Proteomics Informatics – Protein characterization I: post-translational modifications (Week 10)
SLIDE 2 Post-translational modification
- Biologically important post-translational modification
(phosphorylation, acetylation, glycosylation, etc.)
- Introduced on purpose during sample preparation (alkylation,
iTRAQ, TMT etc.)
- Side-products of sample preparation (oxidation, deamidation,
carbamylation, formylation etc.)
SLIDE 3 Post-translational modification
Mann and Jensen, Nature
255 (2003)
SLIDE 4 Unmodified pS18 pT5 b y b y b y"
2 I
2163.024 261.1556
2 I
2243.024 261.1556
2 I
2243.024 421.1862
3 C
2049.94 421.1862
3 C
2129.94 421.1862
3 C
2129.94 520.2546
4 V
1889.909 520.2546
4 V
1969.909 520.2546
4 V
1969.909 621.3022
5 T
1790.841 621.3022
5 T
1870.841 701.3022
5 T
1870.841 718.3549
6 P
1689.793 718.3549
6 P
1769.793 798.3549
6 P
1689.793 819.4025
7 T
1592.741 819.4025
7 T
1672.741 899.4025
7 T
1592.741 920.4502
8 T
1491.693 920.4502
8 T
1571.693 1000.45
8 T
1491.693 1080.481
9 C
1390.645 1080.481
9 C
1470.645 1160.481
9 C
1390.645 1167.513
10 S
1230.615 1167.513
10 S
1310.615 1247.513
10 S
1230.615 1281.556
11 N
1143.583 1281.556
11 N
1223.583 1361.556
11 N
1143.583 1382.603
12 T
1029.54 1382.603
12 T
1109.54 1462.603
12 T
1029.54 1495.687
13 I
928.4923 1495.687
13 I
1008.492 1575.687
13 I
928.4923 1610.714
14 D
815.4083 1610.714
14 D
895.4083 1690.714
14 D
815.4083 1723.798
15 L
700.3814 1723.798
15 L
780.3814 1803.798
15 L
700.3814 1820.851
16 P
587.2974 1820.851
16 P
667.2974 1900.851
16 P
587.2974 1951.891
17 M
490.2447 1951.891
17 M
570.2446 2031.891
17 M
490.2447 2038.923
18 S
359.2042 2118.923
18 S
439.2042 2118.923
18 S
359.2042 2135.976
19 P
272.1722 2215.976
19 P
272.1722 2215.976
19 P
272.1722
175.1195
175.1195
175.1195
Phosphorylation examples
SLIDE 5
Potential modifications
SLIDE 6
Enrichment Strategies for the Detection of Phosphorylated Peptides
SLIDE 7 Enrichment Strategies for the Detection of Phosphorylated Peptides
- Hydrophilic Interaction Chromatography (HILIC)
- Phosphopeptides elute later than their unphosphorylated
counterparts
- Stationary phase is hydrophilic
- Mobile phase is hydrophobic
Unphosphorylated single phosphorylation multiple phosphorylation
SLIDE 8 Time (min)
neutral peptides basic peptides SCX
- Strong Cation Exchange Chromatography
- Stationary phase is negatively charged
- Mobile phase is a buffer that is increasing the pH (if peptide
becomes neutral it elutes)
- Neutral peptides elute earlier: XXpSxxxxxR/K
- Positive peptides elute late: XXXXHXXXXR/K
Enrichment Strategies for the Detection of Phosphorylated Peptides
SLIDE 9
Several Strategies are often combined
SLIDE 10
Loss of the phosphate group
SLIDE 11 0.2 0.4 0.6 0.8 1 1.2 5 10 15 20 25 Number of fragment ions Probability of Localization
Phosphopeptide identification
mprecursor = 2000 Da ∆mprecursor = 1 Da ∆mfragment = 0.5 Da Phosphorylation
Localization of modifications
SLIDE 12 0.2 0.4 0.6 0.8 1 1.2 5 10 15 20 25 Probability of Localization Number of fragment ions
ID 3
Localization (dmin=3)
mprecursor = 2000 Da ∆mprecursor = 1 Da ∆mfragment = 0.5 Da Phosphorylation
dmin>=3 for 47%
peptides
Localization of modifications
SLIDE 13 0.2 0.4 0.6 0.8 1 1.2 5 10 15 20 25 Probability of Localization Number of fragment ions
ID 3 2
Localization (dmin=2)
mprecursor = 2000 Da ∆mprecursor = 1 Da ∆mfragment = 0.5 Da Phosphorylation
dmin=2 for 33% of human tryptic peptides
Localization of modifications
SLIDE 14 0.2 0.4 0.6 0.8 1 1.2 5 10 15 20 25 Probability of Localization Number of fragment ions
ID 3 2 1
Localization (dmin=1)
mprecursor = 2000 Da ∆mprecursor = 1 Da ∆mfragment = 0.5 Da Phosphorylation
dmin=1 for 20% of human tryptic peptides
Localization of modifications
SLIDE 15 0.2 0.4 0.6 0.8 1 1.2 5 10 15 20 25 Probability of Localization Number of fragment ions
ID 3 2 1 1*
Localization (d=1*)
mprecursor = 2000 Da ∆mprecursor = 1 Da ∆mfragment = 0.5 Da Phosphorylation
Localization of modifications
SLIDE 16
Peptide with two possible modification sites
Localization of modifications
SLIDE 17
Peptide with two possible modification sites MS/MS spectrum
m/z Intensity
Localization of modifications
SLIDE 18
Peptide with two possible modification sites MS/MS spectrum
m/z Intensity
Matching
Localization of modifications
SLIDE 19
Peptide with two possible modification sites MS/MS spectrum
m/z Intensity
Matching Which assignment does the data support? 1, 1 or 2, or 1 and 2?
Localization of modifications
SLIDE 20 AAYYQK
Visualization of evidence for localization
AAYYQK
SLIDE 21 Visualization of evidence for localization
AAYYQK AAYYQK
SLIDE 22
Visualization of evidence for localization
3 2 1 3 2 1
SLIDE 23 Estimation of global false localization rate using decoy sites
By counting how many times the phosphorylation is localized to amino acids that can not be phosphorylated we can estimate the false localization rate as a function of amino acid frequency.
0.005 0.01 0.015 0.02 0.05 0.1 0.15 0.005 0.01 0.015 0.02 0.05 0.1 0.15
Amino acid frequency False localization frequency Y
SLIDE 24 S
2 1
S
m 1
How much can we trust a single localization assignment?
If we can generate the distribution of scores for assignment 1 when 2 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment.
S S
m m 2 1 >
∫ ∫ =
∞ 2 1 2 1 2 2 1 2 1 2 2 1
1
dS S F dS S F p
S
m
) ( ) (
1. 2.
SLIDE 25 Is it a mixture or not?
If we can generate the distribution of scores for assignment 2 when 1 is the correct assignment, it is possible to estimate the probability of obtaining a certain score by chance for a given peptide sequence and MS/MS spectrum assignment.
S
1 2
S
m 2
S S
m m 2 1 >
∫ ∫ =
∞ 1 2 1 2 1 1 2 1 2 1 1 2
) ( ) (
2
dS S F dS S F p
Sm
1. 2.
SLIDE 26 ⇒ ≤ ≤
p p p p
th thand 1 2 2 1
1 and 2
⇒ > ≤
p p p p
th thand 1 2 2 1
1
⇒ ≤ >
p p p p
th thand 1 2 2 1
⇒ > >
p p p p
th thand 1 2 2 1
1 or 2 Ø
) (
p p S S
m m
≤
⇒ ≥
1 2 2 1 2 1
Peptide with two possible modification sites MS/MS spectrum
m/z Intensity
Matching Which assignment does the data support? 1, 1 or 2, or 1 and 2?
Localization of modifications
SLIDE 27 Top down / bottom up
Top down
Bottom up
mass/charge intensity
SLIDE 28 Top down Bottom up
Charge distribution
mass/charge intensity mass/charge intensity 1+ 2+ 3+ 4+ 27+ 31+
SLIDE 29 Top down Bottom up
m= 1878 Da
Isotope distribution
mass/charge intensity mass/charge intensity
SLIDE 30 Fragmentation
Top down Bottom up Fragm gmenta tati tion
SLIDE 31
Alternative Splicing
Top down Bottom up
Exon 1 2 3
SLIDE 32
Correlations between modifications
Top down Bottom up
SLIDE 33
The Nucleosome Core Complex
H3 H4 H2A H2B H3 ‘tail’ Luger et al., Nature, 389, 251-260, 1997
SLIDE 34 The N-terminal Tails of Histone H3 and H4
Methylation: mono-, di-, or trimethylation Acetylation Phosphorylation
Ac
H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50
M M M M M P M M Ac M Ac P P P M P
H4 1-SGRGKGGKGLGKGGAKRHRKVLRDNIQGITKPAIRRLARRGGVKRISGLIYE-52
M M Ac Ac Ac Ac Ac P Ac M Ac P
SLIDE 35
Specific post translational modifications (PTMs) of the N-terminal tails of histones function as a scaffold for binding of protein factors leading to transcriptional activation or inactivation. Jenuwein, T., Allis, C.D., Science, 293, 2001
The Histone Code Hypothesis
SLIDE 36 Ac
KSTGGKAPR 9-17 TKQTAR 3-8 KQLATKAAR 18-26 KSAPATGGVKKPHR 27-40 41-50 YRPTVALRE
M Ac Ac Ac
H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50
M P M P P P P
Interdependence of Modifications is lost in Standard Mass Spectrometry Analysis
Ac Ac Ac M Ac M M M M M M M P M M
SLIDE 37 Histone Proteins are a Highly Complex Mixture
ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE ARTKQTARKSTGAKAPRKQLASKAARKSAPATGGIKKPHRFRPGTVALRE
M M M M M Ac M M M M Ac M M M M M
……………… and many many more!
M M M M
SLIDE 38 Protocol
- Isolate m/z ± 0.5 Da
- 60 ms ETD
- ~ 3 min acquisition
Glu-C generated N-terminal H3 peptide (1-50)
m/z
245.2 346.3 982.5 502.4 824.5 892.5 630.5 731.5 1647.9 672.3 1055.6 288.1 571.3 802.5 479.9 958.6 1715.0 1216.7 401.8 1784.1 1129.6 1878.2 1515.4 1255.2 1373.8 1424.8 1937.8 1616.0
LTQ-FTMS LTQ-ETD/PTR
4 9 14 18 23 27 36 N 50 37
m/z
+10 +11 +9 +8 +7 +12
m/z
+ 10 charge states
∆ 1.4 Da ∆ 1.4 Da ∆ 1.4 Da
546.3 547.6 549.1 550.4 551.9 544.9
SLIDE 39 Group ‘4’: 4 Acetyl Groups
c
6
400 800 100
Relative Abundance
c
2 c 3
c
4
c
5
z
2
z
3
z
4
z
5
z
6
z
7
* * * * * * *
1200 1600 2000
m/z
c
9
c
13
c
7
c
8
c
10
c
11
c
12
c
16
c
17
z
9
z
10
z
11
z
12
z
14 z 15
* * * * * * * * * * * * * * * * z
16
A R T K Q T A R K S T G A K A P R K Q L A S K A A R K S A P A T G G I K K P H R F R P G T V A L R E A R T K Q T A R K S T G A K A P R K Q L A S K A A R K S A P A T G G I K K P H R F R P G T V A L R E A R T K Q T A R K S T G A K A P R K Q L A S K A A R K S A P A T G G I K K P H R F R P G T V A L R E
M M M M M M M M M Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac
SLIDE 40 Group ‘5’: 5 Acetyl Groups
400 600 800 1000 1200 1400 1600 1800 2000
m/z
100
Relative Abundance
K4: trimethyl
c
3
c
4
c
5
c
9
c
13
c
6
c
7
c
8
c
10
c
11
c
12
c
16
z
2
z
3
z
4
z
5 z 6
z
7
z
9
z
10
z
11
z
12
z
14
z
15
* * * * * * * * * * * * * * c
2
* * c
14
z
16
z
17 c 17
A R T K Q T A R K S T G A K A P R K Q L A S K A A R K S A P A T G G I K K P H R F R P G T V A L R E
Ac Ac Ac Ac Ac M M M
SLIDE 41
Proteomics Informatics – Protein characterization I: post-translational modifications (Week 10)
