1. Biomolecule analysis with electrospray mass spectrometry Dr. Harry Brumer Wood Biotechnology Laboratory KTH Department of Biotechnology harry@biotech.kth.se

2. Molecular Biotechnology Prof Mathias Uhlén Prof Joakim Lundeberg Prof Per-Åke Nygren Prof Stefan Ståhl Biochemistry Prof Karl Hult Prof Pål Nyrén Dept. of Biotechnology ~200 personer ca 80 pers ca 20 pers Wood Biotechnology Prof Tuula Teeri ca 25 pers Bioprocess technology Prof Sven-Olof Enfors Prof Lena Häggström ca 30 pers Environmental Microbiology Assoc. Prof Gunnel Dalhammar ca 10 pers Structural Biochemistry Prof Torleif Härd ca 15 pers Theoretical Chemistry Prof Hans Ågren Prof Faris Gelmukhanov ca 15 pers Adm ca 5 pers KTH Department of Biotechnology

3. http://www.biotech.kth.se/woodbiotechnology/

4. MS in the Wood Biotechnology Lab Equipment purchase funded by the Wallenberg Consortium North for Functional Genomics Micromass Q-TOF2 orthogonal acceleration quadrupole/time-of-flight MS Orthogonal (Z-spray) electrospray ionisation interface Nanoflow LC system (200 nL/min)

5. MS Components Ionisation source ion optics, including one or more mass analysers detector analyte ionisation mass selection detection EI, CI, FAB, MALDI, ESI mag. sector, quadrupole, TOF, ion trap, FT-ICR faraday cup, dynode, scintillation, MCP

6. Hybrid quadrupole-TOF

9. Electrospray ionisation (ESI) Biological molecules are often non-volatile and/or thermally labile: proteins/peptides nucleic acids (DNA, RNA) carbohydrates EI and CI not applicable w/o derivitisation FAB mass range limited OK for small carbohydrates, peptides MALDI good for biologicals not dynamic, typically gives [M+H] + ions ESI suitable for on-line methods (CE, LC) gives multiply charged ions

10. Electrospray ionisation (ESI)

11. ESI: Ion production

12. “Z-spray” ESI source

13. ESI yields multiply-charged ions Normal quadrupoles can be used for high- mass biomolecules

14. Hybrid quadrupole-TOF

15. Time-of-flight analyzers (TOF) K.E. = 1/2 mv 2

16. Orthogonal acceleration TOF (oaTOF) used in ESI- TOF applications

17. Hybrid quadrupole-TOF

18. Quadrupole mass analyzer Q1 can be tuned to allow only ions of a single m/z to pass

19. Hybrid quadrupole-TOF

20. The collision cell

21. Glu-fibrinogen peptide: TOF MS 0.5 m/z separation of isotopic peaks: [M+2H] 2+

22. Peptide sequencing by MS/MS Amino acids differ in their side chains Predominant fragmentation Weakest bonds

23. Glu-fib ESI-TOF MS/MS Raw data MaxEnt 3 deisotoped, deconvoluted data Glu-Gly-Val-Asn-Asp-Asn-Glu-Glu-Gly-Phe-Ph-Ser-Ala-Arg

24. ESI-TOF MS of bovine insulin, 5736 Da Isotopic peaks sep’d. by 0.16 m/z -> (M+6H) 6+

25. Horse heart myoglobin (HHM) ESI-TOF MS No charge state information available - insufficient resolution

26. HHM - deconvoluted mass From system of equations: [M+n(H + )]/n MaxEnt 1

27. Charge state distributions (M+21H) 21+ (M+9H) 9+ 36749.4 Da ca. 20+ to 50+ 16952 Da ca. 9+ to 21+

28. Monitoring deglycosylation of XET Intact protein mass analysis of a glycoprotein Glycosylation removed enzymatically Kinetics followed by ESI-MS Extent of deglycosylation can be correlated w/ changes in protein function

29. LC-MS & LC-MS/MS

30. LC-MS/MS for glycopeptide discovery Reconstructed chromatogram showing only sugar signature ions (m/z 163, 204, & 366) TOF MS “survey” total ion chromatogram MS/MS spectra acquired in real time

31. Detailed MS/MS analysis Localise site of glycan attachment in the protein

32. Comparison of MS/MS sequence information with other XETs Allows us to define the site of glycosylation for the entire protein family Henriksson, et al. (2003) Biochemical Journal, 375, 61-73. Oligosaccharide MS/MS analysis: Borriss, R., et al. (2003) Carbohydrate Research, 338, 1455-1467. ArabidopsisTCH4 SAGTVTTLYLKSPGTTWDEIDFEFLGNS SGEPYTLHTNVYTQGKGDKEQQFKLWFDPTANFH 140 XET1 SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEFH 150 Kiwi SAGTVTAFYLSSQNSEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKDYH 142 Tomato SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGNREQRIYLWFDPTKGYH 150 Tobacco SAGVVTAFYLSSNNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKGYH 149 Soybean SAGTVTAFYLSSQNAEHDEIDFEFLGNR TGQPYILQTNVFTGGKGDREQRIYLWFDPTKEYH 147 MS/MS seq. YLSSTNNEHDELDFEFLGDR TGQPVLLQTNVFTGGK ***.**::**.* **********. :*:** *:***:* ***::**:: ****** :*