Outline Terminology - University of Minnesota...Column 2: LC-MS Peptide Elution Profiles Mass Spec Absolute Intensity 2 minute intervals Time (sec) Intensity, mAU (214 nm) Liquid Chromatography,

Center for Mass Spectrometry and Proteomics | Phone | (612)625-2280 | (612)625-2279

Outline

• Liquid Chromatography• Electrospray• MS Acquisition • Peptide Fragmentation• PTM Detection• Protein ESI-MS

Terminology

• ESI• Liquid chromatography• Acquisition• DDA (data dependent

acquisition)• MS1 vs MS2• Precursor ion• MS/MS• Product Ions• b-ion, y-ion


Gingras A et al, 2007 Nat Rev Mol Cell Biol 8, 645

A. Obtain protein-complexB. Separate proteins on 1D gelC. Excise protein bands > trypsin digestionD. Liquid Chromatography-ESI-Mass Spectrometry

SDS-PAGE

EXAMPLE Workflow:MIXTURE OF UNKNOWN PROTEINS


Liquid Chromatography (LC): Peptide Separation

Electrospray Ionization (ESI)

Mass Spectrometry (MS)

Example Sample Analysis Workflow


Liquid Chromatography: Separation Technique

Mixtures are separated or partially separated before MS analysis with Chromatographic Technologies


http://www.chemistry.adelaide.edu.au/external/soc-rel/content/lc-col.htm

“… modern Liquid Chromatography (LC), uses a liquid mobile phase to transportthe sample components through a column packed with a solid material - thestationary phase.” Reference: http://www.earl2learn.com

From Tswett’s notebook (1910) on theearly chromatographic experiments:plant pigments were passed throughcalcium carbonate using petroleumether

Mikhail Tswett (1872 – 1919)

Liquid Chromatography Basic Overview


i022_091711_sPRG_pep_digestmix_HC... 9/17/2011 11:27:17 PM

0.00 - 70.01

0 5 10 15 20 25 30 35 40 45 50 55 60 65

0

10

20

30

40

50

60

70

80

90

100

41.21

36.2726.1338.809.43

31.80

24.56

33.72

19.7028.01 48.65

18.35 20.4944.71

30.98

17.65 44.87

55.2515.6450.56 57.679.06

63.0352.05 58.728.53 67.1212.712.57

• Peaks represent analyte elution profiles

• Increased Retention Time = increased peptide hydrophobicity on a C18 column

Liquid Chromatography, Peptide Elution Profile 1D LC

Time (minutes)


Minutes

10 20 30 40 50 60 70

• Column 1 Elution Profile• Use fraction collector – collect peptides in separate tubes

Column 2: LC-MS Peptide Elution Profiles

Mas

s Spe

c Ab

solu

te In

tens

ity

2 minute intervals

Time (sec)

Inte

nsity

, mA

U(2

14 n

m)

Liquid Chromatography, Peptide Elution Profiles 2D LC







Electrospray Ionization (ESI)Produce Analyte Ions

GOAL: eliminate solvent, get analyte into the gas phase; apply high voltage to a liquid to create aerosol

http://www.newobjective.com/electrospray/index.html


Electrospray Ionization

Ref: IJAC Vol 2012 ID 282574

Mas

s Spe

ctro

met

er

Liquid flow







Typical Numbers of Proteins Identified:

• 1D LC-MS: up to 300 • 2D LCMS: up to 5000

(sample and dynamic range dependent)

EXAMPLE Workflow:MIXTURE OF UNKNOWN PROTEINS


EXAMPLE:MIXTURE OF UNKNOWN PROTEINS

Data-Dependent Acquisition Scan Mode (DDA) on the Mass Spectrometer


RT: 0.67 - 73.58

5 10 15 20 25 30 35 40 45 50 55 60 65 70Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

39.08

28.5642.32 46.09 53.64

49.6159.65

24.4532.33 54.1934.17

57.26

17.92 62.4422.91 63.7868.3717.6713.80

1.63 4.17

Next slide: see Full Scan/Survey Scan at 39 minutes

Retention time 39 minutes

1 Dimensional LC-ESI-MS Elution Profile


georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]

500 600 700 800 900 1000 1100 1200 1300 1400m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

851.95

782.40671.68

587.05553.61829.91

504.01

1173.091007.02739.37616.32 929.73

689.84

1136.261063.51

863.44

1239.31 1350.02 1427.47445.12

666.34

477.43

Mass Spectrum at 39.22 minutes shows co-eluting peptides

MS1 Data Acquisition• MS1 spectrum (below)• Peaks below represent unfragmented peptide m/z values


georg_hamel_011713_12503_chymo_tubA_025dda #4335 RT: 39.22 AV: 1 NL: 5.65E7F: FTMS + c NSI Full ms [360.00-1800.00]

500 600 700 800 900 1000 1100 1200 1300 1400m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

851.95

782.40671.68

587.05553.61829.91

504.01

1173.091007.02739.37616.32 929.73

689.84

1136.261063.51

863.44

1239.31 1350.02 1427.47445.12

666.34

477.43

1

2345 6

Data Dependent Acquisition: • Top 6 (most abundant) Peaks Identified• Next 6 Scan Events are MS/MS

Sequential MS/MS scan events are triggered for the 6 most abundant peaks


MS2 Data Acquisition (1st peak triggered in DDA)MS/MS spectrumPrecursor 851.95 m/z

georg_hamel_011713_12503_chymo_tubA_025dda #4329 RT: 39.20 AV: 1 NL: 7.09E6T: FTMS + c NSI d Full ms2 [email protected] [111.00-1715.00]

200 300 400 500 600 700 800 900 1000 1100 1200 1300m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e Ab

unda

nce

279

226187159

727812

911410 628509

280

136

491 794325 826527385 709610 893 10981011956412

241

728

912

x5

peptide fragment ions peak


• Select Precursor Peptide• Isolate Precursor ions in Collision Cell• Fragment precursor ions with Collision

Induced Dissociation (CID)• Measure m/z of Product ions

Tandem Mass Spectrometry (MS/MS)


+LLLYSSQICK+

Collision CellQuadrupole Mass Filter

+ LLL+LLLY

+LLLYSCK+

QICK+SSQICK+

Collision Induced Dissociation1) Electric potential applied to collision cell2) Ions accelerated to high kinetic energy3) Ions collide with neutral gas molecule4) Kinetic energy is converted to internal energy5) Chemical bond breakage occurs

Measure m/z values of product ions+LLLYSSQICK+

N2 collision gas

N2

N2 N2

Tandem Mass Spectrometry1st measurement (MS1) = Intact Peptide m/z2nd measurement (MS2) = Peptide fragment ion m/z

values

PRECURSOR ion 612.8 m/z(LLLYSSQICK) filtered in Q1

Q1 q2 Detector

Collision Induced Dissociation (CID or HCD)


matrixscience.com

peptide bonds

• Peptide Backbone has 3 Bond Types (peptide bond is the weakest of the 3 bonds)• Bond Breakage Yields Complimentary Ion Types, e.g., b- and y-type

Predictable Fragment Ions Types from Peptide Dissociation


Peptide MQIFVKTLTK • 604.8572 m/z precursor ion, monoisotopic• 1208.7077 Intact Peptide Mass, monoisotopic

m/z b series y series m/z1062.6 MQIFVKTLT K 147.1961.6 MQIFVKTL TK 248.1848.5 MQIFVKT LTK 361.3747.4 MQIFVK TLTK 462.3619.3 MQIFV KTLTK 590.4520.3 MQIF VKTLTK 689.5373.2 MQI FVKTLTK 836.5260.1 MQ IFVKTLTK 949.6132.0 M QIFVKTLTK 1077.7

y1y2y3y4y5y6y7y8y9

b9b8b7b6b5b4b3b2b1

Peptide Fragment Ions (or product ions)


Peptide Tandem MS can provide Unambiguous Evidence for Location of

Amino Acid Modifications

Rule: Experimental data must contain fragment ions that provide site localization evidence


MQIFVKTLTK MWmono = 1208.7077MQIFVKTpLTK MWmono = 1288.6740 (phosphorylation +80 Da = HPO3)

Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed


Tandem MS (or MS/MS) for Identification of Amino Acid Post-translational Modification (PTM) Site


+80+80+80+80+80+80

+80+80+80


Mass shifts will occur only in fragments containing the phos-Thr, therefore location of MODIFICATION can be pinpointed

Predictable Mass Shifts for Phosphorylated FragmentsMQIFVKTpLTK


m/z b series y series m/z1142.6 MQIFVKTpLT K 147.11041.5 MQIFVKTpL TK 248.1928.4 MQIFVKTp LTK 361.3747.4 MQIFVK TpLTK 542.3619.3 MQIFV KTpLTK 670.4520.3 MQIF VKTpLTK 769.4373.2 MQI FVKTpLTK 919.5260.1 MQ IFVKTpLTK 1029.6132.0 M QIFVKTpLTK 1157.6

MQIFVKTpLTK (+80 = HPO3); MWmono = 1288.67

Mass shifts will occur only in fragments containing the phos-Thr(bold), therefore location of MODIFICATION can be pinpointed

Predictable Mass Shifts for Phosphorylated Fragments


EXAMPLE: MS2 Spectrum and Confirmed Peptide Match a=3.56742302613418490e-004, t0=4.31796646529983260e+001

100 200 300 400 500 600 700 800 900 1000 1100 1200m/z, amu

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2116

199.1716

86.0972307.1400

136.0725

249.1534 722.3468340.2637

147.1153 420.2225 885.3852 998.4837277.1471

289.1373 635.3242

a1

a2

y8(2+)

b2

y9

b3

y8

a4b4

y7

a5

y6

y5

y4

y3

y2

a3-NH3(2+) y7(2+)

y1

L L L Y S S Q I Ccam KProtein ID: Tyrosine-protein kinase JAK3 (human)


Intact Protein Electrospray Mass Spectrometry


Intact Protein Electrospray - Mass Spectrum

200 1000 2000m/z

+71766.6

+81545.7

+91374.2

+101236.9

+111124.6

+121031.0

+13951.8

+14884.0

+15825.0

+16772.4

+17727.5

0

50

100

Rela

tive

Abun

danc

e PROTEIN CHARGE SERIES

10000mass12500 15000

0

50

100

Rela

tive

Abun

danc

e

12359 +/- 2

Mathematically deconvoluted Data Protein MW


Intact Protein ESI-MS Cytochrome C, 12361 Da

mz = 12361 + 8

8 = 1546

+8

mz = 12361 + 12

12 = 1031+12

1031.0

200 1000 2000m/z

1545.7

0

50

100

Rel

ativ

e Ab

unda

nce

NH3+

COOH

H+

H+H+ H+

H+H+H+

Charge = +8


Intact Protein Electrospray-MSInfusion of Solubilized Relatively Pure Protein

• Relatively pure sample• 20 – 50 µM protein concentration• Detergent and salt free solution• Typical solvent: 50:50, acetonitrile:water, 0.1% formic

acid• Difficult (but possible) to achieve high quality data• Non-covalent interactions are retained with ESI (not

MALDI), usually with neutral/basic buffer system and a lot of trial and error


1600 1800 2000 2200

0

2000

4000

6000

8000

17921730

1858

16721930

1618 2007

18151753 209018821568

m/z

Inte

nsity

, cou

nts Intermediate 1

Protein

Glycosylated Intact Protein ESI Mass Spec


Intermediate 1: Deconvoluted MS DataUsing Bayesian Reconstruct Tool; Theoretical mass = 50140.01 Da; error=4 ppm

5.00e4 5.05e4 5.10e4Mass, amu

50140

50180

5079750229 5049749849

Inte

nsity

, cps

1.0e5

8.0e4

6.0e4

4.0e4

2.0e4

050943

.23

∆ 656

+K

+K

Sialic acid

∆ 291

Fucose

∆146

NAcNeu|

Gal|

GlcNAc

Documents

Outline Terminology - University of Minnesota...Column 2: LC-MS Peptide Elution Profiles Mass Spec Absolute Intensity 2 minute intervals Time (sec) Intensity, mAU (214 nm) Liquid Chromatography,