gas phase ion‐electron reactions for · gas‐phase ion‐electron reactions for carbohydrate and...

Gas Phase Ion Electron Reactions forGas‐Phase Ion‐Electron Reactions for Carbohydrate and Glycopeptide Structural

Ch i iCharacterization

Di Gao, Ning Wang, Wen Zhou, Kristina Håkansson

Department of Chemistry, University of Michigan, Ann Arbor, MI

Identification of Pancreatic Cancer Stem Cells

These areGlycoproteins!

CW Li et al. Cancer Res. 2007, 67, 1030-1037.

Tandem Mass Spectrometry of Glycans

Positive ion mode

(+)

Negative ion mode

(‐)

Vibrational excitation• Collision Activated Dissociation:

Vibrational excitation• Collision Activated Dissociation:

Sialic acid, sulfonate loss Rearrangement

More cross‐ring cleavages Improved ionization for acidic species

Ion‐electron/ion reactions• Electron Capture Dissociation

Ion‐electron/ion reactions• Electron Detachment Dissociation

M l i l i i l h d

Complementary info; Retention of labile groups;

Electron Capture DissociationElectron Transfer Dissociation: Negative Electron Transfer

Dissociation

Complementary infoMultiply positively charged Multiply negatively charged

OSO3HOH

ESI of NOT‐4S with 20 µM CaCl2

O

OSO3H

OHOOO

OHO

O

OH

OCH2OH O

OSO3H

OH

CH2OH

O

O

OSO3H

OHOOO

OH

O O

OH

OCH2OH O

3

OH

CH2OH

O

O

OH

(NOT-4S + 3Ca - 4H)2+

OH

(NOT-4S + 4Ca - 5H)3+(NOT 4S 4Ca 5H)

120011001000900800700600500400300200m/z

HC Liu, K Håkansson. Int. J. Mass Spectrom. 2011, 305, 170-177.

ECD of Ca2+‐Adducted ‐Carrageenan Sulfated Oligosaccharidesg

Mn+ + e- M(n - 1)+• fragments

OOH

OOOO

HOO

OCH2OH O

OSO3H

OH

CH2OH

O OH

Z3(2Ca) Y2(2Ca) Z2(2Ca) Y1(2Ca)

O

OSO3H

OHOOO

OHO

O

OH

OCH2OH O

OSO3H

OH

CH2OH

O OH

Z3(Ca/2Ca)Y3(2Ca) Y2(2Ca)

OHOOO

OH

HO

OH

OH

2,4A4(2Ca)

OH

OHOH

C2(2Ca) C3(2Ca) 2,4A4(2Ca)0,3X3(2Ca)

OSO HOSO3H

H OH O

OSO3H

OOCH2OH O

OSO3HCH2O

H

Y7(4Ca)Z7(4Ca)

Z2(Ca)Y2(Ca)

Y6(4Ca)Z6(Ca)

Z3(Ca)

O

OSO3H

OHOOO

OH

OHO

O

OH

OCH2OH O

OH

CH2OH

O

O

OHOOO

OH

O O

OH

C 2

OHO

O

OH

C7(4Ca)B2(Ca) C6(4Ca)B5(Ca) B6(Ca)

HC Liu, K Håkansson. Int. J. Mass Spectrom. 2011, 305, 170-177.

C2(Ca)

SolariX FT‐ICR Mass Spectrometer

Hollow G t V lCI Source

QuadrupoleICR Cell

Hollow Cathode

Dual-stage Ion

Gate ValveCI Source

Funnel

Dual Octopole

Collision cell (Hexapole)

ElectrosprayIon source

7T MagnetIon Transfer Octopole

ETD: Mn+ + A-• M(n - 1)+• + A fragments

ECD vs. ETD of LNDFH I

ECD ETDMg

Ca

D Gao

ECD vs. ETD of NA2

ECD ETDMg

Ca

D Gao

ECD vs. ETD of Triply Charged La‐adducted NA2

ECD

ETD

D Gao

Isomer Differentiation by Metal‐Assisted ECD/ETD/

LSTa A2, A3X XX3, X2

Could not be differentiated by

LSTb A2X2

CAD.

LSTc A2, A3LSTc 2 3X2

D Gao

ECD of Doubly‐Charged La‐Adducted LSTa

D Gao

Summary of Electron‐Based Activation MethodsMethods

= EID

F Kjeldsen, OA Silivra, IA Ivonin, KF Haselmann, M Gorshkov, RA Zubarev. The 52nd ASMS Conference on Mass Spectrometry and Allied Topics, 2004.

EID of Singly‐Protonated Glycane

100

90

80

70

B3α(a) CAD

CAD

Abu

ndan

ce 60

50

40

30

20

B4/Y3α (C4/Z3α) (B3/Y3) (B3/Y4)

Z3α

Y3α

B4C4

Y3β(Y4)

[M + H]+B3/Y3α (C3/Z3α)

m/z1,000900800700600500400300200100

20

10 3 (658)3 (1000) M ‐ H2O

B(b) EID

e

20

15

Internal B/Y/Y (GlcNAc)

B4/Y3α /Y3 (B3 /Y4/Y3)(C3 /Y4/Z3)

B3α [M + H]+

~X5

EID

Abu

ndan

ce

10

5(658) Z2

3 (1000)

Y2

B3/Y3α (C3/Z3α)

B4/Z3α(B3 /Z3)(B3 /Z4)

B4/Y3α (C4/Z3) (B3/Y3) (B3/Y4)

Y3α

C4

Y3 (Y4)

M ‐ H2O

m/z1,000900800700600500400300200100

3 (658) 2 C4

D Gao, K Håkansson, submitted.

EID of Singly‐Protonated Derivatized Glycance

100

90

80

70

Y2

Migration 654 Y +Fuc

(a) CAD

CAD

Abu

ndan

ce 60

50

40

30

20

Y1

Migration 492 Y1+Fuc

Y2+Fuc

Y3α/Y3β

Y3α

Y4/Y3βY3β (Y4)


B3α /Y4(C3α /Z3β)(C3α/ Z4) [M + H]+


m/z1,2001,1001,000900800700600500400300200100

10 3

20Internal B/Z/Y GlcNAc

B3α/Y3αY2

Y1 [M + H]+(b) EID

nce

20

15 9FL

Y3β (Y4)

B3α/Y3α

(C3α/Z3α)

B4/Y3α /Y3β

(B3α/Y4/Y3β)B3α

Z3β(Z4)


Z3α/Z3β

~X5B3α /Y4(C3α /Z3β)(C3α/ Z4)

Migration1003Y3+Fuc

EID

Abu

ndan

10

5

Y3α

Y3α/Y3β


Migration 492: Y1+Fuc

Y4/Y3βZ1

B2

1,5X1 Z2 1,5X2 1,5X3α

1,5X3β (1,5X4 )

3

Y3α/Z3β

Z3αY4/Z3β

1,5X4/Z3β (Z4/1,5X3β)

m/z1,201,000800600400200

β3

1,200

D Gao, K Håkansson, submitted.


= EID


EDD vs. IRMPD of Disialylated N‐Glycan from Fetuin

[M – nH]n‐ + e‐ (~20 eV) [M – nH](n ‐ 1)‐• + 2e‐

C1 Y6, Z6Y5, Z5 C3 Y4, Z4 B4 Y3, Z3

B6

1,5X 1,5X 1,5X3 EDD

B4 Y3, Z3

1,5A61,5A7

Y5 Z5C1 Y6, Z6

, X5, X4 X3

C3 Y4 Z4

EDD

4 3 3Y5, Z5 C3 Y4, Z4

B1 Y6 B2, C2 B3, C3 B4, C4

B6C6 Y1Z11,5A5

W. Zhou

IRMPD

6 6 1 1

0 2A 2 4A

1,5A20,2A3

1,3A3

5

0,2A 2,4A 0,2A7, 2,4A7,1,3A7B1 Y6 B2, C2 B3, C3 B4, C4

0,2A6, 2,4A6,1,3A6 (1,3A3 = 1,3A6 = 1,3A7 = 0,2A7)

EDD of Chloride‐Adducted Glycans

CH2OH

B C3

ZC4 C5O

O

O

O

O

CH2OH

CH2OH CH2OH

OHOH

OH

O

CH2OH

OH

OH

B1

1,3A3C2 Z4 C3

Y3Z3

Y4Y5 Z5

OO

O

O O

CH2OH

NHCOCH3

OH

OHOHOH

OH

OHO

0,2A2

A31,5X4

1,5X5

Y3Z3

2,4A60,2A5OO

O

O

O

CH2OHCH2OHNHCOCH3 NHCOCH3

OH

OH OHOH

OH

20,2A4

B12,5A6

C2 Z4

Y3

Y4Y5 Z5

O

NHCOCH3

OH

OH

OH

0,2A2

1,3A31,5X41,5X5

2

JR Kornacki, JT Adamson, K Håkansson. J. Am. Soc. Mass Spectrom. 2012, 23, 2031.

Site‐Specific Glycosylation

Asn297

Oli h idOligosaccharide

R Jefferis. Nat. Rev.Drug Disc. 2009, 8, 226.

ECD is Complementary to IRMPD

IRMPD ECD

m/z1,7001,6001,5001,4001,3001,2001,1001,000900800700600500400300200

m/z1,5001,4001,3001,2001,1001,000900800700600500400300200 400 600 800 1000 1200 1400

m/z200 400 600 800 1000 1200 1400

m/z1600

= Mannose

= N -Acetyl l i

R QHMDSSTSAA SSSNYCNQMM KSRNLTKDRC KPVNTFVHE

Glucosamine

JT Adamson, K Håkansson. J. Proteome Res. 2006, 5, 493.

EDD of Tryptic N-glycopeptide

SKPAQGYGYLGVFNNSK

JT Adamson, K Håkansson. 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, June 5-9, 2005.

(M - 2H)2-(M - 3H)3-

’’y3

EDD of a pronase-derived O-glycopeptide from Fetuin ( )

AGPTPS (GPTPSA)

B1βC1β

B2βC2β

y4’

Y1α Z1α

AGPTPS (GPTPSA)

B1βC1β

y5’

Y1α

y3

b4’

IRMPDB1βC1βY2β Z2β

B2α C2αY3α

B1βC1βY2β Z2β

1α

Y3α Z3α

B1α C1α

y5 y3 y4’x5’y4

B1α C1α

y4’y4

5 glycosidic, 0 cross‐ring, 3 backbone 8 glycosidic, 0 cross‐ring, 1 backbone

AGPTPS (GPTPSA)

Y1β 0,3X1β

1,4X1β 2,5X1β

3,5X1β

a5 a5’ b5’b4

AGPTPS (GPTPSA)

Y1β Z1β1,5X1β

a5 c5 a5’b4

EDDB1βC1β Y2β Z2β

Y2α Z2α

B3α Y1α Z1α

0,3X2α1,4X2α

2,5X2α3,5X2α

B C

0,3X2β1,3X2β

3,5X2β

B1βC1β Y2β Z2β

Y2α Z2α

B3α Z1α

B C

0,3X2β1,3X2β

3,5X2β 1,5X2α

0,3X3α1,3X3α

2,5X3α 3,5X3α

B1α C1αY3α 0,3X3α

1,3X3α2,5X3α

3,5X3α

B1α C1αY3α

9 glycosidic, 8 cross‐ring, 9 backbone 7 glycosidic, 5 cross‐ring, 6 backbone

Negative Ion Electron Capture Dissociation (niECD)

[M ‐ H]‐Singly deprotonated Angiotensin I

[M – nH]n‐ + e‐ [M – nH](n + 1)‐• ( )

3

[M ‐ H]2‐•

Singly deprotonated Angiotensin I

4.5 eV electrons for 20 seconds

2

[M ‐ H]

1

m/z2,0001,5001,000500

HJ Yoo, N Wang, S Zhuang, H Song, K Håkansson. J. Am. Chem. Soc. 2011, 133,16790‐16793.


[M ‐ H]‐ ECD of angiotensin I (IIE-ECDlens=1.5 eV)_[M-H]2-oElectron Capture Efficiency as Si l d d A i i I

( )[M – nH]n‐ + e‐ [M – nH](n + 1)‐•

3

[M H]

[M H]2 • 3 00

3.50

Electron Capture Efficiency as Function of Electron EnergySingly deprotonated Angiotensin I


2

[M ‐ H]2‐•

2.00

2.50

3.00

(%)

1 0.50

1.00

1.50

Effi

cien

cy

1

0.00 0 1 2 3 4 5 6 7 8 9 10

Electron energy (eV)

m/z2,0001,5001,000500



[M ‐ H]‐Si l d d A i i I

[M – nH]n‐ + e‐ (2.5‐7.5 eV) [M – nH](n + 1)‐• fragments (c’/z•)

( )

3

[M H]

[M H]2 • H O

Singly deprotonated Angiotensin I


z•

HN

HN OH

R1 O R3 O R5

2

[M ‐ H]2‐• ‐ H2O

z5‐•*

H2N NH

NH

O R2 O R4 O

c’

1 c’3‐c’8‐

z7‐•c’ ‐

c’92‐(c’8 ‐ H2O)‐

1z9‐•

z7z6‐•c 5

m/z2,0001,5001,000500


FT-ICR Mass Spectra of Human Apo-TransferrinPronase Digest

30P iti i dP iti i dce

20

Positive ion modePositive ion mode(+)(+)

bund

anc

0

10Ab

0

20

30 Negative ion modeNegative ion mode((--))da

nce

10

20 (( ))

Abu

nd

0 200 400 600 800 1000 1200 1400 1600 1800 2000Ning Wang

niECD of an O-Glycopeptide

+ APSAVPD

25[M ‐ H]‐

[M NeuAc H]‐

[M ‐ NeuAc ‐ 2H]2‐

nce

20

15

[M ‐ NeuAc ‐ H]

c’4‐

[M H O H]2‐•[M ‐ CO2 ‐ H]2‐•

[NeuAc ‐ H] ‐[M ‐ 2NeuAc ‐ H]‐

Abu

ndan 15

10c’6‐c’3‐[M ‐ H]2‐•

[M‐ H2O ‐ H]

c’62‐y’4‐

5 z’3‐

m/z1,6001,4001,2001,000800600400200

Ning Wang

Conclusions

Both ECD and ETD provide complementary structuralinformation compared with CAD/IRMPD but ETD shows amore pronounced charge state effect.

EID allows analysis of singly charged glycans with most EID allows analysis of singly charged glycans with mostinformation for tagged glycans in negative ion mode.

EDD provides extensive information about glycans andpronase‐derived peptides.

niECD appears promising for acidic glycopeptide analysis.

Acknowledgements

$$$ NIH (1R01GM107148-01)

NSF (CHE 11-52531)

NIH (1R21CA138331)

gas phase ion‐electron reactions for · gas‐phase ion‐electron reactions for carbohydrate and...

Documents