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ASMS 2003

Using Mascot to characterizeProtein modifications

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ASMS 2003

Post-translationalModifications (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 isbeta-Casein variant CnH.

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ASMS 2003

The other protein is beta-casein precursor.

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ASMS 2003

When we look at the detail, we see that Serine or Threonine is phosphorylated.

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ASMS 2003

With Peptide Mass Fingerprint, it is not possible to map the modification sites. Oneneeds 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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ASMS 2003

Four peptides identified in this slide have scores higher than 34, the significancethreshold. 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 significancethreshold.

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ASMS 2003

Let’s focus on Query 34, where the ion score was 101. We see a rich MS/MSspectrum.

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ASMS 2003

Most of the Y ions have been identified.

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ASMS 2003

From the error graph, one can see that the calibration is quite good. I would believethat this peptide is deamidated.

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ASMS 2003

This example shows a phosphorylated peptide. There are two possiblephosphorylation sites, but the score for phospho-serine (102) is much, much greaterthan that for phospho-threonine. I such cases, there is no doubt about the location ofthe modification

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ASMS 2003

The spectrum shows a strong run of y ions that have lost phosphate as a neutralloss

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ASMS 2003

Background

• Eukaryotic chromosomes: DNA is associated withhistone proteins to form nucleosomes, buildingblocks of chromatin

• PTMs to histones regulate the access to DNA inchromatin

• These mods can change the net charge andstructure of histones

• Histone H3 is modified on the N-terminus (rich inArg and Lys)

• Protease cleavage results in hydrophilic peptides(poorly retained on a RP-HPLC column)

In eukaryotic chromosomes, large amounts of DNA are compacted by associationwith histone proteins to form nucleosomes, the building blocks of chromatin. Accessto DNA in chromatin is regulated by post-translational modifications (PTMs)(methylation, phosphorylation, etc.) to histones. Such modifications can change thehistone’s net charge and structure thus playing a pivotal role in the control ofchromatin structure and function. The majority of histone H3 modifications occuron 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 andpoorly retained on a RP-HPLC column.

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ASMS 2003

Background continued…

• CAD mass spectra of highly multiply chargedpeptides-interpretation is difficult.

• Peptides + Propionic anhydride converts N-terminiand Lysines to propyl amides. This results in adecrease in net charge of the peptides and increasedhydrophobicity.

• Now the peptides can be retained on RP-HPLCcolumns and their CAD spectra are simpler.

The collisionally-activated dissociation (CAD) mass spectra of highly multiplycharged peptides are very difficult to interpret. Their research efforts are placed ondeveloping methods to better analyze histones by HPLC and mass spectrometry.Treatment of peptides with propionic anhydride converts free amine groups onunmodified Lysines or Lysine residues containing 1 methyl group modification andN-termini to propyl amides. The consequence of the above strategy is a decrease inthe net charge of the peptides, as well as increased hydrophobicity thus facilitatingtheir analysis by increasing retention times on an HPLC column and simplifyingtheir CAD mass spectra.

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ASMS 2003

Methods

• Histone protein + Glu-C…isolate 1-50 residue N-terminus by off-line HPLC.

• Second digestion with Chymotrypsin of the 1-50fragment generates appropriate length peptides forMS/MS.

• Derivatization with propionic anhydride.• On-line RP-HPLC with direct elution into an

electrospray ionization quadrupole ion trap massspectrometer.

• MS/MS followed by peptide and PTM identificationusing Mascot.

Their methods begin with an enzymatic digestion of the histone protein with Glu-Cand isolation of the 1-50 residue amino terminus by off-line HPLC. A seconddigestion with Chymotrypsin is performed on the 1-50 fragment to produce peptidesof suitable lengths for tandem mass spectrometry experiments. Peptides are thenderivatized by the addition of the propionic anhydride reagent. The mixture ofpeptides are separated by on-line RP-HPLC before direct elution into anelectrospray ionization quadrupole ion trap mass spectrometer. Tandem massspectrometry experiments are then performed to fragment the ions and determinepost-translational modification sites after searching with Mascot.

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ASMS 2003

Results• Glu-C digestion gave the 1-50 piece• Chymotrypsin digestion produced 1-5, 1-19, 1-20, 6-

19, 6-20, 20-39, 21-39, 23-39, 24-39, 21-41, 40-50, 42-50 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 multiplemodifications!

The digestion of the H3 protein with Glu-C cleaved the protein to produce the 1-50piece further isolated by off-line HPLC. A Chymotrypsin digestion of the 1-50 pieceprimarily produced peptide residues of 1-5, 1-19, 1-20, 6-19, 6-20, 20-39, 21-39, 23-39, 24-39, 21-41, 40-50, 42-50 and other random pieces. The primary modificationsfound were on Lys and Ser residues. However, modifications could also be presenton Arg and Thr residues. Ser and Thr can be modified by the addition of aphosphate group. Lys can be modified by the addition of mono, di or tri-methylgroups or by the addition of an acetyl group. Arg residues can be modified by theaddition of one or two methyl groups. In addition, adding propionic anhydridecreates propyl amide groups on the amino terminus of peptides and peptidescontaining unmodified Lys residues or Lys residues modified by one methyl group.Lys residues containing di, tri-methyl or acetyl groups are not modified by thepropionic anhydride reagent. Thus, as can be easily seen, a vast amount ofmodifications and combination of modifications can be expected when analyzinghistone peptides. Currently, Mascot is one of the few software programs availablethat is able to search data possibly containing multiple modifications. Modificationsof N-terminal (Pr) and Lys modifications of propyl amide (Pr), propyl amide andmethyl (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 thedatabase.

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ASMS 2003

b10

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z05

101520253035404550556065707580859095

100

Rel

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e A

bund

ance

y2

y4 y5

y7

y8

y9

y10

y11

y10+2

b3

y8+2b4

b5 b6 b7

b9 b12

b18+2

170 241 342 526 597 668 824 980 1067 1138 1235 1306 1407 1464 1521 1620 1804 1988 2085 2240Leu Ala Thr Lys Ala Ala Arg Lys (diMe) Ser Ala Pro Ala Thr Gly Gly Val Lys Lys Pro His2240 2071 2000 1899 1715 1644 1573 1417 1261 1174 1103 1006 935 834 777 720 621 437 253 156

170 241 342 512 583 654 810 1008 1095 1166 1263 1334 1435 1492 1549 1648 1804 1988 2085 2240Leu Ala Thr Lys (Ac) Ala Ala Arg Lys (Me) Ser Ala Pro Ala Thr Gly Gly Val Lys (diMe) Lys Pro His2240 2071 2000 1899 1729 1658 1587 1431 1233 1146 1075 978 907 806 749 692 593 437 253 156

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z05

101520253035404550556065707580859095

100

Rel

ativ

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bund

ance

y2 y4y5

y7

y8

y9

y10

y11

y12

y10+2

y8+2

b5

b6 b7b9

b10

b11

b18+2

200 400 600 800 1000 1200 1400 1600 1800 2000

m/z05

101520253035404550556065707580859095

100

Rel

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x5 x10

y2

y4y7b6

y12

y10

y11b4

b5

b8 b17

b10 [b17 + OH]+2

y3 b9

b18+2

b17+2

b16+2

b14+2

b10+2

170 241 342 526 597 668 824 1008 1095 1166 1263 1334 1435 1492 1549 1648 1804 1988 2085 2240Leu Ala Thr Lys Ala Ala Arg Lys Ser Ala Pro Ala Thr Gly Gly Val Lys (diMe) Lys Pro His2240 2071 2000 1899 1715 1644 1573 1417 1233 1146 1075 978 907 806 749 692 593 437 253 156

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.

N-term- PrK23-PrK27-PrK36-diMeK37-Pr

N-term- PrK23-PrK27-diMeK36-PrK37-Pr

N-term- PrK23-AcK27-MeK36-diMeK37-Pr

This slide shows three peptides with the same parent mass, but they differ in thePTM site(s). There are 4 Lysines that can be modified, there is the amino terminusand a Ser and an Arg residue. The possible combination of PTM sites is large. Thebottom spectrum demonstrates how four different PTMs can be identified byMascot. Mascot was used to identify combination of mods on histone peptides.

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S B 0 7 3 9 0 2 0 3 #3 2 1 9 RT: 9 6 . 9 2 AV: 1 N L : 8 . 6 7 E 5T : + c d F u l l m s 2 1 0 0 6 . 7 9 @ 3 5 . 0 0 [ 2 6 5 . 0 0 - 2 0 0 0 . 0 0 ]

3 0 0 4 0 0 5 0 0 6 0 0 7 0 0 8 0 0 9 0 0 1 0 0 0 1 1 0 0 1 2 0 0 1 3 0 0 1 4 0 0 1 5 0 0 1 6 0 0 1 7 0 0 1 8 0 0 1 9 0 0m /z

0

5

1 0

1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

9 5

1 0 0

Rel

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bund

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1 7 6 0 . 0 0

8 9 5 . 5 6

1 7 6 0 . 9 81 1 1 7 . 6 2

1 1 1 8 . 7 11 3 7 7 . 7 6 1 5 9 2 . 9 8

8 6 7 . 6 0 1 0 6 3 . 6 41 5 7 5 . 8 56 3 5 . 4 8

1 1 6 4 . 6 49 6 7 . 0 0 1 5 9 3 . 9 07 3 7 . 5 3 1 3 4 9 . 6 75 3 9 . 4 11 7 3 1 . 8 93 8 3 . 2 2 1 5 4 8 . 8 71 2 7 5 . 6 0 1 7 7 2 . 0 13 2 0 . 1 7 5 2 0 . 1 6 5 5 1 . 9 4 1 4 7 5 . 8 5 1 6 2 2 . 9 4

241 312 383 539 737 824 895 992 1063 1164 1221 1278 1377 1575 1759 1856 2011Lys Ala Ala Arg Lys (Me) Ser Ala Pro Ala Thr Gly Gly Val Lys (Me) Lys Pro His2011 1771 1700 1629 1473 1275 1188 1117 1020 949 848 791 734 635 437 253 156

Mascot correctly identified the endogenous Lys methylations on K27 and K36, aswell 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 byTandem Mass Spectrometry”

Benjamin A. Garcia1, Scott A. Busby1, A. CelesteDunsmoor1, Jeffrey Shabanowitz1,

Cynthia M. Barber2, C. David Allis2, Donald F. Hunt1,3

Departments of Chemistry1 and Pathology3,University of Virginia, Charlottesville, VA;Department of Biochemistry and MolecularGenetics2, University of Virginia Health ScienceCenter, Charlottesville, VA