LECTURE 20 MASS SPECTROMETRY SOFT IONIZATION CONCLUDING REMARKS

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LECTURE 20 MASS SPECTROMETRY SOFT IONIZATION
CONCLUDING REMARKS
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What Tripeptide? Ala // Leu // Val
Tripeptide C14H27N3O4
302.2
ESI
151.6
intensity
324.3
101.4
m/z
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ESI A POPULAR METHOD
M
POS
C36H44N4O MW 548
small doublet
P296
C28H48SO10 MW 576
NEG
MH 549 ? M 548 M2H/2 550/2 275
14 POSSIBLE FORMULAS
M- - H1O3
P296
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(mn)!
(12C)m(13C)n
(m-n)!(n)!
11.10.60.220.06
Note C100 ?(100)(1.1) size of m1 more
accurate analysis Apdx B (533)
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(mn)!
(12C)m(13C)n
(m-n)!(n)!
11.10.60.220.06
Note C100 ?(100)(1.1) size of m1 more
accurate analysis Apdx B (533)
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(mn)!
(12C)m(13C)n
(m-n)!(n)!
11.10.60.220.06
Note C100 ?(100)(1.1) size of m1 more
accurate analysis Apdx B (533)
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MH 1141 ? M 1140 M2H/2 1142/2
571
C60H92N12010 1140
See pg 313
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10 10(1696.4) 10 16954 20 20(848.4)-20
16949 ? all ionavg 16952.4
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FOUR WAYS TO ANALYZE IONS

• MAGNETIC FIELD
• ELECTRIC FIELD
• MS-MS
• ION TRAP (ORBI-TRAP)
• 3. TOF
• 4. FT-ICR

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P302
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What Tripeptide? Ala // Leu // Val
Tripeptide C14H27N3O4
302.2
ESI
151.6
intensity
324.3
101.4
m/z
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MS2 on 302.2
203.1
100.1
intensity
171.1
132.1
m/z
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No 118 90
MS2 on 302.2
No 72 114
203.1
100.1
intensity
171.1
132.1
m/z
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P 299
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P300
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EXPAND THE MOLECULAR ION PEAK
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• MATRIX
• MATRIX INTO ION SOURCE
• LASER
• ACCELERATE IONS
• DETECTION IONS
• WORK-UP DATA

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EXPAND THE MOLECULAR ION PEAK
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Electrospray Ionization Fourier Transform Ion
Cyclotron Resonance Mass SpectrometryLTQ
ORBITRAP
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LTQ ORBITRAP
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LTQ ORBITRAP
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Electrospray Ionization Fourier Transform Ion
Cyclotron Resonance Mass Spectrometry

• Electrospray Ionization
• Multiple charging allows study of large intact
  biomolecules
• Soft ionization allows study of intact
  noncovalent complexes
• FTICR Mass Spectrometry
• High and ultra-high resolution mass measurements
  (gt109)
• essential for characterizing complex mixtures
• High precision mass measurements (low or sub ppm)
• Measurements over extended m/z range
• Very high sensitivity

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Bruker 9.4 Tesla ESI-FTMS
9.4 T shielded magnet
analyzer stage
transfer stage
source chamber
ESI source
10-10 Torr
Laser window
ion guide
cell
ESI needle
capillary
turbo-drag pump
turbo pump
turbo pump
turbo pump
IR Laser
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9.4T ESI-FTICR Mass Spectrometer
Plate stacker
Plate conveyer
Barcode reader
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ESI Preserves Specificity of Noncovalent
Complexes Formed in Solution
Consider four oligonucleotides which form 2
complementary duplexes
A 5-GCACGTACCGCC-3 B 3-CGTGCATGGCGG-5 X
5-GCCCAAGCTGGCA-3 Y 3-CGGGTTCGACCGT-5
By complementarity A B AB X Y XY B
Y B Y B X Y B XY etc...
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ESI-FTMS of Specific DNA Duplex
(B-3H)3-
(XY-5H)5-
B 5-GGCGGTACGTGC-3 X 5-GCCCAAGCTGGCA-3 Y
3-CGGGTTCGACCGT-5
Na
(Y-3H)3-
(X-3H)3-
Na
1300
1400
1500
1600
1200
m/z
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High Precision ESI-FTICR Mass Measurement
use of unbound RNA as internal mass standard
provides low ppm mass measurement errors
(RNA-5H)5-
theoretical isotope distributions
Relative Abundance
D (m/z) 123.059 D MW 615.297 ( 0.006)
measured isotope distributions
M/DM 100,000 (FWHM)
(RNAParomomycin-5H)5-
1740
1760
1780
1800
1820
1840
m/z
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SUMMARY

• Analysis of peaks in ESI require more thought!
• E.S.I. look for MH, MNa, or M-H-, or
  M2H. F.A.B. look for MH, MNa.
• Expand m/z cluster to look at a 1 amu scan use
  head count of peaks to ID charge (two
  pks 2) (three peaks 3)
• Four ways to analyze ions (ALWAYS EXPAND M.I.
  CLUSTER)
• ESI detection best done by quad, tof or icr
• MS/MS is a useful analytical technique
• Problem solving should combine MS NMR data

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Homework Assignment
Chapter 7 1 - 6 8 10 15