mass and the mass spectrum - university of alabama at ... · 1/13/2014 · january 14, 2014 what...
TRANSCRIPT
1/13/2014
1
Mass and the Mass Spectrum
Matt Renfrow
MCLM 570
January 14, 2014
What does a mass spectrometer do?
1. It measures mass better than any other technique.
2. It can give information about chemical structures.
What are mass measurements good for?g
To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers
Assigning numerical value to the intrinsic property of “mass” is based on using carbon-12, 12C, as a reference point.
One unit of mass is defined as a Dalton (Da).
How is mass defined?
One Dalton is defined as 1/12 the mass of a single carbon-12 atom.
Thus, one 12C atom has a mass of 12.0000 Da.
Isotopes
+Most elements have more than one stable isotope.
For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da.
Wh d ?+Why do we care?
Mass spectrometers can “see” isotope peaks if their resolution is high enough.
If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved.
1/13/2014
2
Element Mass AbundanceH 1.0078
2.014199.985%0.015
C 12.000013.0034
98.891.11
N 14 0031 99 64
Stable isotopes of most abundant elements of peptides
N 14.003115.0001
99.640.36
O 15.994916.999117.9992
99.760.040.20
1981.84
1982.84
Mass spectrum of peptide with 94 C-atoms (19 amino acid residues)
No 13C atoms (all 12C)
One 13C atom
“Monoisotopic mass”
1983.84Two 13C atoms
4361.45
Isotope pattern for a larger peptide (207 C-atoms)
m/z
4360.45
Mass spectrum of insulin
13C
2 x 13C
12C : 5730.61
Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.
1/13/2014
3
Monoisotopic mass
Monoisotopic masscorresponds tolowest mass peak
When the isotopes are clearly resolved the monoisotopic massis used as it is the most accurate measurement.
Average massAverage mass corresponds to the centroid of the unresolved peak cluster
When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.
Poorer resolution
Better resolution
What if the resolution is not so good?
At lower resolution, the mass measured is the average mass.
6130 6140 6150 6160 6170Mass
100
60
70
80
90
100
ns
ity
ISO:CH3
15.0229M
FWHM = M
R = M/M
How is mass resolution calculated?
15.01500 15.01820 15.02140 15.02460 15.02780 15.03100
Mass (m/z)
0
10
20
30
40
50
% I
nte
n FWHM = M
1/13/2014
4
Mass measurement accuracy depends on resolution
6000
8000Resolution =18100
15 ppm error
High resolution means better mass accuracy
0
2000
4000
6000
Co
un
ts
2840 2845 2850 2855
Mass (m/z)
Resolution = 14200
Resolution = 4500
24 ppm error
55 ppm error
Two peptides - same nominal mass - simulation
Peptide mixture: [Val5]-Angiotensin II Lys-des-Arg9-Bradykinin
Sequence: DRVYVHPF KRPPGFSPF
Formula: C49H69N13O12 C50H73N13O11
Exact mass: [M+2H]2+ = 516.76671 [M+2H]2+ = 516.78490
m (mmu): 18.2 mmu18.2 mmu
RP = 18,000RP = 18,000
516 77581 ( b d)
RP = 56,700RP = 56,700
Inte
ns
ity
(%)
0
20
40
60
80
100
m/z516.65 516.70 516.75 516.80 516.85 516.90
516.77581 (observed)
516.76671(correct)
516.78490(correct) In
ten
sit
y (%
)
0
20
40
60
80
100
m/z516.65 516.70 516.75 516.80 516.85 516.90
516.78490516.76671
2 ppm
1 ppm(4)
Theoretical Delta Delta RDB CompositionMass [ppm] [mmu]
516.76671 0.0 0.0 21.0 C49 H71 O12 N13
516.76647 0.5 0.2 15.0 C49 H79 O11 N9 S2
516.76638 0.6 0.3 12.0 C41 H75 O14 N15 S1
516.76705 -0.7 -0.3 11.5 C43 H77 O15 N12 S1
516.76604 1.3 0.7 16.0 C48 H75 O16 N9
516.76738 -1.3 -0.7 20.5 C51 H73 O13 N10
516 76604 1 3 0 7 21 5 C H O N
Is Mass Accuracy Important ?Results for error limit up to 5 ppm
5 ppm(23)
2 ppm(10)
516.76604 1.3 0.7 21.5 C47 H69 O11 N16
516.76580 1.8 0.9 15.5 C47 H77 O10 N12 S2
516.76772 -2.0 -1.0 16.5 C44 H73 O11 N16 S1
516.76773 -2.0 -1.0 11.0 C45 H79 O16 N9 S1
516.76805 -2.6 -1.3 25.5 C52 H69 O9 N14
516.76537 2.6 1.3 16.5 C46 H73 O15 N12
516.76807 -2.6 -1.4 7.0 C38 H79 O14 N15 S2
516.76513 3.0 1.6 10.5 C46 H81 O14 N8 S2
516.76513 3.1 1.6 16.0 C45 H75 O9 N15 S2
516.76839 -3.3 -1.7 16.0 C46 H75 O12 N13 S1
516.76479 3.7 1.9 20.0 C52 H75 O11 N9 S1
516.76872 -3.9 -2.0 25.0 C54 H71 O10 N11
516.76470 3.9 2.0 17.0 C44 H71 O14 N15
516.76874 -3.9 -2.0 6.5 C40 H81 O15 N12 S2
516.76446 4.3 2.2 11.0 C44 H79 O13 N11 S2
516.76897 -4.4 -2.3 12.5 C40 H73 O16 N16
516.76907 -4.6 -2.4 15.5 C48 H77 O13 N10 S1
489 490 491 492 493 494 495 496m/z
489 490 491 492 493 494 495 496m/z
Mass Resulution = m / ∆m 50%
489 490 491 492 493 494 495 496m/z
FT-ICR MS
RP = 491.2594 / 0.0055 amu= 89,319
489 490 491 492 493 494 495 496m/z
LTQ MS
RP = 491.45 / 0.69 amu= 712
ah 20121221 ORBI ST6Gal2 CID#3136 RT: 1284 AV: 1 NL: 136E7ah_20121221_ORBI_ST6Gal2_CID #3136 RT: 12.84 AV: 1 NL: 1.36E7T: FTMS + p NSI Full ms [300.00-1800.00]
100
70
80
90
390.1954R=69101
z=2
50
60
tive
Ab
un
da
nce 796.1542
R=47400z=4637.1249
R=53100z=5
7473320
z 2
20
30
40
Re
la
460.2390R=62601
z=1587.3278R=55601
z=2
660.3551R=52201
z=2
840.6559R=46600
z=3
747.3320R=49200
z=4
917.8965R=44500
1260.4801R=3760011681613
300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800m/z
0
10
R=44500z=2
R=37600z=2
1168.1613R=38800
z=31061.5383R=41000
z=3
1339.6492R=35004
z=?
1570.5303R=27904
z=?
1482.6348R=27101
z=1
1672.8555R=25804
z=?
1748.709R=1800
z=?
m/z
ah 20121221 ORBI ST6Gal2 CID#3136 RT: 1284 AV: 1 NL: 330E6ah_20121221_ORBI_ST6Gal2_CID #3136 RT: 12.84 AV: 1 NL: 3.30E6T: FTMS + p NSI Full ms [300.00-1800.00]
100470.9296R 62201
70
80
90 R=62201z=3
471.7597R=61504
z=?
50
60
tive
Ab
un
da
nce
443.2123R=63501
z=1
20
30
40
Re
la
461.2423R=63004
z=1
465.1943R=62504
z=?474.9147R=62101
z=3444.2155 481.16834422553 4635780
430 435 440 445 450 455 460 465 470 475 480 485m/z
0
10
z=3R=61504z=1
R=56904z=?
442.2553R=58004
z=?
453.2485R=62400
z=3
430.2285R=64504
z=?
485.0027R=54004
z=?
457.7580R=58804
z=?
466.1977R=56204
z=?
436.2570R=66001
z=3
463.5780R=59804
z=?450.6086R=47504
z=?
489.49R=383
z=?
m/z
ah_20121221_ORBI_ST6Gal2_CID #3117 RT: 12.76 AV: 1 NL: 6.21E6F: FTMS + p NSI Full ms [300.00-1800.00]
ah_20121221_ORBI_ST6Gal2_CID #3136 RT: 12.84 AV: 1 NL: 2.82E6T: FTMS + p NSI Full ms [300.00-1800.00]
470.929680
90
100
90
100
471.2639R=64504 40
50
60
70
ela
tive
Ab
un
da
nce
60
70
80
nce
R 64504z=3
10
20
30
Re
470.9139R=78604
z=?
470.9389R=88404
z=?
40
50
60
Re
lativ
e A
bu
nd
a
471.5980R=62404
470.86 470.88 470.90 470.92 470.94 470.96 470.98 471.m/z
0z=?
10
20
30
R
z=3
474.91R=621
z=3471.7597R=61504
z=? 474.7621470.3247
469.0 469.5 470.0 470.5 471.0 471.5 472.0 472.5 473.0 473.5 474.0 474.5m/z
0
10R=29404
z=?R=36704
z=?
ah_20121221_ORBI_ST6Gal2_CID #3117 RT: 12.76 AV: 1 NL: 7.97E6F: FTMS + p NSI Full ms [300.00-1800.00]
100
80
90 470.9294R=62001
z=3
50
60
70
e A
bu
nd
an
ce
637.3253R=52700
z=5564.7407R=56601
z=2548.3080R=57901
20
30
40
Re
lativ
e
z=1538.2928R=59301
z=2
611.8011R=54001
z=2477.2923R=61901
z=2
705.8903R=50100
z=2660.3550R=52301
580.3182R=54701
747.0812R 49700
650.6547R=51804
z=?531.3499R=56104
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 7400
10
20 R=52301z=2
R=54701z=2
R=49700z=4
460.2389R=61601
z=1
497.9135R=60001
z=3
718.5354R=48600
z=5
625.3066R=50501
z=2513.6211R=56100
z=3
604.3214R=52501
z=3
672.8903R=50704
z=?
z=?
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740m/z
ah 20121221 ORBI ST6Gal2 CID#3136 RT: 1284 AV: 1 NL: 631E6ah_20121221_ORBI_ST6Gal2_CID #3136 RT: 12.84 AV: 1 NL: 6.31E6T: FTMS + p NSI Full ms [300.00-1800.00]
100637.9271R=51804
70
80
90R=51804
z=5
50
60
tive
Ab
un
da
nce
660.3551R=52201
64157766362248
20
30
40
Re
la z=2641.5776R=52700
z=4650.0007R=53000
z=3657.3304R=50000
z=3
645.0815R=52700
4
622.0747R=53500 662.7886
R=52901674.3255R 46400
633.3589 65335576673177
636.2248R=45604
z=?
620 625 630 635 640 645 650 655 660 665 670 675m/z
0
10
z=4z=4 R=52901z=2
R=46400z=4
R=53501z=2
653.3557R=51400
z=3
667.3177R=43800
z=4
629.9045R=45104
z=?
669.6773R=42404
z=?
625.8212R=29504
z=?
m/z
ah 20121221 ORBI ST6Gal2 CID#3136 RT: 1284 AV: 1 NL: 980E5ah_20121221_ORBI_ST6Gal2_CID #3136 RT: 12.84 AV: 1 NL: 9.80E5T: FTMS + p NSI Full ms [300.00-1800.00]
100
70
80
90
674.3255
705.2003R=52004
z=?
50
60
tive
Ab
un
da
nce R=46400
z=4711.8686R=50801
z=2
697.3212R=49901
z=2687.8203R=39304
z=?6673177
706.8941R=47404
z=2
712.8719R=50304
z=2
20
30
40
Re
la 667.3177R=43800
z=4688.5815R=44104
z=?
694.8643R=45201
z=2
703.0815R=50200
z=4
718.7360R=49304
z=?
z 2
683.7191R=49804
z=?708.8068R=42104
z=?
675.0031R=55604
z=?
698.3261R=49004
z=2
681.3226R=56204
670.5719R=46604
700.3504R=46701
717.7227R=43604
670 675 680 685 690 695 700 705 710 715 720m/z
0
10
z ?z=?
R 46604z=?
R 46701z=2
R=43604z=?
m/z
GalNAc
GalFT-ICR-MS
Gal
1450 155015001400 1600 1650 m/z
quadrupoleConcept of q p+ SWIFTisolated
Concept of MS/MS
MS21450 155015001400 1600 1650 m/z
MS2
GalNAc
Gal
Gal
GalNAcGal
GalNAcIRMPD
MS / MS
1450 155015001400 1600 1650 m/z
ah_20121221_ORBI_ST6Gal2_CID #3117 RT: 12.76 AV: 1 NL: 7.97E6F: FTMS + p NSI Full ms [300.00-1800.00]
100
80
90 470.9294R=62001
z=3
50
60
70
e A
bu
nd
an
ce
637.3253R=52700
z=5564.7407R=56601
z=2548.3080R=57901
20
30
40
Re
lativ
e
z=1538.2928R=59301
z=2
611.8011R=54001
z=2477.2923R=61901
z=2
705.8903R=50100
z=2660.3550R=52301
580.3182R=54701
747.0812R 49700
650.6547R=51804
z=?531.3499R=56104
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 7400
10
20 R=52301z=2
R=54701z=2
R=49700z=4
460.2389R=61601
z=1
497.9135R=60001
z=3
718.5354R=48600
z=5
625.3066R=50501
z=2513.6211R=56100
z=3
604.3214R=52501
z=3
672.8903R=50704
z=?
z=?
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740m/z
ah 20121221 ORBI ST6Gal2 CID#3102 RT: 1272 AV: 1 NL: 786E4ah_20121221_ORBI_ST6Gal2_CID #3102 RT: 12.72 AV: 1 NL: 7.86E4T: ITMS + p NSI d Full ms2 [email protected] [155.00-1235.00]
100791.3508
70
80
90
678.2643
549.1775
50
60
tive
Ab
un
da
nce
20
30
40
Re
la 432.2576524.6768
989.3562
234.16881048.4411961.4388
860.4360414.2571
200 300 400 500 600 700 800 900 1000 1100 1200m/z
0
10301.0873 656.3471 904.4372
467.1752 773.3503216.1683 1188.77831099.3592
m/z
ah 20121221 ORBI ST6Gal2 CID#3108 RT: 1273 AV: 1 NL: 201E4ah_20121221_ORBI_ST6Gal2_CID #3108 RT: 12.73 AV: 1 NL: 2.01E4T: ITMS + p NSI d Full ms2 [email protected] [155.00-1235.00]
100
70
80
90
50
60
tive
Ab
un
da
nce 516.1766
20
30
40
Re
la
527.1769509.1764
531.1770
500 505 510 515 520 525 530 535 540m/z
0
10520.0933510.1764 543.3440540.1772500.1761 513.1765 533.1770 535.1771505.2596 523.2601
m/z
ah 20121221 ORBI ST6Gal2 CID#3237 RT: 1324 AV: 1 NL: 447E6ah_20121221_ORBI_ST6Gal2_CID #3237 RT: 13.24 AV: 1 NL: 4.47E6T: ITMS + p NSI d Full ms2 [email protected] [195.00-1530.00]
100
70
80
90
493.2581
7493485
50
60
tive
Ab
un
da
nce
749.3485 907.4361
584.3440
836.4342 931.3534
20
30
40
Re
la 699.3471
608.2613380.2551 1193.4439
816.4336
421.3395322.2535 1340.531310944412
200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500m/z
0
10
1094.4412674.3464
1135.5257
280.25231250.5288 1498.86891382.6158
m/z
ah_20121221_ORBI_ST6Gal2_CID #3117 RT: 12.76 AV: 1 NL: 7.97E6F: FTMS + p NSI Full ms [300.00-1800.00]
100
80
90 470.9294R=62001
z=3
50
60
70
e A
bu
nd
an
ce
637.3253R=52700
z=5564.7407R=56601
z=2548.3080R=57901
20
30
40
Re
lativ
e
z=1538.2928R=59301
z=2
611.8011R=54001
z=2477.2923R=61901
z=2
705.8903R=50100
z=2660.3550R=52301
580.3182R=54701
747.0812R 49700
650.6547R=51804
z=?531.3499R=56104
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 7400
10
20 R=52301z=2
R=54701z=2
R=49700z=4
460.2389R=61601
z=1
497.9135R=60001
z=3
718.5354R=48600
z=5
625.3066R=50501
z=2513.6211R=56100
z=3
604.3214R=52501
z=3
672.8903R=50704
z=?
z=?
440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740m/z
Myoglobin
c18ziptip80bump_tunebyhand_3ms #2-11 RT: 0.06-0.56 AV: 10 NL: 3.36E4T: FTMS + p ESI Full ms [500.00-1500.00]
100
RXR-Ligand binding domain
c18ziptip80bump_tunebyhand_3ms #2-11 RT: 0.06-0.56 AV: 10 NL: 6.45E4T: FTMS + p ESI Full ms [500.00-1500.00]
100 9401850 60
70
80
90
da
nce
779.1834R=53019
z=35779.0677R=47940
z=35
779.2957R=50590
80
90
940.1850R=40385
z=?802.0407R=50180
z=34
20
30
40
50
Re
lativ
e A
bu
nd
z=35
778.9847R=47212
z=35779.3549R=61305
z=35779.7514R=61040
z=?
779.6134R=44343
z=?
778.9245R=52076
z=35
60
70
bu
nd
an
ce 779.1248R=53062
z=35
1009.7550R=40362
z=?
778.8 779.0 779.2 779.4 779.6 779.8 780.0m/z
0
10778.8336R=53839
z=?
780.0625R=77856
z=?
30
40
50
Re
lativ
e A
b
1090.4523R=36345
z=25
757.5110R=56767
z=36 1185.2789R=43943
1238.7905R=26032
10
20
30z=23
R=26032z=?
1362.8720R=17608
z=?
700 800 900 1000 1100 1200 1300 1400m/z
0
total crude lipids
FT-ICR mass spectra of total crude lipids derived from Mtb∆whiB3 growing in macrophages demonstrate the absence of SL-1. (A) Total crude lipid extracts were prepared from Mtb growing inside macrophages and analyzed in the negative ( ) p p p g g p g y gion mode by FT-ICR MS. We observed the presence of Ac2PI species at m/z 835.5261 and 851.5566, which corresponds to their reported theoretical masses. The dimannose specie esterified to three acyl chains (Ac3PIM2) corresponds to mass m/z 1413.8888 was also detected in all the strains tested. Note that the multiple lipoforms of SL-1 were absent in Mtb∆whiB3. (B) The SL-1 region of FT-ICR mass spectrum showed a complete absence of this class of lipids (~m/z 2300 to 2600) in Mtb∆whiB3 (C) FT-ICR mass spectra of the PDIM region.
1/13/2014
1
How does a mass spectrometer work?
Sample
Ion source:makes ions
Mass analyzer: separates ions
Mass spectrum:presents
information
I M D t
High Vacuum System
Mass Spectrometer Block Diagram
InletIonsource
Mass Analyzer Detector
DataSystem
I M D t
High Vacuum System
Mass Spectrometer Block DiagramTurbo molecular pumps
InletIonsource
Mass Analyzer Detector
DataSystem
I M D t
High Vacuum System
Sample Introduction
Inlet IonSource
Mass Analyzer Detector
DataSystem
HPLCFlow injectionCharged vapor streamSample plate
1/13/2014
2
I M D t
High Vacuum System
Ion Source
Inlet IonSource
Mass Analyzer
Detector DataSystem
MALDIESIFABLSIMSEICIDART
I M D t
High Vacuum System
Mass Analyzer
InletIonsource
Mass Analyzer Detector
DataSystem
Time of flight (TOF)QuadrupoleIon TrapMagnetic SectorFT-ICR MSOrbitrap MS
I M D t
High Vacuum System
Detector
InletIonsource
Mass Analyzer Detector
DataSystem
Microchannel PlateElectron MultiplierMass analyzer/ion trap AC image
I M D t
High Vacuum System
Data System
InletIonsource
Mass Analyzer Detector
DataSystem
PC