Download Using Mascot to characterise protein modifications

Using Mascot to characterize
Protein modifications
ASMS 2003
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Post-translational
Modifications (PTMs)
• Help understand complex biological systems
• Phosphorylation is one of the most important
protein PTMs
• Mascot allows up to 9 variable modifications to
be specified at a time
• Use variable modifications sparingly, follow it by
error tolerant search
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This slide shows that two proteins have scores above the threshold. One of them is
beta-Casein variant CnH.
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The other protein is beta-casein precursor.
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When we look at the detail, we see that Serine or Threonine is phosphorylated.
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With Peptide Mass Fingerprint, it is not possible to map the modification sites. One
needs tandem mass spectrometry to do that.
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This slide shows at least 5 proteins with scores above the threshold.
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Four peptides identified in this slide have scores higher than 34, the significance
threshold. Deamidation seems to be a common modification for all of them.
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Here we see four other peptides that have scores higher than the significance
threshold.
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Let’s focus on Query 34, where the ion score was 101. We see a rich MS/MS
spectrum.
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Most of the Y ions have been identified.
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From the error graph, one can see that the calibration is quite good. I would believe
that this peptide is deamidated.
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This example shows a phosphorylated peptide. There are two possible
phosphorylation sites, but the score for phospho-serine (102) is much, much greater
than that for phospho-threonine. I such cases, there is no doubt about the location of
the modification
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The spectrum shows a strong run of y ions that have lost phosphate as a neutral
loss
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Background
• Eukaryotic chromosomes: DNA is associated with
histone proteins to form nucleosomes, building
blocks of chromatin
• PTMs to histones regulate the access to DNA in
chromatin
• These mods can change the net charge and
structure of histones
• Histone H3 is modified on the N-terminus (rich in
Arg and Lys)
• Protease cleavage results in hydrophilic peptides
(poorly retained on a RP-HPLC column)
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In eukaryotic chromosomes, large amounts of DNA are compacted by association
with histone proteins to form nucleosomes, the building blocks of chromatin. Access
to DNA in chromatin is regulated by post-translational modifications (PTMs)
(methylation, phosphorylation, etc.) to histones. Such modifications can change the
histone’s net charge and structure thus playing a pivotal role in the control of
chromatin structure and function. The majority of histone H3 modifications occur
on the 1-50 residue N-terminal tail, which is rich in Arginine and Lysine residues.
This tail produces peptides upon protease cleavage that are very hydrophilic and
poorly retained on a RP-HPLC column.
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Background continued…
• CAD mass spectra of highly multiply charged
peptides-interpretation is difficult.
• Peptides + Propionic anhydride converts N-termini
and Lysines to propyl amides. This results in a
decrease in net charge of the peptides and increased
hydrophobicity.
• Now the peptides can be retained on RP-HPLC
columns and their CAD spectra are simpler.
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The collisionally-activated dissociation (CAD) mass spectra of highly multiply
charged peptides are very difficult to interpret. Their research efforts are placed on
developing methods to better analyze histones by HPLC and mass spectrometry.
Treatment of peptides with propionic anhydride converts free amine groups on
unmodified Lysines or Lysine residues containing 1 methyl group modification and
N-termini to propyl amides. The consequence of the above strategy is a decrease in
the net charge of the peptides, as well as increased hydrophobicity thus facilitating
their analysis by increasing retention times on an HPLC column and simplifying
their CAD mass spectra.
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Methods
• Histone protein + Glu-C…isolate 1-50 residue Nterminus by off-line HPLC.
• Second digestion with Chymotrypsin of the 1-50
fragment generates appropriate length peptides for
MS/MS.
• Derivatization with propionic anhydride.
• On-line RP-HPLC with direct elution into an
electrospray ionization quadrupole ion trap mass
spectrometer.
• MS/MS followed by peptide and PTM identification
using Mascot.
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Their methods begin with an enzymatic digestion of the histone protein with Glu-C
and isolation of the 1-50 residue amino terminus by off-line HPLC. A second
digestion with Chymotrypsin is performed on the 1-50 fragment to produce peptides
of suitable lengths for tandem mass spectrometry experiments. Peptides are then
derivatized by the addition of the propionic anhydride reagent. The mixture of
peptides are separated by on-line RP-HPLC before direct elution into an
electrospray ionization quadrupole ion trap mass spectrometer. Tandem mass
spectrometry experiments are then performed to fragment the ions and determine
post-translational modification sites after searching with Mascot.
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Results
• Glu-C digestion gave the 1-50 piece
• Chymotrypsin digestion produced 1-5, 1-19, 1-20, 619, 6-20, 20-39, 21-39, 23-39, 24-39, 21-41, 40-50, 4250 and other random pieces
• Primary modifications…found on Lys & Ser
• Mods could also be on Arg & Thr
• Ser & Thr can be phosphorylated
• Lys can have mono, di or tri-methyl groups or an
acetyl group
• Arg can have mono or di methyl groups
• Propyl amide can be on the N-terminus or on
unmodified Lys or Lys with mono-methyl group
• Mascot is one of the few software programs
available for searching data with multiple
modifications!
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The digestion of the H3 protein with Glu-C cleaved the protein to produce the 1-50
piece further isolated by off-line HPLC. A Chymotrypsin digestion of the 1-50 piece
primarily produced peptide residues of 1-5, 1-19, 1-20, 6-19, 6-20, 20-39, 21-39, 2339, 24-39, 21-41, 40-50, 42-50 and other random pieces. The primary modifications
found were on Lys and Ser residues. However, modifications could also be present
on Arg and Thr residues. Ser and Thr can be modified by the addition of a
phosphate group. Lys can be modified by the addition of mono, di or tri-methyl
groups or by the addition of an acetyl group. Arg residues can be modified by the
addition of one or two methyl groups. In addition, adding propionic anhydride
creates propyl amide groups on the amino terminus of peptides and peptides
containing unmodified Lys residues or Lys residues modified by one methyl group.
Lys residues containing di, tri-methyl or acetyl groups are not modified by the
propionic anhydride reagent. Thus, as can be easily seen, a vast amount of
modifications and combination of modifications can be expected when analyzing
histone peptides. Currently, Mascot is one of the few software programs available
that is able to search data possibly containing multiple modifications. Modifications
of N-terminal (Pr) and Lys modifications of propyl amide (Pr), propyl amide and
methyl (Pr-Me), di-methyl (di-Me) and tri-methyl (tri-Me), Ser (phosphorylation)
and Arg modification of (Me) were created and Mascot was used to search the
database.
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Relative Abundance
170 241 342 526 597 668 824 1008 1095 1166 1263 1334 1435 1492 1549 1648 1804 1988 2085 2240
Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly Val Lys (diMe) Lys Pro His
2240 2071 2000 1899 1715 1644 1573 1417 1233 1146 1075 978 907 806 749 692
593
437 253 156
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
200
b10
y10+2
y10
b18+2
y7
y8
b5
y4 by6
y8+2b
4
b7
400
600
y11
b9
y9
5
y2 b3
800
1000
b12
1200
1400
1600
1800
m/z
N-term- Pr
K23-Pr
K27-Pr
K36-diMe
K37-Pr
2000
Relative Abundance
980
1067 1138 1235 1306 1407 1464 1521 1620 1804 1988 2085 2240
170 241 342 526 597 668 824
Leu Ala Thr Lys Ala Ala Arg Lys (diMe) Ser Ala Pro Ala Thr Gly Gly Val Lys Lys Pro His
2240 2071 2000 1899 1715 1644 1573
1417
1261 1174 1103 1006 935 834 777 720 621 437 253 156
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
200
x5
x10
b18+2
b17+2
b10+2
y2
b5
y3 b4
400
y10
+2
b16
b14+2
b
y7
y4 6
b8
600
800
b10 y
11
b9
1000
1200
m/z
y12
[b17 + OH]+2
b17
1400
1600
1800
2000
N-term- Pr
K23-Pr
K27-diMe
K36-Pr
K37-Pr
Relative Abundance
170 241 342
512
583 654 810
1008 1095 1166 1263 1334 1435 1492 1549 1648
1804
1988 2085 2240
Leu Ala Thr Lys (Ac) Ala Ala Arg Lys (Me) Ser Ala Pro Ala Thr Gly Gly Val Lys (diMe) Lys Pro His
593
437 253 156
2240 2071 2000 1899 1729 1658 1587 1431 1233 1146 1075 978 907 806 749 692
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
0
200
y10+2
y10
b10
y7
y8+2
y2
400
b6
b7
b5 y5
y4
600
y8 b18
+2
y9
800
1000
b9
y
1
y12 b11
1200
m/z
1
1400
1600
N-term- Pr
K23-Ac
K27-Me
K36-diMe
K37-Pr
1800
2000
The complexity of analyzing histone peptide samples is shown by comparison of
three mass spectra of 20-39 residue peptides all having the same parent
m/z of 748.2 Da (charge state = +3), but having different PTM sites. Modifications
were identified using the Mascot software program. Pr = chemical modification of propionyl
group, Ac = endogenous acetylation, Me = endogenous methylation, diMe = endogenous
dimethylation.
ASMS 2003
This slide shows three peptides with the same parent mass, but they differ in the
PTM site(s). There are 4 Lysines that can be modified, there is the amino terminus
and a Ser and an Arg residue. The possible combination of PTM sites is large. The
bottom spectrum demonstrates how four different PTMs can be identified by
Mascot. Mascot was used to identify combination of mods on histone peptides.
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241 312 383
539
737
824 895 992 1063 1164 1221 1278 1377
1575
1759 1856 2011
Lys Ala
Ala
Arg Lys (Me) Ser
Ala Pro Ala Thr Gly Gly
Val Lys (Me) Lys Pro His
2011 1771 1700 1629
1473
1275 1188 1117 1020 949 848 791 734
635
437 253 156
S B 0 7 3 9 0 2 0 3 # 3 2 1 9 R T : 9 6 . 9 2 AV: 1 N L : 8 . 6 7 E 5
T: + c d Full ms2 [email protected] [ 265.00-2000.00]
1760.00
100
95
90
85
80
75
895.56
70
Relative Abundance
65
60
55
1760.98
1117.62
50
45
40
35
30
25
1118.71
20
15
867.60
10
320.17 383.22
520.16
1592.98
1575.85
1164.64
967.00
737.53
539.41
5
1377.76
1063.64
635.48
1593.90
1349.67
1275.60
551.94
1475.85
1548.87
1731.89
1622.94
1772.01
0
300
400
500
600
700
800
900
1000
1100
m /z
1200
1300
1400
1500
1600
1700
1800
1900
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Mascot correctly identified the endogenous Lys methylations on K27 and K36, as
well as our chemical modifications of propionyl on K37 and K23 (amino terminus).
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Poster
Tuesday (#941) entitled “Analysis of Human
Histone H3 Post-Translational Modification Site
Patterns from Cells Arrested During Mitosis by
Tandem Mass Spectrometry”
Benjamin A. Garcia1, Scott A. Busby1, A. Celeste
Dunsmoor1, Jeffrey Shabanowitz1,
Cynthia M. Barber2, C. David Allis2, Donald F. Hunt1,3
Departments of Chemistry1 and Pathology 3,
University of Virginia, Charlottesville, VA;
Department of Biochemistry and Molecular
Genetics2, University of Virginia Health Science
Center, Charlottesville, VA
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