mass and the mass spectrum - university of alabama at ... · 1/13/2014 · january 14, 2014 what...

1/13/2014

1

Mass and the Mass Spectrum

Matt Renfrow

MCLM 570

[email protected]

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.

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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


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=?


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


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


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


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


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


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


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


DataSystem

I M D t

High Vacuum System

Sample Introduction

Inlet IonSource


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


DataSystem

Time of flight (TOF)QuadrupoleIon TrapMagnetic SectorFT-ICR MSOrbitrap MS

I M D t

High Vacuum System

Detector

InletIonsource


DataSystem

Microchannel PlateElectron MultiplierMass analyzer/ion trap AC image

I M D t

High Vacuum System

Data System

InletIonsource


DataSystem

PC

mass and the mass spectrum - university of alabama at ... · 1/13/2014 · january 14, 2014 what...

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