me 330.804: mass spectrometry in an “omics” world...• records an entire mass spectrum • best...
TRANSCRIPT
10/21/2012
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ME 330.804: Mass Spectrometry in an “Omics” World
Lecture 2WED 24 OCT, 2012R CotterMass Analysis and MS/MS
MAMS bioaerosolmass spectrometer
1ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
MALDI is generally used on a time-of-flight MS
1948 Cameron and Eggers: velocitron
1953 Wolff and Stephens: constant momentum TOF
1955 Katzenstein & Friedland:drawout pulse
1955 Wiley and McLaren:time-lag focusingcommercialized by Bendix
Early milestones
Wiley, W.C.; McLaren, I.H., Time-of-Flight Spectrometer with Improved Resolution, Rev. Sci. Instr. 26 (1955) 1150-1157
2ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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160
180
200
220
240
0S 10uS 20uS 30uS
Substance P, 1348 Da
Methionine enkephalin-Arg-Gly Leu 900 Da
Peptide sequencing standard, 1639 Da
Parathyroid hormone 28-48, 2148 Da
Beta-melanocyte, 2660 Da
Hepatitus B virus pre-S region 120-145, 3008 Da
Diabetes-associated peptide 8-37, 3200 Da
ACTH 7-38, 3659 Da
ACTH 1-39, 4541 Da
Pancreatic polypeptide, 4182 Da
Biocytin beta-endorphin, 3819 Da
160
180
200
220
240
0S 10uS 20uS 30uS
Substance P, 1348 Da
Methionine enkephalin-Arg-Gly Leu 900 Da
Peptide sequencing standard, 1639 Da
Parathyroid hormone 28-48, 2148 Da
Beta-melanocyte, 2660 Da
Hepatitus B virus pre-S region 120-145, 3008 Da
Diabetes-associated peptide 8-37, 3200 Da
ACTH 7-38, 3659 Da
ACTH 1-39, 4541 Da
Pancreatic polypeptide, 4182 Da
Biocytin beta-endorphin, 3819 Da
DeV
mt
2/1
2
A simple “linear” time-of-flight mass spectrometer
A time-of-flight mass spectrum of a mixture of peptides
The time-of-flight mass spectrometer
3ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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02/1
0
2/12/1
02/1
0
2/1
2
22t
eEsU
DmUeEsU
eE
mt
time in ion source time in flight tube
initial kineticenergy distribution
distribution of initial position inthe source
turn-around time distribution in
time of ion formation
The real time-of-flight equation
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How is the effect of an initial kinetic energy distribution improved?
(a) low laser power
(b) high accelerating voltage
(c) reflectron
Ions formed with different initial kinetic energies
20 2
1mvUeV
The ion will leave the source with a larger kinetic energy
a higher velocity, and a shorter flight time, causing lower mass resolution.
5ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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How is the effect of an initial spatial distribution improved?
(a) thin sample
(b) detector at D = 2s
(c) dual stage extraction to move the space-focus plane
Ions are formed in different locations
Ions leave the source with energies eEs’ dependent upon their location
where s’ = s-∆s
]'2['2
2/1
DseEs
mt
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Wiley, W.C.; McLaren, I.H., Rev. Sci. Instrument. 26 (1955) 1150-1157.
Ions differing in initial position in the source, initial kinetic energies and different directions of their initial velocities are focused in the Wiley-McLaren TOF mass spectrometer.
Time-lag focusing
E0
s0 s1
E1
Drawout pulsePushout pulse
1
2
3
4
7ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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Whittal, R.M.; Li, L., Anal. Chem. 67 (1995) 1950-1954Brown, R.S.; Lennon, J.J., Anal Chem. 67 (1995) 1998-2003.Vestal, M.L.; Juhasz, P.; Martin, S.A, Rapid Commun. Mass Spectrom. 9 (1995) 1044-1050.
U0
U1
U2
U0
U1
U2
20 kV (constant)
18 kV (constant)
Ground (constant) Ground (constant)
2 kV pulse20 kV
18 kV
20 kVNegative pulse
0 V
delay delay
timetime
ABI, Kratos and others Bruker and others
Delayed extraction and MALDI
Time-lag focusing and “delayed extraction”
8ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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t = m
2eV [ L + L + 4d]
1/ 2
1 2
Reflectrons correct for the effects of kinetic energy spread after the ions have left the source
Single-stage reflectron
Dual-stage reflectron
Mamyrin, B. A.; Karataev, V. I.; Shmikk, D. V.; Zagulin, V. A, Mass reflectron. New nonmagnetic time-of-flight high-resolution mass spectrometer. ZhurnalEksperimental'noi i TeoreticheskoiFiziki (1973) 64, 82-89.
9ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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0
10
20
30
40
50
60
70
80
90
100
%Int.
5731 5732 5733 5734 5735 5736 5737 5738 5739 5740 5741
Mass/Charge
1[c]
573
5.6
4
573
4.6
3
573
6.6
3
57
33.6
5
573
7.6
4
573
2.5
6
573
8.6
3
57
31.4
3
57
39.7
2
Insulin B-chain using delayed extraction/time-lag focusing on a linear instrument (no reflectron)
Bovine insulin using delayed extraction/time-lag focusing on a reflectron instrument
Delayed extraction on linear and reflectron TOFs
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What does it do?
• improves mass resolution: ions are extracted at right angles to the initial velocity (kinetic energy) distribution
• improves duty cycle: ions are stored between pulsed extraction cycles
Dawson, J.H.J.; Guilhaus M., Rapid Commun. Mass Spectrom. 3 (1989) 155.
Dodenov, A.F.; Chernushevich, I.V/; Laiko, V.V., in Time-of-Flight Mass Spectrometry, in Cotter, R.J., Ed,; ACS Symposium Series 549, Washington DC (1994) pp. 108-123.
Orthogonal acceleration mass spectrometers
To drift region
Pushout pulse
EI source Focusing lenses
x
y
z
11ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
oa-TOF with an ion guide and reflectron
Commercial orthogonal acceleration TOFs can have mass resolutions up to 60,000
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What’s out there? JEOL, USA
AccuTOF™ DART® Direct Analysis in Real Time Time-of-Flight Mass Spectrometer
AccuTOF™ LC Liquid Chromatograph Time-of-Flight Mass Spectrometer
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Bahr, U.; Karas, M., Rapid Commun. Mass Spectrom. 13 (1999) 1052-1058.
Orthogonal acceleration and mass accuracy
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http://www.chem.vt.edu/chem‐ed/ms/quadrupo.html
Quadrupole mass spectrometers
http://huygensgcms.gsfc.nasa.gov/MS_Analyzer_1.htm
15ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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http://www.waters.com/WatersDivision/ContentD.asp?watersit=EGOO-66MNYR&WT.svl=1#quads
dc voltage: Urf voltage: V0cos(t)
If U/V0 = 0.167, ions of m/z are transmitted (have stable trajectories) when:
m/z = 0.136V0/r02f2
where f is 1MHz
rf-only mode transmits all ions and is used for:• collision chamber• quadrupole injection into a trap or oaTOF (ion guide)
How it works: the stability diagram
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Mass filter
• passes a single mass at any time; throws all other masses away
• best example is a quadrupole
• can be used as a mass spectrometer by scanning
• high duty cycle for monitoring single ions; low duty cycle for acquiring a mass spectrum
Mass spectrometer
• records an entire mass spectrum
• best duty cycle is a mass analyzer with the multiplex recording advantage, i.e records all ions of all masses
• best examples are the TOF, ion trap and FTMS
The quadrupole is a mass filter. How does that differ from a mass spectrometer?
17ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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S D
DC and RF voltages set to pass a single mass
RF-only mode to pass all masses; used as a collision chamber
DC and RF voltages scanned to record all masses in succession
Because both mass analyzers are mass filters, the duty cycle for scanned spectra is low
The quadrupole is a mass filter. To record a mass spectrum it must be scanned
The triple quadrupole: a tandem MS
18ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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MS1 Activation Region
MS2S D
vacuum system
Tandem mass spectrometers have…
a mass analyzer (MS1) that separates ions by mass and selects ions of a single mass …. mass filter
an activation region for fragmenting mass-selected ions … generally by collision-induced dissociation
a mass analyzer (MS2) for recording the mass spectrum of the fragments of the selected mass …. mass spectrometer
Tandem mass spectrometers
19ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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S D
Triple quadrupole mass spectrometers
dc
volt
age
dc
volt
age
dc
volt
age
time time time
Normal (product ion) scan
Multiple reaction monitoring (MRM)
dc
volt
age
dc
volt
age
dc
volt
age
time time time
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Q1 is an RF-only quadrupole filter that collimates ions from high pressure source.
Q3 is an RF-only quadrupole filter used as a collision chamber.
Collisions are low energy.
Q2 is a quadrupole mass filter that selects the precursor mass
The TOF makes it possible to analyze product ions with higher m/z than their precursors
Electrospray ionization on a QTOF
21ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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What are the advantages?
• MS1 is a mass filter for selection of a single precursor from a continuous beam; MS2 is a multiplex recorder. Optimal configuration for product ion MS/MS spectra.
• better mass accuracy from the TOF than a third quadrupole on a triple quadrupole mass spectrometer
What are the disadvantages?
• storage/extraction has limited duty cycle
• low energy collisions in q3; not as effective for some post-translational modifications
Why use a quadrupole/TOF?
22ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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ABI 5600 TripleTOF
Resolution, MS High MassUp to 40,000 (FWHM) at m/z 956 using a 10 ms accumulation time.Resolution MS/MS Low MassUp to 25,000 (FWHM) at ~100 m/z using a 10 ms accumulation time.Resolution MS/MS, High mass Up to 30,000 (FWHM) on a fragment ion from [Glu]-Fibrinopeptide B above precursor m/z using a 10 ms accumulation time.
What’s out there?
23ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
What’s out there?
Waters Synapt G2-S
“Step-wise” ion funnelETDMSE
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Collision energy is derived from the relative energy between the ion and the target gas (in the center-of-mass frame) and is related to the kinetic energy in the laboratory frame
Low energy CID uses multiple collisions, raises the internal energy slowly until the weakest bond breaks.
Low energy CID is not particularly favorable for determining post-translational modifications, especially phosphorylation or glycosylation.
High energy CID is usually carried out on a tandem time-of-flight (TOF/TOF) mass spectrometer
High vs. low energy CID
LABMn
nrel E
mm
mE
ELAB is 10-60 eV
ELAB is 1-20 keV
25ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
ion source
mass selection gate
retarding lens
collision cell
2nd source pulsed extraction
ion source
mass selection gate
collision cell “lift” cell
ion source mass selection gate
collision cell
a
b
c
20 keV1 keV
19-20 keV
8 keV
15-23 keV
20 keV
0-20 keV
0-8 keV
ion source
mass selection gate
retarding lens
collision cell
2nd source pulsed extraction
ion source
mass selection gate
collision cell “lift” cell
ion source mass selection gate
collision cell
ion source mass selection gate
collision cell
a
b
c
20 keV1 keV
19-20 keV
8 keV
15-23 keV
20 keV
0-20 keV
0-8 keV
Applied Biosystems
Bruker Daltonics
Shimadzu AXIMA TOF2
Tandem time-of-flights available commercially
Curved-field reflectron
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ultrafleXtreme™
1 Hz-1 kHz rep rate laserR= 40,000
What’s out there?
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Bruker DaltonicsABI Sciex
5800 TOF/TOF
1 kHz rep rate laserMS/MS Resolution (Precursor Glu1-Fib)175.1195 RES > 2800684.3469 RES > 45001056.4750 RES > 55001441.6348 RES > 6500
27ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
What’s out there?
Shimadzu (Kratos)AXIMA TOF2
Collision energy: 20 keVR (linear mode): 5,000R (reflectron mode): 20,00
28ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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Unimolecular, low and high energy collisions
Courtesy of G
uenter Allm
aier, Martina
Marchetti-D
eschmann and E
rnst Pittenauer
29ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
High energy CID increases side chain fragmentation
Courtesy of G
uenter Allm
aier, Martina
Marchetti-D
eschmann and E
rnst Pittenauer
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ring electrode
endcap electrode
endcap electrode
supplemental RF voltage
fundamental RF voltage (1.1 MHz)
signal out
electron filament –70V
gas or volatile sample in
helium bath gas in
vacuum chamber
Scan amplitude to obtain all mass spectra
• resonance ejection mode mass scan
• high amplitude RF for ion ejection, isolation or selection
• low amplitude RFfor ion activation
Quadrupole ion trap: “tandem in time”
31ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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20
20
20 )2(
8
zrm
eVqz
20
20
20 )2(
16
zrm
eUaz
zqzr
V
z
m20
20
20 )2(
8
Mathieu parameters:
Mass selective instability mode:if dc voltage on the endcaps is zero, then scan along the az line (by varying the rf voltage); ion ejection occurs at the stability boundary when az = 0.908
The mass ejected is then given by:
Where z is the number of charges and 0 is the angular drive frequency(0/2 = 1.1 MHz)
Williams, J.D.; Cox, K.A.; Schwartz, J.C.; Cooks, R.G., in Practical Aspects of Ion Trap Mass Spectrometry, Volume II, Cairns, T., Ed., CRC Press, Boca Raton (1995), pp. 3-50
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ring electrode
endcap electrode
endcap electrode
fundamental RF voltage (1.1 MHz)
signal out
The “mass-selective instability” mode
mass scan
ion trapping
ion cooling
ionization
33ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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Mass
Inte
nsit
y
220
4
rq
eVm
z
z0
r0
)(
)(
RFVq
DCUa
z
z
matrixscience.org
How do ion traps work?
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Resonance ejection mode:
A supplementary rf voltage is applied to the endcaps
The fundamental rf voltage on the ring electrode is scanned
Ions are ejected “through a hole in the stability region”
Extension of mass range through axial modulation
Supplementary rf = 69.9 kHzqeject = 0.182m/z = (0.91/0.182) x 650 = 5 x 650 = 3,250
Supplementary rf = 35.2 kHzqeject = 0.091m/z = (0.91/0.091) x 650 = 10 x 650 = 6,500
35ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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ring electrode
endcap electrode
endcap electrode
supplemental RF voltage
The “resonance ejection” mode
mass scan
ionizationfundamental RF voltage (1.1 MHz)
signal out
supplemental RF voltage
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I-III. trapping cycle: fundamental (1.1 MHz) RF voltage on ring electrode
IV. mass isolation cycle (MS1): resonant ejection of all but selected ion, using high amplitude supplementary RF on ring electrode
V. excitation cycle (low energy CID): low amplitude supplementary RF voltage on endcaps
VI. mass analysis cycle (MS2):resonance ejection mode, high amplitude supplementary RF voltage on endcaps while scanning the amplitude of the fundamental RF voltage on the ring electrode
The trap as a tandem instrument
37ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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Isolation and excitation can be carried out by scanning the resonant frequency or by a stored-waveform inverse Fourier transform (SWIFT) pulse.
Doroshenko, V.M.; Cotter, R.J., Rapid Commun. Mass Spectrom, 10 (1996) 65-73.
Mass isolation
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Capillary needle (3KV)
N2
Differentially pumped regions
Increasing vacuum
Capillary interface
Quadrupole, hexapole or octopole ion guides
Finnigan (Thermo) LCQ DUO/DECA; Bruker Esquire 4000/6000; Agilent LC/MSD 3D ion traps
Electrospray ionization and quadrupole ion trap
ESI is an atmospheric method, so that ions have to be transmitted into the vacuum and the trap
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Quadrupoles or hexapoles
Pulsed UV or IR laser
XY sample stage (3KV)
Extended capillary
Moyer, S. C. and Cotter, R.J., Atmospheric Pressure MALDI, Anal. Chem. 74 (2002) 468A–476A.
Mass Technologies AP MALDI source
Atmospheric pressure MALDI (APMALDI)
The capillary interface and RF-only multipole ion guides enable the use of any atmospheric pressure source
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800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z0
100
Re
lativ
e A
bund
ance
1420.4
1274.1
1079.5
2117.3
1296.6787.1
2101.1
[M+Na]+
[M+Na]+
[M+Na]+
MAN6
A1F
M3N2F
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z0
100
Re
lativ
e A
bund
ance
1420.4
1274.1
1079.5
2117.3
1296.6787.1
2101.1
800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 m/z0
100
Re
lativ
e A
bund
ance
1420.4
1274.1
1079.5
2117.3
1296.6787.1
2101.1
[M+Na]+
[M+Na]+
[M+Na]+
MAN6
A1F
M3N2F
650 700 750 800 850 900 950 1000 1050 m/z0
100
Rel
ativ
e A
bund
ance
1061.4
933.3712.2
1062.3
978.4
979.5
714.4 917.4 1063.5980.4
916.2 969.2 1049.7730.0 1077.5686.7 1009.6899.9832.1 959.3629.1 772.0 801.8
MS/MS 1079Da
M3N2F -Fuc
-Fuc
-GlcNAc
-GlcNAc
650 700 750 800 850 900 950 1000 1050 m/z0
100
Rel
ativ
e A
bund
ance
1061.4
933.3712.2
1062.3
978.4
979.5
714.4 917.4 1063.5980.4
916.2 969.2 1049.7730.0 1077.5686.7 1009.6899.9832.1 959.3629.1 772.0 801.8
650 700 750 800 850 900 950 1000 1050 m/z0
100
Rel
ativ
e A
bund
ance
1061.4
933.3712.2
1062.3
978.4
979.5
714.4 917.4 1063.5980.4
916.2 969.2 1049.7730.0 1077.5686.7 1009.6899.9832.1 959.3629.1 772.0 801.8
MS/MS 1079Da
M3N2F -Fuc
-Fuc
-GlcNAc
-GlcNAc
MS/MS 1079Da
M3N2F -Fuc
-Fuc
-GlcNAc
-GlcNAc
650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 m/z0
100
Re
lativ
e A
bun
danc
e
1198.5
1401.4
1318.41216.5
995.2 1036.51257.4
1095.5875.3 1115.3 1321.3723.1 833.2772.2 1157.3
MS/MS 1420Da
MAN6-Man
-Man-Man
-GlcNAc-GlcNAc
-GlcNAc-Man
650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 m/z0
100
Re
lativ
e A
bun
danc
e
1198.5
1401.4
1318.41216.5
995.2 1036.51257.4
1095.5875.3 1115.3 1321.3723.1 833.2772.2 1157.3
650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 1400 m/z0
100
Re
lativ
e A
bun
danc
e
1198.5
1401.4
1318.41216.5
995.2 1036.51257.4
1095.5875.3 1115.3 1321.3723.1 833.2772.2 1157.3
MS/MS 1420Da
MAN6-Man
-Man-Man
-GlcNAc-GlcNAc
-GlcNAc-Man
AP/IRIS MS spectrum of 3-oligosaccharide mixture (8 pmol of each oligosaccharide)
Taranenko N.I., Atmospheric Pressure Infrared Ionization from Solutions (AP/IRIS), Proceedings of the 51st ASMS Conference on Mass Spectrometry and Allied Topics, Montreal, 2003.
Atmospheric pressure MALDI on an ion trap: examples of MS, MS/MS and MSn
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B. A. Collings, W. R. Stott and F. A. Londry, Resonant excitation in a low-pressure linear ion trap, J. Am. Soc. Mass Spectrom. 14 (2003) 622-634
Linear ion traps: the LTQ and the Q-trap
The Q-trap (MDS Sciex or Agilent) can be used as the third quadrupole in a triple quad (for MRM experiments) or as an ion trap for full scanned MSn spectra.
Axial ejection of ions
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Figure 1. Schematic portrayal of the experimental apparatus based on the ion path of a triple quadrupole mass spectrometer. Q3 can be operated as either an RF/DC quadrupole or a linear ion trap with mass selective axial ejection.
Figure 2. Generic scan function used for filling and scanning the Q3 linear ion trap mass spectrometer.
James W. Hager *, J. C. Yves Le Blanc, Product ion scanning using a Q-q-Qlinear ion trap (Q TRAPTM) mass spectrometer, Rapid Commun. Mass Spectrom. 17 (2003) 1056-1064
The Q-trap
43ME 330.804: MS2012Mass Spectrometry in an “Omics” World http://www.hopkinsmedicine.org/mams/
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Linear ion traps: the Thermo LTQ
A major advantage of linear (2D) ion traps is that they can hold more ions than quadrupole (3D) ion traps.
This improves dynamic range, and reduces charge repulsion and shielding effects on mass resolution
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Linear ion traps: the Thermo LTQ
The LTQ loads ions axially,
but ejects or scans them out radially.
This becomes important for the addition of electron transfer dissociation (ETD), an orbitrap or an FTMS, all of which utilize axial movement of ions
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60 m3/hr 300 L/s 400 L/sec 210 L/sec 210 L/sec
Actively Shielded7 Tesla Magnet
Cold ECD< 2 eV
IRMPD Laser
15 L/s
ESI source
Transfer capillary and skimmer
Octapole ion guide
Quadrupole lenses
Octapole ion transfer lens
Linear ion trap
The Fourier-transform mass spectrometer
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In an ion cyclotron resonance (ICR) mass spectrometer, ions are confined in crossed electric and magnetic fields, where their resonant frequency depends upon the mass: ω = qB/m
Fourier transform mass spectrometry
210 L/sec 210 L/sec
Actively Shielded7 Tesla Magnet
Ions → Figure 1: Illustration of the Lorentz force (F=qvxB) as it acts on a positive ion (left) and a negative ion (right), in the presence of a constant magnetic field [ B].
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In the FTMS ions confined in an ICR cell are excited by a pulse representing a range of frequencies
8 MHz 100 kHz
RF frequency sweep
1. RF signal is applied to a set of excitation plates
2. Amplitude of ion oscillation increasesMagnetic field B
time
ω
Fourier transform mass spectrometry
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Only those frequencies that are absorbed resonantly will be returned as image currents
3. Image currents are detected on a pair of receiver plates
4. The transient decreases as the ions return to the center
5. The time-domain signal is Fourier transformed to a frequency domain mass spectrum
time
frequency (mass)
FFT
Fourier transform mass spectrometry
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The FTMS provides very high mass resolution
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Mass selection: ions are ejected from the FTMS using high amplitude RF signal; mass selection is accomplished with a “notched” frequency sweep
8 MHz 100 kHz
RF frequency sweep
time
Excitation: low energy CID can be accomplished using low-level RF excitation
SORI: sustained off-resonance irradiation
MS/MS and MSn on the FTMS
Other excitation methods can also be used: infrared multi-photon dissociation (IRMPD) and electron capture dissociation (ECD)
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Cold ECD (< 2 eV)electron source
IRMPD Laser
IRMPD and ECD can be used on an FTMS
210 L/sec 210 L/sec
Actively Shielded7 Tesla Magnet
Ions →
IR laser radiation and/or thermal electrons are usually introduced opposite the ion introduction:
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Glycosylation sites:
Top Panel, Conventional CAD MS/MS results in no information about the location of O-GlcNAc.
Bottom Panel, ECD FTMS readily allows for site mapping O-GlcNAc.
ECD and post-translational modifications
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• ECD produces mainly c and z-series ion
• ECD requires multiply-charged ions
How does ECD work?
1. Molecular ions are formed and trapped in the ICR cell2. One multiply-charged +ve ion species is mass selected3. Thermal electrons are focused into the ICR cell
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60 m3/hr 300 L/s 400 L/sec 210 L/sec 210 L/sec
Cold ECD< 2 eV
IRMPD Laser
15 L/s
ESI source
Transfer capillary and skimmer
Octapole ion guide
Quadrupole lenses
Octapole ion transfer lens
Linear ion trap
The LTQ FTMS: low energy CID, ECD and IRMPD
Actively Shielded7 Tesla Magnet
ThermoFinnigan LTQ FTMS
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The “orbitrap” mass analyzer
Figure 1. Cutaway view of the Orbitrap mass analyzer. Ions are injected into the Orbitrap at the point indicated by the red arrow. The ions are injected with a velocity perpendicular to the long axis of the Orbitrap (the z-axis). Injection at a point displaced from z = 0 gives the ions potential energy in the z-direction. Ion injection at this point on the z-potential is analogous to pulling back a pendulum bob and then releasing it to oscillate.
Hu Qizhi; Noll Robert J; Li Hongyan; Makarov Alexander; Hardman Mark; Graham Cooks R, The Orbitrap: a new mass spectrometer, J. Mass Spectrom 40 (2005) 430-43.
• invented by Alexander Makarov
• a purely electrostatic trap with no magnetic fields or RF electric fields
• image current detection and FT
• no MS/MS in the orbitrap
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The LTQ/orbitrap
1. mass selection for MSn‐1 stages takes place in the LTQ
3. the C‐trap focuses a linear ion beam to a point in the trap
2. Final product ions are ejected axially
4. The MSn spectrum is read out in high resolution on the orbitrap
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Figure 3. (a) Typical transient acquired to record the mass spectrum of bovine insulin. The transient acquired is equivalent to the free induction decay of FT NMR experiments. Top shows an expanded portion of the transient.
Hu Qizhi; Noll Robert J; Li Hongyan; Makarov Alexander; Hardman Mark; Cooks, R Graham, The Orbitrap: a new mass spectrometer, J. Mass Spectrom 40 (2005) 430-43.
The orbitrap is also an FTMS!
The image current signal is obtained in the time domain and is then converted by Fourier transform to the frequency domain and into a mass spectrum.
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ETD does not use free electrons but employs radical anions such as anthracene or azobenzene:
Electron transfer dissociation (ETD)
http://en.wikipedia.org/wiki/Electron_transfer_dissociation
60 m3/hr 300 L/s 400 L/sec15 L/s
ESI source
Transfer capillary and skimmer
Octapole ion guide
Quadrupole lenses
Linear ion trap
Anions can be brought into the trap through the same or opposite side as the positive (sample) ion source
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Ion activation in the LTQ/orbitrap configuration
ETD negative ion source
• Electron transfer dissociation (ETD)
• Collision induced dissociation (CID)
Ion activation and dissociation takes place in the LTQ
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Ion activation in the LTQ/orbitrap configuration: CID, higher energy CID (HCD) and ETD
Diagram of the Thermo LTQ Orbitrap VELOS with HCD collision cell
HCD collision cell provides higher energy collisions than the quadrupole cell. Advantage for iTRAQ global quantitation analyses
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