1. Identification of Post Translational Modifications
Beatrix Ueberheide
March 25th 2019

2. Homework from last lecture

3. Assigned Paper

5. Ubiquitination - MS

6. Jackson Paper: USP4 ubiquitination
Catalytic dead USP4 (Cys311A) no longer interacts with CtIP and MRN
Could it be that ubiquitination of USP4 is blocking the interaction with CtIP and MRN and that inactive USP4 can no longer de-ubiquitinates itself and hence prevents interaction?
Co-expressed USP4 with HA tagged ubiquitin:
GFP-USP4 WT; GFP-USP4 CD; GFP
Performed affinity purification using the HA tag and Western Blot using the GFP handle
Performed GFP affinity purification followed by LC-MS
Found a sh%^$ load of ubiquitin

7. Table S4

8. Sites of ubiquitination on USP4CD WT
STLVCPECAK [M+1H]+1

9. Ubiquitinated spectrum

10. Which cysteine is ubiquitinylated?

11. Let’s check public databases:

12. From The GPM

13. From The GPM

14. Could it be this?

15. Easy Problem to have avoided

16. Figure Legend

17. Figure Legend

18. Figure Legend

19. Figure Legend

20. Characterizing PTMs

21. Detecting PTMs
In theory quite simple, only the mass of the amino acid changes
In practice difficult for several reasons
Peptide is too short or too long to be detected
Not enough sequence coverage to assign the site of modification
Modification is present only in a small percentage
Peptide is never selected for MS/MS
Several amino acids in the peptide could be modified
Modification might interfere with fragmentation
Not enough sequence coverage to identify the peptide

22. Biological Mass Spectrometry
Proteolytic digestion
Protein(s)
Peptides
Base Peak Chromatogram MS
500
1000
1500
m/z
Time (min)
Mass Spectrometer
200
600
1000
m/z
MS/MS
Database Search
Manual Interpretation

23. Searching Proteomics Data
GSFLYEYSRRHPEYAVSVLLRLAKEYEATLEECCAKDDPHACYSTVFDKLKHLVDEPQNLIKQNCDQFEKGEYGFQNALIVRYTRKVPQVSTPTLVEVSRSLGKVGTRCCTKPESERMPCTEDYLSLILNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALTP
Protein
Digestion
LFTFHADICTLPDTEK
RPCFSALTPDETYVPK
MPCTEDYLSLILNR
VPQVSTPTLVEVSR
DDPHACYSTVFDK
Peptide Mass
Measurement
500
1000
1500
m/z MS
Peptide Fragmentation
200
600
1000
m/z
MS/MS

24. Proteomics is more than Mass Spec
Enrichment
Fractionation
Sample
Protein Extraction
Protein Digestion
Data Analysis
LC-MS
Peptide Clean up
Enrichment
Fractionation

25. Inherent difficulty of proteome characterization
Proteolytic digestion
Protein(s)
Peptides
Size of peptides depends entirely on the protein sequence (K, R, D, E, Y, F,….)
Peptides too short or too long are not detectable
If those peptides carry PTMs, they go undetected as well

26. Inherent difficulty of proteome characterization
Proteolytic digestion
Protein(s)
Peptides
Protein A
Protein B
Protein C
Protein D
Protein E
One protein group is reported!

27. Inherent difficulty of proteome characterization
Proteolytic digestion
Protein(s)
Peptides
Protein A Cancer
Protein A Healthy

28. The Central Dogma Proteomics Metabolomics seconds to minutes
hours (seconds)
Genomics
Transcriptomics
Proteomics
Metabolomics
Adapted from Patti et al (2011) Nature Reviews| Molecular Cell Biology

29. Proteomics

30. Proteomics Protein Production/Changes Protein PTM modification/Changes
Protein-Protein Interaction/Changes
Protein Structural Changes

31. Dynamic Range 200ng HeLa lysate: ~2500 proteins >8000 proteins10 20 30 40 50 60 70 80 90
100
110
120
Time (min)
Relative Abundance
444.74
477.31
408.73
492.79
652.36
472.77
425.72
599.76
416.25
590.81
566.77
567.78
420.79
533.32
644.73
757.40
895.95
446.91
655.85
584.80
680.37
533.27
726.05
911.40
455.32
899.46
200ng HeLa lysate:
~2500 proteins
>8000 proteins 10 20 30 40 50 60 70 80 90
100
110
120
130
140
150
160
Time (min)
Relative Abundance
575.31
395.24
476.23
671.82
682.70
480.79
492.75
467.26
518.21
547.32
663.58
829.38
489.96
371.10
518.03
637.98
749.80
669.59
756.43
992.46
623.58
593.83
840.41
738.06
871.38
200ng Human Plasma:
~250 proteins

32. Success Rate of a Proteomics Experiment
Distribution of
Protein Amounts
Number of Proteins
Fractionation
Enrichment
Proteins
Detected
Log (Protein Amount)
DEFINITION: The success rate of a proteomics experiment is defined as the number of proteins detected divided by the total number of proteins in the proteome.
J. Eriksson, D. Fenyö, "Improving the success rate of proteome analysis by modeling protein-abundance distributions and experimental designs". Nature Biotechnology 25 (2007)

33. Fractionation and Enrichment
One sample becomes 10 or more Fractions
Protein identification increases from ~2000 to ~8000 ( 2 hours versus 60 hours instrument time)

34. Fractionation and Enrichment increases the MS instrument time

35. Multiplexing for Quantitation
SILAC
TANDEM MASS TAGS

36. SILAC

37. Multiplex Analysis (TMT)

38. iTRAQ

39. Multiplex Analysis (TMT)

40. TMT (iTRAQ)

41. MS2 of a peptide Peptide: AVDTWSWGER
Protein: Galectin 3 binding protein
May stimulate host defense against viruses and tumor cells

42. Zoom in

43. Quantitative multiplexed proteomic analysis of TNKS DKO cells
7,254 proteins quantified:
608 showed significant change in abundance
287 increased in the DKO
74 of the 287 contained a TNKS-binding site
23 of the 74 tankyrase targets are shown in the heatmap
Highlighted proteins were selected for validation
Bhardwaj A., Yang, Y., Ueberheide, B., Smith, S., Whole proteome analysis of human tankyrase knockout cells reveals targets of tankyrase-mediated degradation, Nature Communications, 8:2214 (2017)

44. Characterizing PTMs

45. Modified Peptide is not abundant enough to be selected for MS/MS
enriching for the modification
Phospho specific enrichment (IMAC, TiO2)
Antibody based enrichment (ubiquitin, phosphotyrosine)

46. Fractionation and Enrichment
Total Cell Lysate
1824 Protein Groups
12389 Peptides
26 phosphorylated peptides
100 % S & T 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
105
110
115
120
125
130
135
Time (min) 5 10 15
Relative Abundance
Total cell lysate 1 HILIC Fraction
917 Protein Groups
1208 Peptides
95 phosphorylated peptides
100 % S & T
0 % Y 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Time (min) 5 10 15 60 65 70 75 80 85 90 95
100
Relative Abundance

47. Fractionation and Enrichment20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Time (min) 5 10 15 60 65 70 75 80 85 90 95
100
Relative Abundance
Total cell lysate 1 HILIC Fraction
917 Protein Groups
1208 Peptides
95 phosphorylated peptides
100 % S & T
0 % Y
Total Cell Lysate 1 HILIC Fraction TiO2 Enrichment
347 Protein Groups
419 Peptides
387 phosphorylated peptides
99.2 % S & T
0.8 % Y 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 62 64
Time (min) 5 10 15 25 35 45 55 65 70 75 80 85 90 95
100
Relative Abundance

48. Enrichment for pY Pre-Enrichment Post-Enrichment NL: 2.44E95 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
105
110
115
120
125
130
135
140
145
Time (min)
Relative Abundance
NL: 2.44E9
NL: 2.50E7
Pre-Enrichment
Post-Enrichment

49. Enrichment for pY Pre-Enrichment Post-Enrichment 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95
100
105
110
115
120
125
130
135
140
145
Time (min)
Relative Abundance
NL: 2.44E9
NL: 2.50E7
Pre-Enrichment
Total number of Proteins
Total number of Peptides
Total Phospho Peptides
Tyrosine Phospho Peptides
Pre-Enrichment
2364
18826
221 5
Post-Enrichment
988
2919
583
574
Post-Enrichment

50. Enrichment Strategies for the Detection of Phosphorylated Peptides
Unphosphorylated
single phosphorylation
multiple phosphorylation
Hydrophilic Interaction Chromatography (HILIC)
Phosphopeptides elute later than their unphosphorylated counterparts
Stationary phase is hydrophilic
Mobile phase is hydrophobic
Peptide elute when it is more soluble in the solvent, the higher the charge the less soluble in the organic phase
Works for PO4 but other modifications have other ways to enrich

51. Enrichment Strategies for the Detection of Phosphorylated Peptides
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

52. Several Strategies are often combined

53. Multiplexed Mouse Study
10mg of protein for each of the 27 samples

54. Multiplexed Mouse Study
49 x 3 hr LC-MS analysis (>147 hours of instrument time)

55. Is the protein or the phosphorylation changed?

56. What if the enrichment worked, but you still don’t ‘see’ much?

57. MS/MS is not informative
m/z

58. MS/MS is not informative
Peptide with two possible modification sites

59. MS/MS is not informative
Peptide with two possible modification sites
MS/MS spectrum
m/z

60. MS/MS is not informative
Peptide with two possible modification sites
Matching
MS/MS spectrum
m/z

61. MS/MS is not informative
Peptide with two possible modification sites
Matching
MS/MS spectrum
m/z

62. MS/MS is not informative
Peptide with two possible modification sites
Matching
MS/MS spectrum
m/z

63. Change the way the peptide get’s dissociated in the mass spectrometer
What if enough of the modified peptide is present, but still no ID is possible?
Change the way the peptide get’s dissociated in the mass spectrometer

64. Tandem MS - Dissociation Techniques
CAD: Collision Activated Dissociation (b, y ions)
increase of internal energy through collisions

65. Tandem MS - Dissociation Techniques
CAD: Collision Activated Dissociation (b, y ions)
increase of internal energy through collisions
ETD: Electron Transfer Dissociation (c, z ions)
bombardment of peptides with electrons (radical driven fragmentation)

66. Tandem MS - Dissociation Techniques
CAD: Collision Activated Dissociation (b, y ions)
selective fragmentation
ETD: Electron Transfer Dissociation (c, z ions)
more random fragmentation

67. Detecting PTMs
In theory quite simple, only the mass of the amino acid changes
In practice difficult for several reasons
Peptide is too short or too long to be detected
Not enough sequence coverage to assign the site of modification
Modification is present only in a small percentage
Peptide is never selected for MS/MS
Several amino acids in the peptide could be modified
Modification might interfere with fragmentation
Not enough sequence coverage to identify the peptide

68. Searching for PTMs

69. CAD versus ETD x x Low charge
Modified from Joshua Coon, Analytical Chemistry, 81, (2009)

70. Doubly charged peptides

71. CAD versus ETD x Low charge Labile modifications
Modified from Joshua Coon, Analytical Chemistry, 81, (2009)

72. Phosphopeptides

73. O-Sulfonated Peptides

74. CAD versus ETD x x Low charge Labile modifications Intact proteins
Modified from Joshua Coon, Analytical Chemistry, 81, (2009)

75. Intact Histone Protein
CAD
ETD

76. Intact Histone Protein
CAD M M P Ac Ac Ac P M P Ac M Ac M M M P P M M
H3 1-ARTKQTARKSTGGKAPRKQLATKAARKSAPATGGVKKPHRYRPTVALRE-50
ETD

77. Large highly charged peptides
1:1000
m/z 11 12 13 14
Time (min)
415 m/z