HUPO 2017 Dublin - Bruker Lunch Seminar presentation: "Clinical proteomics: beyond peptide profiling".

1. Clinical proteomics: beyond peptide
profiling
Bruker Daltonics HUPO2017 seminar
Hans JCT Wessels, PhD
Translational Metabolic Laboratory
Radboudumc, Nijmegen
Tel. +31-(0)24-3616933
Email: hans.wessels@radboudumc.nl
'917.5396
10+
'1019.3769
9+
'1146.6736
8+
'1310.3404
7+
'1528.5633
6+
'1834.0761
5+
9165 9170 9175 Da

2. The Proteome
Intact protein profiling by LC-Q TOF MSn 2
Proteins are highly versatile macromolecules that enact a wide range of biological
functions such as catalysis, regulation, communication, mechanical support,
movement, and transport. At least several million unique proteins (proteoforms)
exist in the human body which are, surprisingly, encoded by only fifteen thousand
genes.
Images taken from “The Inner Life of the Cell” produced by XVIVO for Harvard University

3. Proteoforms
3Intact protein profiling by LC-Q TOF MSn
Proteoform:
Designates all of the different molecular forms in which the protein product of a single
gene can be found, including changes due to genetic variations, alternatively spliced RNA
transcripts and post-translational modifications
Proteoform: a single term describing protein complexity.
Smith LM, Kelleher NL; Consortium for Top Down Proteomics. Nat Methods. 2013 Mar;10(3):186-7. doi: 10.1038/nmeth.236

4. Protein glycosylation
• More than 90% of all proteins are glycosylated in plasma
• Glycosylation modulates protein biology
• Abberant glycosylation is observed in many common human diseases
Complex Hybrid High Mannose
X 2
N-Glycan classes
Glucose Mannose Galactose HexNAc Sialic acid Fucose

5. Protein glycosylation
Microheterogeneity (glycan diversity at a single glycosylation site)
Macroheterogeneity (unique combinations of glycans at multiple sites)
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
N432 N630
Glucose Mannose Galactose HexNAc Sialic acid Fucose

6. Glycosylation creates tremendous protein diversity
Gene
Protein
N-Glycosylation

7. Analytical glycoprotein challenge
Glycoprotein biomarkers

8. <Titel van de presentatie> 8
Schematic view on protein glycosylation
Glycosylation in cellular mechanisms of health and disease.
Ohtsubo K, Marth JD. Cell. 2006 Sep 8;126(5):855-67

9. 9
Result sample 2
Tryptic peptides sample 2
Result sample 1
Tryptic peptides sample 1
Proteins sample 2Proteins sample 1
<Titel van de presentatie>
Bottom-up (shotgun) proteomics
90%
10%
20%
80%
No difference
100% 100%
100% 100% 100%100% 100% 100%
Protein inferenceProtein inference

10. <Titel van de presentatie> 10
Top-Down proteomics
Result sample 2Result sample 1
90%
10%
? ?
? ?
? ?
? ?
20%
80%
Differential proteoforms identified but structural
differences (micro- & macroheterogeneity) remain unclear
Proteins sample 2Proteins sample 1
90%
10%
20%
80%

11. <Titel van de presentatie> 11
Glycopeptide profiling
Tryptic glycopeptidesTryptic glycopeptides
Differential glycopeptides identified but connectivity between
sites (and other PTMs/sequence variants) is lost
Result sample 1Result sample 1
90% 90%
10% 10%
20%20%
80%80%
Proteins sample 2Proteins sample 1
90%
10%
20%
80%

12. <Titel van de presentatie> 12
Complementary methods to get the full picture
Top-Down proteomics
90%
10%
? ?
? ?
Glycopeptide profiling
90%
10%
Combined result
90%
10%
90%
10%

13. <Titel van de presentatie> 13
Protein N-glycan synthesis pathway (gene defects in red)

14. Need for structural information in CDG-II diagnostics
4
3
2
1
0
Control
CDG-IIpatient1
CDG-IIpatient2
#sialicacids
?
?
Transferrin
Iso Electric Focussing

15. <Titel van de presentatie>
Transferrin Q-ToF MS (ISO-certified diagnostics)
Asn Asn
- - - -
Charge deconvolution
ESI-MS
Transferrin
capture
High-resolution mass spectrometry glycoprofiling of intact transferrin for diagnosis and subtype identification in the congenital disorders of glycosylation.
van Scherpenzeel M, et al. Transl Res. 2015 Dec;166(6):639-649.e1. doi: 10.1016/j.trsl.2015.07.005. Epub 2015 Aug 8.

16. Full plasma glycopeptide profiling

17. <Titel van de presentatie> 17
Transferrin: B4GALT1-CDG
Transferrin Q-ToF MS Glycopeptide profiling
Transferrin N432
Transferrin N630
B4GALT1 ST
Asn Asn Asn
High-resolution mass spectrometry glycoprofiling of intact transferrin for diagnosis and subtype identification in the congenital disorders of glycosylation.
van Scherpenzeel M, et al. Transl Res. 2015 Dec;166(6):639-649.e1. doi: 10.1016/j.trsl.2015.07.005. Epub 2015 Aug 8.

18. <Titel van de presentatie> 18
Transferrin: MAN1B1-CDG site-specific defect
Transferrin Q-ToF MS Glycopeptide profiling
Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency.
Van Scherpenzeel M, et al. Brain. 2014 Apr;137(Pt 4):1030-8. doi: 10.1093/brain/awu019. Epub 2014 Feb 24

19. <Titel van de presentatie> 19
Transferrin: NANS-CDG protein-specific defect
Glycopeptide profiling
Transferrin N630
Transferrin N432
NANS-CDG
Transferrin Q-ToF MS
NANS-mediated synthesis of sialic acid is required for brain and skeletal development.
van Karnebeek CD, et al. Nat Genet. 2016 Jul;48(7):777-84. doi: 10.1038/ng.3578. Epub 2016 May 23

20. Advantage of holistic approach over targeted methods
PLS-DA PCA score plot
Protein X – N…

21. <Titel van de presentatie> 21
timsTOF Pro to unlock the full potential of glycopeptide profiling

22. <Titel van de presentatie> 22
Towards Top-Down plasma proteomics in glyco- and biomarker sciences
132
127125
11031
30 108
113 12170 10674 10573 85825414 18 6621 52 80 10063 8439 8917 49 9220 9825 91 123222427 34 9426 9646 789 103574743414 10211 44
134
60
131
68
130
6
120
11661 763716
111 126115
51 1332
11811232
12855 10977 8713 29 11711459 69 7265 12215 11986 10481 996248 755323 958328 58 12988 1076719 9740 90643335 101 12479361 71 934512 563 38107 42 5085
0
1
2
3
6x10
Intens.
5 10 15 20 25 30 35 Time [min]
Sample: Plasma
Mass spectrometer: Impact II
Gradient length: 35 min
Compounds detected: 2700
> 30 proteoforms in this peak
(7 - 46 kDa)
16Kda 35Kda
120
126125 127
118
128
117
122119
121
124
123
0.00
0.25
0.50
0.75
1.00
1.25
6x10
Intens.
31.5 32.0 32.5 33.0 33.5 Time [min]
560.3346
755.0565
1273.1534
1455.0323
1659.8122
1926.0672
2253.8843
2506.4807
2666.3939
+MS, 33.37-33.47min #1986-1992
0.0
0.5
1.0
1.5
2.0
2.5
4x10
Intens.
500 750 1000 1250 1500 1750 2000 2250 2500 m/z
'16580.0519
Mr
'16596.0448
Mr
+MS, 33.37-33.47min, Deconvoluted (MaxEnt, 316.88-3004.94, *0.0697917, 60000)
0.0
0.5
1.0
1.5
5x10
Intens.
16580 16585 16590 16595 16600 16605 16610 m/z
'34629.9854
Mr
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
34640 34650 34660 34670
Towards a routine application of Top-Down approaches for label-free discovery workflows.
Schmit PO, et al. J Proteomics. 2017 Aug 27. pii: S1874-3919(17)30264-6. doi: 10.1016/j.jprot.2017.08.00
16 kDa 35 kDa

23. Analysis of proteoforms in membrane protein complexes by Top-Down
proteomics
23
Acknowledgements
Translational Metabolic Laboratory
Alain van Gool
Anouk Suppers
Dirk Lefeber
Esther Willems
Hans Wessels
Jolein Gloerich
Koen Rademaker
Maurice van Dael
Monique van Scherpenzeel
Nurulamin Bin Abu Bakar
Ron Wevers
Center for Molecular and Biomolecular Informatics
Wynand Alkema
Bruker Daltonics
Gary Kruppa
Kristina Marx
Markus Lubeck
Patrick van Houts
Pierre-Olivier Schmit
Scarlet Beck
Stuart Pengelley