Biotechnology for the Pulp and Paper Industry

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Biomolecule analysis with electrospray mass
spectrometry
Dr. Harry Brumer Wood Biotechnology
Laboratory KTH Department of Biotechnology harry_at_b
iotech.kth.se
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KTH Department of Biotechnology
Biochemistry Prof Karl Hult Prof Pål Nyrén
ca 20 pers
Molecular Biotechnology Prof Mathias Uhlén
Prof Joakim Lundeberg Prof Per-Åke Nygren
Prof Stefan Ståhl
ca 80 pers
Wood Biotechnology Prof Tuula Teeri
ca 25 pers
Dept. of Biotechnology 200 personer
Bioprocess technology Prof Sven-Olof Enfors
Prof Lena Häggström
ca 30 pers
Structural Biochemistry Prof Torleif Härd
Adm
ca 15 pers
ca 5 pers
Environmental Microbiology Assoc. Prof Gunnel
Dalhammar
Theoretical Chemistry Prof Hans Ågren Prof Faris
Gelmukhanov
ca 10 pers
ca 15 pers
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http//www.biotech.kth.se/woodbiotechnology/
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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)

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MS Components

• Ionisation source
• ion optics, including one or more mass analysers
• detector

mass selection
analyte
ionisation
detection
EI, CI, FAB, MALDI, ESI
mag. sector, quadrupole, TOF, ion trap, FT-ICR
faraday cup, dynode, scintillation, MCP
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Hybrid quadrupole-TOF
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Hybrid quadrupole-TOF
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Hybrid quadrupole-TOF
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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 MH ions
• ESI
• suitable for on-line methods (CE, LC)
• gives multiply charged ions

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Electrospray ionisation (ESI)
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ESI Ion production
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Z-spray ESI source
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ESI yields multiply-charged ions

• Normal quadrupoles can be used for high-mass
  biomolecules

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Hybrid quadrupole-TOF
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Time-of-flight analyzers (TOF)

• K.E. 1/2 mv2

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Orthogonal acceleration TOF (oaTOF)

• used in ESI-TOF applications

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Hybrid quadrupole-TOF
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Quadrupole mass analyzer

• Q1 can be tuned to allow only ions of a single
  m/z to pass

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Hybrid quadrupole-TOF
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The collision cell
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Glu-fibrinogen peptide TOF MS

• 0.5 m/z separation of isotopic peaks M2H2

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

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ESI-TOF MS of bovine insulin, 5736 Da

• Isotopic peaks sepd. by 0.16 m/z -gt (M6H)6

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Horse heart myoglobin (HHM)ESI-TOF MS

• No charge state information available -
  insufficient resolution

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HHM - deconvoluted mass

• From system of equations Mn(H)/n
• MaxEnt 1

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Charge state distributions
(M21H)21
16952 Da ca. 9 to 21
(M9H)9
36749.4 Da ca. 20 to 50
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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

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LC-MS LC-MS/MS
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LC-MS/MS for glycopeptide discovery

• TOF MS survey total ion chromatogram

• MS/MS spectra acquired in real time

• Reconstructed chromatogram showing only sugar
  signature ions (m/z 163, 204, 366)

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Detailed MS/MS analysis

• Localise site of glycan attachment in the protein

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Comparison of MS/MS sequence information with
other XETs
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 ..
.

• 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.

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