1. Full protein characterization by
Top-Down and Bottom-Up
proteomics
Rolduc, 14 April 2014
Hans Wessels
Radboud Centre for Proteomics, Glycomics & Metabolomics
Radboudumc

2. • Background
• Radboud Centre for Proteomics, Glycomics & Metabolomics
• Need for combined Top-Down and Bottom-Up proteomics
• Experimental setup
• Case: characterization of Complex I subunits in Y. lipolytica
Contents

3. Proteomics MetabolomicsGlycomics
Research Biomarkers Diagnostics
• Mass spectrometry / NMR-based (20 dedicated fte)
• Part of core diagnostic laboratory (Department Laboratory Medicine, 310 people)
• Close interaction with Radboudumc scientists, technology centers and external partners
Radboud Centre for Proteomics, Glycomics & Metabolomics

4. Bottom-up proteomics
• Protein identification
• Differential protein expression profiling
• Protein-Protein interactions
Established technology (>200 projects)
Top-down proteomics
• Clinical diagnostics
• Protein characterization
Emerging technology (8 projects ongoing)
Targeted proteomics
• Biomarker quantitation
Emerging technology (5 projects ongoing)
Whole proteome analysis De novo protein identification
Protein complex isolation and characterization
Proteomics 2009 Cell Metab. 2010 Proteomics 2012 PLoS One 2013
EMBO Journal 2010 Analytical Chemistry 2011 Expert Rev. Proteomics 2012 J Proteomics 2013
Nature 2010 Nature 2011 Nucleic Acids Res. 2012 N Engl J Med. 2014
Proteomics expertise

5. Diagnostics of glycosylation disorders by Top Down MS
• 12 families with liver disease and dilated cardiomyopathy (5-20 years)
• Initial clinical assessment didn’t yield clear cause of symptoms
• Specific sugar loss of serum transferrin identified via glycoproteomics
• Genetic defect in glycosylation enzyme (PGM1) identified via exome sequencing
{Tegtmeyer et al, NEJM 370;6: 533 (2014)}

6. Diagnostics of glycosylation disorders by Top Down MS
• Outcome 1: Explanation of disease
• Outcome 2: Dietary intervention as succesful personalized therapy
• Outcome 3: Glycoprofile transferrin applied as diagnostic test

7. Output:
• Exact mass of intact protein X
• Sequence of N- and C-termini
• Unknown PTMs (+28 Da shift)
Combined strength of Top Down and Bottom Up proteomics
Output:
• Sequence of fragments of protein X
• Methylated Lysine (K150)
• Methylated Arginine (R170)
AA18 AA271
Uncharacterized protein
PROCESSED PROTEIN FORM X
MetMetAA18 AA271

8. Experimental overview

9. Data analysis overview
Experimental data
Protein
form
Protein
form 1
Protein
form 2
Protein
form 3
In silico data

10. UHR ESI Qq-ToF with ETD capability
Bruker Daltonics maXis 4G ETD

11. Characterization of mitochondrial
complex I subunits in Yarrowia
lipolytica by Top-Down and
Bottom-Up proteomics

12. Complex I deficiency leads to severe multi-systemic disorders
• Elucidation of the CI structure is required to understand the effect of mutations
• Enzyme deficiency can only be explained in ~50% of CI deficient patients
• Hypothesis: abberrant subunit processing may explain unsolved patients

13. • 42 established subunits (7 mtDNA, 35 nDNA)
• Unknown mature subunit forms
• Unknown and dynamic post-translational modifications
• Study: Combine Top-Down and Bottom-Up characterization of all subunits
Mitochondrial complex I of Y. lipolytica as a model for human CI
Abdrakhmanova,A. et al. (2004). BBA-Bioenergetics 1658:148-156.
Morgner,N. et al. (2008) BBA-Bioenergetics 1777:1384-1391.
Angerer et al. (2011) Biochem. J 437:279-288
Hunte, Zickermann & Brandt (2010) Science 329:448-451

14. Survey View
500
1000
1500
2000
2500
m/z
10 20 30 40 50 60 70 Time [min]
LC-MS ion map of the 42-subunit protein Complex I
10 20 30 40 50 60 70 Time [min]
500
1000
1500
2000
2500
m/z

15. ESI spectrum of 1 subunit of Complex I
Survey View
500
1000
1500
2000
2500
m/z
10 20 30 40 50 60 70 Time [min]
'1009.7168
10+
'1121.7954
9+
'1261.8938
8+
'1442.0208
7+ '1682.1905
6+
'2018.4295
5+
+MS, 56.8-58.7min #3408-3522
0
1
2
3
4
5
4x10
Intens.
1000 1200 1400 1600 1800 2000 2200 m/z
5+
6+
7+
8+
9+
10+
5+
6+7+
8+
9+
10+
Mr 10087 Da

16. 3 case examples of Complex I subunit characterisation
• Protein without import sequence cleavage: NIDM
• Protein with import sequence cleavage: NUMM
• Protein in two isoforms: with and without leader Met truncation: N2BM

17. '729.2271
15+
'781.2432
14+
'841.2617
13+
'874.8977
15+
'911.2832
12+
'937.3187
14+
'994.0360
11+
'1093.3391
10+
'1214.7095
9+
'1366.4235
8+
'1412.0093
7+
'1516.8848
7+
'1561.4838
7+
'1647.1765
6+
'1821.5622
6+
'1873.6333
7+
+MS, 11.2-12.1min #673-728
0.0
0.5
1.0
1.5
2.0
2.5
3.0
5x10
Intens.
800 1000 1200 1400 1600 1800 2000 m/z
NIDM: ESI UHR Qq-TOF mass spectrum
Chargestate envelope of NIDM
800 2000 m/z1000 1200 1400 1600 1800

18. '729.2271
15+
'781.2432
14+
'841.2617
13+
'874.8977
15+
'911.2832
12+
'937.3187
14+
'994.0360
11+
'1093.3391
10+
'1214.7095
9+
'1366.4235
8+
'1412.0093
7+
'1516.8848
7+
'1561.4838
7+
'1647.1765
6+
'1821.5622
6+
'1873.6333
7+
+MS, 11.2-12.1min #673-728
0.0
0.5
1.0
1.5
2.0
2.5
3.0
5x10
Intens.
800 1000 1200 1400 1600 1800 2000 m/z
NIDM: ESI UHR Qq-TOF mass spectrum
Chargestate envelope of NIDM
841.7 m/z
13+
800 2000 m/z1000 1200 1400 1600 1800

19. '841.2617
13+
'842.0707
28+842.2976
2+
+MS, 11.2-12.1min #673-728
0
2
4
6
4x10
Intens.
840.5 841.0 841.5 842.0 842.5 843.0 m/z
NIDM: ESI UHR Qq-TOF mass spectrum (zoom-in on z=13+)
841.7 m/z

20. NIDM: ETD MS/MS mass spectrum of m/z 841.2 (z=13+)
700.3461
1+
828.4028
1+
1052.4863
2+
1135.5799
1+
1321.6325
3+
'1413.0075
3+
'1564.3353
7+
'1824.8896
6+
'2019.7285
4+
'2189.6673
5+
'2737.5847
4+
'3649.7803
3+
+MS2(ETD 842.9055), 10.0eV, 6.146-10.103min #370-611, Smoothed (0.03,6,SG)
0
100
200
300
400
500
Intens.
500 1000 1500 2000 2500 3000 3500 4000 m/z
500 1000 1500 2000 2500 3000 3500 4000 m/z

21. NIDM: ETD MS/MS mass spectrum of m/z 841.2 (z=13+)
700.3461
1+
828.4028
1+
1052.4863
2+
1135.5799
1+
1321.6325
3+
'1413.0075
3+
'1564.3353
7+
'1824.8896
6+
'2019.7285
4+
'2189.6673
5+
'2737.5847
4+
'3649.7803
3+
+MS2(ETD 842.9055), 10.0eV, 6.146-10.103min #370-611, Smoothed (0.03,6,SG)
0
100
200
300
400
500
Intens.
500 1000 1500 2000 2500 3000 3500 4000 m/z
500 1000 1500 2000 2500 3000 3500 4000 m/z

22. '1155.5726
4+
1161.5736
1+
1163.5907
2+
'1165.8294
4+
'1168.9086
9+
'1170.4378
5+
'1172.8267
4+
'1176.8306
4+
1179.0341
2+
1180.8363
2+
'1183.5754
8+
'1187.8358
4+
'1192.5667
3+
'1194.5875
4+
'1201.0944
4+
'1203.3824
5+
'1205.3429
4+
'1210.2575
9+
'1211.9224
3+
'1215.0362
9+
'1216.9279
9+
'1220.5760
2+
'1222.6080
2+
'1226.2501
3+
+MS2(ETD 842.9055), 10.0eV, 6.146-10.103min #370-611, Smoothed (0.03,6,SG)
0
20
40
60
Intens.
1160 1170 1180 1190 1200 1210 1220 m/z
NIDM: ETD MS/MS mass spectrum of m/z 841.2 (z=13+)
Complex MS/MS
High resolution
1160 1170 1180 1190 1200 1210 1220 m/z

23. NIDM: MASCOT virtual precursor ion search in BioTools

24. Top Down proteomics
Bottom Up proteomics
m/z z MASCOT Score Residues Fragmentation Modifications
841.2617 13 128 S2-K92 ETD Truncated Met, Acetyl: 1
MS/MS sequence coverage: 85.7%
MS sequence coverage: 100%
Combined MS/MS sequence coverage 53.3%
Combined MS sequence coverage: 58.7%
m/z z MASCOT Score Fragmentation Residues Modifications
829.412 3 65 CID S2-K21 Truncated Met, Acetyl: 1
872.042 3 29 ETD S2-K22 Truncated Met, Acetyl: 1
872.042 3 40 CID S2-K22 Truncated Met, Acetyl: 1
NIDM: Top Down & Bottom Up database search results
ETD
CID

25. NIDM: deconvoluted experimental and simulated spectra
'10923.3198
Mr
'10947.2792
Mr
'10961.2630
Mr
CI filtered Captive 3ul 05FA_Tray02-E1_01_1071.d: +MS, 11.2-12.1min, Deconvoluted (MaxEnt, 503.10-2187.28, *0.063125, 50000)
CIfilteredCaptive₃ul₀₅FA_Tray₀₂-E₁₀1₁071.d:C₄₈₀H₇₄₃N₁₃₉O₁₅₂S₄, , 11014.3560
CIfilteredCaptive₃ul₀₅FA_Tray₀₂-E₁₀1₁071.d:C₄₇₇H₇₃₄N₁₃₈O₁₅₂S₃, , 10923.3105
0.0
0.2
0.4
0.6
0.8
1.0
6x10
Intens.
0.0
0.2
0.4
0.6
0.8
1.0
1.2
6x10
0.0
0.2
0.4
0.6
0.8
1.0
1.2
6x10
10920 10940 10960 10980 11000 11020 m/z
Measured spectrum
Simulated spectrum - unprocessed form
(database entry)
Simulated spectrum - hypothesized form
(according to MS/MS results)

26. Mr 10923.3198 Da
Mass error: 0.0088Da (0.81 ppm)
NIDM: Overlay of deconvoluted experimental and simulated spectra

27. • AA Sequence: S2-K92
• N-terminal Methionine truncation
• Acetylation of Ser at protein N-terminus
• No predicted import sequence cleavage (MITOPROT)
• Methionine truncation in line with previous work1,2
NIDM: summary
1 Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels. Sokolova L, Wittig I, Schägger H,
et al. Proteomics 2010, 10, 1401-1407
2 A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.
Angerer H, Zwicker K, Wumaier Z, et al. Biochemical Journal 2011, 437, 279-288

28. m/z z MASCOT Score Residues Fragmentation Modifications
874.8315 15 59 K19-H136 ETD N-term truncation
MS/MS sequence coverage: 61.0%
MS sequence coverage: 100%
Combined MS/MS sequence coverage 49.3%
Combined MS sequence coverage: 50.7%
m/z z MASCOT Score Fragmentation Residues Peptide Sequence Modifications
1036.03 2 73 CID S20-K37 K.SIISYNGNTIEIPEEYTK.Q
1036.03 2 35 ETD S20-K37 K.SIISYNGNTIEIPEEYTK.Q
NUMM: Top Down & Bottom Up database search results
Top Down proteomics
Bottom Up proteomics

29. NUMM: Overlay of deconvoluted experimental and simulated spectra
Mr 13107.3636 Da
Mass error: 0.0049 Da (0.4 ppm)

30. • AA Sequence: K19-H136
• N-terminal truncation: M1-S18
• Unexpected signal cleavage with respect to previous work1,2
• Import sequence predicted by MITOPROT algorithm (single AA difference)
MITOPROT: MLSRFVSKRAFSSTQVSK
Experimental: MLSRFVSKRAFSSTQVS
NUMM: Summary
1 Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels. Sokolova L, Wittig I, Schägger H,
et al. Proteomics 2010, 10, 1401-1407
2 A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.
Angerer H, Zwicker K, Wumaier Z, et al. Biochemical Journal 2011, 437, 279-288

31. m/z z MASCOT Score Residues Fragmentation Modifications
972.5070 7 25 M1-H61 ETD
MS/MS sequence coverage: 43.3%
MS sequence coverage: 100%
Combined MS/MS sequence coverage 60.0%
Combined MS sequence coverage: 63.3%
m/z z MASCOT Score Fragmentation Residues Peptide Sequence Modifications
656.943 2 17.2 CID 1 - 11 -.MAPQLKDPWAR.R
495.667 3 41.8 ETD 1 - 12 -.MAPQLKDPWARR.E Met oxidation
591.48 2 32.1 CID 2 - 11 M.APQLKDPWAR.R Met truncation
446.695 3 37.4 ETD 2 - 12 M.APQLKDPWARR.E Met truncation
Top Down proteomics
Bottom Up proteomics
NB2M: Top Down & Bottom Up database search results

32. '6800.4980
Mr
'6824.4391
Mr
'6838.4356
Mr
'6898.4550
Mr
'6931.5310
Mr
+ Met
+MS, 15.7-16.7min, Deconvoluted (MaxEnt, 503.10-1600.10, *0.06375, 50000)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
6x10
Intens.
6800 6820 6840 6860 6880 6900 6920 6940 6960 m/z
∆m=131.0330 Da
Met=131.0405 Da
Error=0.0075 Da
+MS, 15.7-16.7min, Deconvoluted (MaxEnt, 503.10-1600.10, *0.06375, 50000)
C₃₁₅H₄₆₃N₉₁O₈₀, , 6800.4953
0.0
0.2
0.4
0.6
0.8
1.0
6x10
Intens.
6796 6798 6800 6802 6804 6806 6808 6810 6812 6814 m/z
+MS, 15.7-16.7min, Deconvoluted (MaxEnt, 503.10-1600.10, *0.06375, 50000)
C₃₂₀H₄₇₂N₉₂O₈₁S₁, , 6931.5358
0.0
0.5
1.0
1.5
5x10
Intens.
6930 6932 6934 6936 6938 6940 6942 6944 m/z
Mr 6800.4980 Da
Mass error: 0.0048Da
(0.69 ppm)
Mr 6931.5310 Da
Mass error: 0.0027Da
(0.40 ppm)
NB2M: Overlay of deconvoluted experimental and simulated spectra

33. • AA Sequence: M1-H60 and A2-H60
• Both Met truncated and unprocessed form can be present in Complex I
• No signal peptide cleavage predicted by MITOPROT
• Met truncation reported previously1,2
Summary for NB2M
1 Laser-induced liquid bead ion desorption-MS of protein complexes from blue-native gels. Sokolova L, Wittig I, Schägger H,
et al. Proteomics 2010, 10, 1401-1407
2 A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.
Angerer H, Zwicker K, Wumaier Z, et al. Biochemical Journal 2011, 437, 279-288

34. LC-MS: detection of small to large subunits in a single analysis
9 kDa subunit (deconvoluted)
75kDa subunit (deconvoluted)49kDa subunit (deconvoluted)
'9603.9448
Mr
'9617.9600
Mr
'9631.9697
Mr
'9644.9081
Mr
'9654.9367
Mr
'9669.9202
Mr
'9685.8928
Mr
+MS, 51.9-52.6min, Deconvoluted (MaxEnt, 503.09-2410.26, *0.10625, 50000)
0.0
0.5
1.0
1.5
5x10
Intens.
9550 9600 9650 9700 9750 m/z
49989.6584
+MS, 54.6-56.9min, Smoothed (0.07,3,SG), Deconvoluted (MaxEnt, 498.39-2528.81, *0.664063, 8000)
2
4
6
8
4x10
Intens.
49400 49600 49800 50000 50200 50400 50600 m/z
74340.9883
75196.3196
76237.1362
+MS, 37.9-41.1min, Deconvoluted (MaxEnt, 503.09-2472.80, *0.664063, 8000)
0
1
2
3
4
5
6
4x10
Intens.
73500 74000 74500 75000 75500 76000 76500 77000 77500 m/z
20kDa subunit (deconvoluted)
'20707.5208
Mr
'20725.4879
Mr
'20744.4732
Mr
'20755.4811
Mr '20763.4648
Mr
'20781.4432
Mr
+MS, 43.0-44.3min, Deconvoluted (MaxEnt, 503.09-2421.67, *0.10625, 50000)
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
20680 20700 20720 20740 20760 20780 20800 m/z

35. • Top Down and Bottom Up proteomics is a powerful combination to
characterize proteins
• Several subunits of CI in Y. lipolytica have different forms with respect to
current knowledge
• Resolution and sensitivity of the methodology enable analysis of dynamic
PTMs (e.g. phosphorylation, oxidation, etc)
• Current analysis on 42-subunit in 100 fmol of purified complex. Further
development of Top Down proteomics methodology ongoing to increase
sensitivity and throughput to analyze more complex samples
Summary

36. Top-Down Proteomics
Blue-native gel electrophoresis
Complexome Profiling3-5
• Composition of native protein complexes (complexome profiling)
• Stoichiometry of subunits (complexome profiling with spike-in of peptide standards)
• Exact molecular form of subunits (Top Down proteomics)
Outlook: In-depth characterization of protein complexes
3 LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes. Wessels HJ et al. Proteomics 2009
4 Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex. Heide H et al. Cell Metab. 2012
5 Analysis of 953 human proteins from a mitochondrial HEK293 fraction by complexome profiling. Wessels HJ et al. PLoS One. 2013

37. Acknowledgements
Radboud Centre for Proteomics, Glycomics & Metabolomics
Alain van Gool
Dirk Lefeber
Jolein Gloerich
Monique van Scherpenzeel
Hans Wessels
Maurice van Dael
Ming Liang Wu
Radboud University Nijmegen
Mike Jetten
Huub op den Camp
Nijmegen Centre for Mitochondrial Disorders
Ulrich Brandt
Jan Smeitink
Bruker Daltonics
Pierre-Olivier Schmit
Stuart Pengelley
Patrick van Houts
Goethe-Universität Frankfurt am Main
Volker Zickermann
Synaffix
Floris van Delft
E hans.wessels@radboudumc.nl