top-down” protein identification and characterization...ms configurations for top-down protein...

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Yu XiaDepartment of Chemistry

Purdue University, West Lafayette, IN

July 7th, 2011

“Top-Down” Protein Identification and Characterization

Early ‘Top-Down’--Dissociation Fingerprints

Smith, Loo, Barinaga, Edmonds, Udseth, JASMS (1990) 1, 53.

Horse Heart Bovine Pigeon

Tuna Rabbit Chicken

Rat Dog Yeast

CID of Cytochrome c - (M+15H)15+ on QQQ

Used for MS Short Course at Tsinghua by R. Graham Cooks, Hao Chen, Zheng Ouyang, Andy Tao, Yu Xia and Lingjun Li

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Key Enabling Technologies for Proteomics

Reid, Ion Trap Fall Workshop, 2006, San Diego, CA

Protein Extraction

Bioinformatics

Mass Spectrometry

Matrix effect on ionization Dynamic range Dissociation chemistry Mass analysis

algorithm development coupling with other information

Separation

“Bottom-Up” vs. “Top-Down” in Protein Identification

Intact cell or tissue

cell lysis

2-D gel

HPLC

HPLC ESI/MALDI

MS/MSdigestion

MS/MSESI

Requires solution to z-state ambiguity problem: mass resolution or chemistry


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N C

N C

- PTM

100% atoms represented

5-40% atoms represented(intact mass info absent)

Bottom up

Top down

Enzyme digestion

Gas-phase dissociation

Top-Down Approaches

• Obtain intact molecular weight information• Detect/analyze modifications

- measuring large ions, z state challenge

Requirements of characterizing whole protein ions

- Capability of fragmenting protein ions

Currently, top-down works better for small proteins, < 50 kDaMiddle-down is another option.


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726.56

726.46

726.35 726.67

726.78726.24

726.89

727.00726.13

100

Re

lativ

e A

bu

nda

nce

z= 9, M = 6530

726.56

726.46

726.35 726.67

726.78726.24

726.89

727.00726.13

100

Re

lativ

e A

bu

nda

nce

z= 9, M = 6530

726.87100

Re

lativ

e A

bu

nda

nce

722 723 724 725 726 727 728 729 730 731 732m/z

726.87100

Re

lativ

e A

bu

nda

nce

722 723 724 725 726 727 728 729 730 731 732m/z

Measure big ions…

Measure intact protein mass… Measure product ion mass

For ESI MS: the “Z” needs first be determined

Less problem for MALDI MS: singly charged ions

how big the ions can be analyzed determined by

how well the charge can be resolved

Physical way: resolve isotopic spacing, FT-ICR can do the best

How to Infer Mass - Method #1 - Isotopes

mass m /z z z 1.007276 Da

1. Find charge state from isotope spacing

2. Determine mass (average or monoisotopic) according to equation:

z = 1/(isotope spacing)= 1/(726.67-726.56)= 1/0.11 = 9.09 = 9

Mass (mono) = (726.13 x 9) - (9 x 1.007276) = 6526.10 Da

726.56

726.46

726.35 726.67

726.78726.24

726.89

727.00726.13

z= 9, M = 6530

726.56

726.46

726.35 726.67

726.78726.24

726.89

727.00726.13

z= 9, M = 6530

For [M+nH] n+

Mass (avg) = (726.56 x 9) - (9 x 1.007276) = 6529.97 Da


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8008

13C0

724722720

RP = 5000

Large Molecule Isotopic Distributions

ResolvingPower (RP):

122121120

= 1000mm

80168000

RP = 30,000

Small Molecule(120 Da)

Small Peptide(720 Da)

Small Protein(8000 Da)

13C113C3

13C5

13C2

13C1

13C0

13C0

“monoisotopic”peak (C = 12.000)

“chemist’s average”

(C = 12.011)

“most abundant” isotope peak (C = 13.003)

How to Infer Mass - Method #1 - Isotopes

How to Infer Mass - Method #2 - Charge States

z1 m /z 1 z2 m /z 2

1. Pick two adjacent charge states

2. Assume:

3. Derive:

m /z 1m /z 2

m /z 1 z2

(since z1 = z2+1)4. Use same eq. as before to get mass

1102.114.16854.1838

4.16852

z

Mass (avg) = (1838.4 x 11) - (11x 1.007276) = 20211.3 Da


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How to Infer Mass Method #3 – Ion/Ion Proton Transfer Rxns

Sequential single proton transfer reactions.Reduce multiply charged ions to +1. Resolve “Z” problem

Greatly simplify spectrum caused by multiply charging

Applies to both MS1 and MS/MS experiments

Require instrument capable of ion/ion reactions

Mass Analyzers with relatively low mass resolution (ion trap & time-of-flight) can be used for protein analysis

MS of Yeast (Saccharomyces cerevisiae) LC Fraction 8.4-8.6 Min

Abu

ndan

ceA

bund

ance

m/z

m/z

ESI MS

Post Ion/Ion MS

Mixtures: parent ions (3-D ion trap)

2000200

5000 50000

>20 proteins


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9000 11000 13000 15000 1700010000 12000 14000 16000

y137+

V/T

y152+

V/Ay129

+

L/G y140+

A/Q

y154+

D/D

y130+ -

y136+

y2382+

b111+

L/G

b104+ - b110

+

b88+

V/A

b86+

D/D

b89+ -

b97+

b100+

A/Q

y143+ - y151

+

20

40

30

m/z7000 9000 11000 13000 15000 17000 19000 21000 23000 25000 27000

10

50

y238+

V/Gy228+

W/P

2+

b103+

V/T

y174+

D/G

7000

Post-ion/ion rxn

MS/MS

500 700 900 1100 1300 1500 1700

100

200

300

400

m/z

Mixtures: product ions (19+ of porcine elastase – 25.9 kDa)

500 2000

McLuckey & Hunt R. BrownMcLaffertyNemeth-Cawley & Rouse

MS Configurations for Top-down Protein Analysis


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MS Configurations for Top-down Protein Analysis

Ionization Method

Mass Spectrometer

Fragmentation Methods

Resolving Power

Mass Accuracy

Mass Range

ESI QQQ CID 103 100 ppm <104

ESI Ion Trap CID, ETD 103 50-100 ppm <105

MALDITOF,

TOF/TOFISD, CID 102-104 5-50 ppm >105

ESI Q-TOFCID, IRMPD,

ETD104 5 ppm <104

ESI LTQ-Orbitrap CID, ETD 6*104 1-5 ppm ~103

ESI Q-FTMSCID, IRMPD,

ECD105-106 1-5 ppm >104

Kelleher et al., Anal. Chem. 2004, 200A, McLuckey et al. Chem. Reviews 2001, 571-606

Collision-Induced Dissociation (CID)

CID Beam-type Trapping

Instrument TOF/TOF Q-TOF Ion Trap/ ICR

Collision Energy 2-10 keV 1-200 eV 1-20 eV

Activation Time 1-10 µs 0.5-1 ms 10-100 ms

Efficiency <10% 5-50% 50-100%

H2N—CH—C—NH—CH—C—NH—CH—COH

R1 O R2 O R3 O

— — ——— —— ——

y2

b1

z2

c1

x2

a1

y1

b2

z1

c2

x1

a2

H2N—CH—C—NH—CH—C—NH—CH—COH

R1 O R2 O R3 O

— — ——— —— —— —— —— ——

y2

b1

z2

c1

x2

a1

y1

b2

z1

c2

x1

a2

• During collisions, kinetic energy is converted internally to vibrational activation.


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Charge State Dependent Fragmentation of Protein Ions under Ion Trap CID

8+

4+

12+

Ion Trap CID of Ubiquitinlow charge states:Limited structure informationC-terminal of Asp, GluSmall molecule losses

medium charge states:More structure informationNon-specific cleavages

high charge states:Limited structure informationN-terminal of Pro

I. Structural information from entire molecule FT-ICR Orbitrap Q-TOF Ion trap MSn (zoom scan) Ion/ion reactions in ion traps and hybrids

Two Categories of “Top Down” Approaches:

Methods that provide intact mass and structural information in a single “experiment”

II. Structural information from termini only T3 (TOF/TOF) Ion trap MSn

Q-TOF


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Advantages of FT-ICR MS

• Resolution/Resolving Power

– up to 106, typically 105

– isotopes

• Mass Accuracy

– 10 ppm or better

• Ion trapping experiments

• Complex mixture analysis

• Downsides: speed, cost


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LTQ-Orbitrap for Top-Down

Mass accuracy: ~1ppmProtein size: ~25 kDa

CID of +15 β-lactoglobulin, Rs: 30,000, 12 sec acquisition

Mol. Cell. Proteomics 2006, 5, 949-958

Lin, Campbell, Mueller, Wirth, RCMS (2003) 17, 1809-1814.

MALDI TOF/TOF (ABI 4700, Rs: 6500)

High Energy CID: 2keV of [M+H]+


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Mohring, kellman, Jurgens, Schrader, JMS (2005) 40, 214-216.

ESI-Q-TOF (QSTAR, Rs: 8000-10,000)low energy CID of 9+ ubiquitin

Amide bond cleavage: 70%

b29, y100, y69 : y102, y16, b30

y9913+13+

y96y9713+

b63-647+

b314+

12+

y9712+

y163+

b25

5+

y14 : b43+ +

y122+

b25, y61, y554+ 9+ 8+

y69 : b2610+ 4+

b24, b95+ 2+

2+ 4+4+ 9+ 13+13+A100

b2+

2+b31, y13

5+

y3+

b3+

y126, b12617+ 17+

400 600 800 1000 1200 1400 1600 1800 2000m/z

% R

elat

ive

Abu

ndan

ce

y96

794 796 798 800

b264+

m/z m/z

CB D

y679+

y99

13+

b294+

y84

11+y68

9+

850 854 866862858 870794 796 798 800

y6910+

m/z

b264+

163 Da

UltrazoomRs = 18114

Zoom Rs = 5534

High Resolution Linear Ion Trap Amide bond cleavages

observed (Sequence coverage %)

Scan Mode MS/MS

‘Enhanced’ 17 (13.4%)

‘Zoom’ 30 (23.6%)

‘Ultrazoom’ 36 (28.3%)

combined 64/127 (50.4%)

UltrazoomRs = 18114

Scherperel, Yan, Wang, Reid Analyst (2006) 131, 291-302.

CID of the [M+18H]18+ WT SaDHNA.


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m/z

Abu

ndan

ce

4000 5000 6000 7000 8000300020001000

1500

m/z

Abu

ndan

ce

4000 5000 6000 7000 8000300020001000

1500

“Top-Down” Strategy on Ion Trap Instruments

++

+ +++

+

m/z

Abu

ndan

ce

4000 5000 6000 7000 8000300020001000

1500

m/z

Abu

ndan

ce

4000 5000 6000 7000 8000300020001000

1500

Protein Identification

Protein Database search

++

+

+

+++

++

+

++++ +

+

++

Protein Mixtures Ionization

ESI

Dissociation

+

+ ++

+

+

+

+

Precursor isolation

Ion/Ion chemistry

Ion/Ion chemistry

++

++

Charge statereduction

Ion/Ion chemistry

Q0 Q2

Q1

+HV

- HV

+

-

-

--

-++

++

++ +

++-

---

--- --

--

----

LIT Ion/Ion Reactor TOF AnalyzerDual Ion Source

Ion/Ion Rxns on Q-q-TOF (Q Star)

QSTAR XL

~~

Xia et al. Anal. Chem. 2006, 78, 4146-4154


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m/z

Rel

ativ

e A

bund

ance

, %Post Ion/Ion Ion-Trap CID of +8 Ubiquitin

100

b11-18+

b9+

b3-6+

[M+H]+

y37+

b39+

b52+

b51+

y24+

b32+

b33+

b58+y58-65

+

y70-74+y40

+

y42-44+

[M+2H]2+

b36+

y18+

0 2000 4000 6000 80000

1247.7 2727.5 4992.6 8566.5

b11+ y24

+ y44+ [M+H]+

Mass accuracy: ~ 20 ppm rfwhm : 6000-8000

200 62001200 4200 82002200 3200 5200 7200

ETD + PT Reactions of +12 Ubiquitin

1+X 3 X 9

2+c2-4

c5-17z4- 17,

z3

c59-74, z62-753+

c73/z74

c75

c57c45

z60*

z52c51

c47

c44

c41

z45*-OH

c23-28, c30-33, z32*

M Q I F V K T L T G K T I T L E V E P S D T I E N V K A K I Q D K E G I P P D Q

Q R L I F A G K Q L E D G R T L S D Y N I Q K E S T L H L V L R L R G G

105 15 20 25 30 35 40

45 50 55 60 65 70 75

H2N -

-COOH

Rel

ativ

e A

bund

ance

100 ms ET +50 ms PT in Q2


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Relative Informing Power of ESI based Top-Down Approaches

m/z

+10+20

+30

ESI

m/z

High Resolution

m/z

+1 +1 +1

Proton Transfer I/I Rxn

Liu, Chrisman, Erickson, McLuckey, Anal. Chem., 79 (2007) 1073-1081

TOF + Ion/Ion

MALDI-ISD-TOF/TOF MS: A Pseudo-MS3 ApproachT3 (Terminus-Specific TOF/TOF) Sequencing

Suckau, Resemann, Anal. Chem. (2003) 75, 5817-5824.

High energy CID

CID of y12

ISD of RNase B


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Q-TOF MS/MS – Examination of low m/z fragments

Nemeth-Cawley, Rouse J. Mass Spectrom. (2002) 37, 270.

Coon, Ueberheide, Syka, Dreyser, Ausio, Shabanowitz, Hunt PNAS (2005) 102, 9463-9468.

Ion Trap ETD + PT with No Mass Extension on LTQ

+13 ubiquitin

•-

-

ETD

proton transfer


top-down” protein identification and characterization...ms configurations for top-down protein...

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