Intro to Mass Spec-part 4-tandem MS-Pao-Chi Liao.pdf

7/28/2019 Intro to Mass Spec-part 4-tandem MS-Pao-Chi Liao.pdf

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Intro to Mass Spec-part 4-Pao-Chi Liao 1

(Introduction to Mass Spectrometry)

(Tandem Mass Spectrometry, MS/MS)

[email protected]
mailto:[email protected]:[email protected]


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What is Tandem Mass Spectrometry?

Dictionary definition of tandem:An arrangement of two or more people or

things placed one behind the other.

For examples, tandem bicycle

Tandem Mass Spectrometry:

An arrangement of two or more mass

spectrometers (tandem-in-space) or massspectrometry experiments (tandem-in-time)

placed one behind the other.

Acronym: MS/MS; MS3; MSn


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ionization source

mass analyzer I

mass analyzer II

fragmentation

analytes A, B, C, D

precursor ion

product ions A1+ , A2

+ , A3+ , A4

+

A+

A+ , B+ , C+ , D+ I

m/z

A+

B+

C+

D+

I

m/z

A+

A1+

A2+

A3+

A4+

An Example of Tandem Mass Spectrometry

Experiment: Product Ion Scan


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Activation (Dissociation, Fragmentation)

Methods in MS/MS

Metastable decay

Collision-induced dissociation (CID), or

collisionally activated dissociation (CAD).

High energy vs . low energyCID.

Surface induced dissociation, photo-dissociation, electron excitation.


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Rationales for (Why) Performing

Tandem Mass Spectrometry

To obtain chemical structure-related

information.

For examp le, dif ferent iat ion of isobar

compounds or isomers .

To increaseselectivity and S/N ratio in

quantitative measurement.For example, metabo l i te quat itation in

com plex bio logical matr ices.


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MS/MS by fragmentation enhances selectivity

isobar

distinct fragments

MW: 278 Da for both


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C

N

C C

C

O

NH2

NH+

N

CH

CH2

CH2

OH

NH

m/z 196 m/z

C

N

C

NH

C

C

O

NH2

NH2+

N

CH

152

196m/z

C

N

C

NH

C

C

O

+NH3

NH

N

CH

CH2

CH2

OH

C

N

C

NH

C

C

O

N

N

CH

CH2

CH2

OH

C

N

C

NH

C

C

O

N

N

CH

CH2CH2

OH

C

N

C

NH

C

C

O

NH

N

CH

+

m/z 135

+

+

NH3CH2CH2O

CH2CH2O

C

N

C

NH2

C

C

O

NH2

N

N

CH

CH2

CH2

OH

m/z

+

196

C

N

C

NH2

C

C

O

NH2

N

N

CH

CH2

CH2

OH

C

NH2

C

C

O

N

N

CH

CH2

CH2

OH

+ NCNH2 CH2CH2O

C

NH2

C

C

O

NH

N

CH

+

+

m/z 110

C

N

C C

C

O

NH2

NH+

N

CH

CH2

CH2

OH

NH

m/z 196

CH2

CH2

OH+

m/z 45

Fragment Ions Derived from N7-HEG

by CID-MS/MS experiment

Precursor

Ion

Product

Ion


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4 fmol 16 fmol

128 fmol 1.02 pmol

Matrix effect consideredWithout matrix

LC-MS/MS

LC-MS

High Sensitivity and Specificity Provided

by LC-MS/MS for N7-HEG Detection


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128 fmol 1.02 pmol

Matrix effect consideredWithout matrix

LC-MS/MS

LC-MS

128 fmol 1.02 pmol

High Sensitivity and Specificity Provided

by LC-MS/MS for N7-HEG Detection


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Common Applications of

Tandem Mass Spectrometry

Examination ofion-molecule reactions

Molecular structural confirmation

Pharmacokinetic investigations

Neonatal screening for newborn diseases

Abused drug screening when LC-MS is used

Biological monitoring of toxicant exposure

Protein identification, de novosequencing,and post-translational modification analysis

in proteomics study


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Mass analyzer

scan

scancollision-induced

dissociation (CID)

select ion

772.913Full MS scan

Product ion scan (product of 772.9)

Peptide Fragmentation Pattern Obtained by

Product Ion Scan of Peptide Ion


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Time-of-Flight (TOF)-Based

Tandem Mass Spectrometers

Post source decay (PSD) analysis using a

reflectron

Quadrupole-time-of-flight (Q-TOF) tandemmass spectrometer

Tandem time-of-flight (TOF/TOF) massspectrometer

(F S i d k M S t t


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(From Suizdak, Mass Spectrometry

for Biotechnology, 1996)

x10

x10

High Resolution!


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Effect of high resolution onresolving isotopic peaks

High resolution/

resolved isotopic peaks:

Use monoisotopic mass

Low resolution/

unresolved isotopic peaks:

Use average mass


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Post Source Decay (PSD) Analysis

Using a Reflectron

PSD is a meta-stable decay process takes

place after ions have exited source region.

Observation of PSD ofMALDI ions was firstly

reported in 1992 (Spengler et al., J Phys Chem, 96,9678).

It was realized that MALDI ions are not as

soft as many had expected.

First commercial mass spectrometer that

performed PSD analysis was introduced ~

1994.


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(From Schematic Diagram of PE-SCIEX Voyager Elite MALDI-TOF mass spectrometer)


Using a Reflectron


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of a Charged Peptide Using a Reflectron


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Quadrupole-Time-of-Flight (Q-TOF)

Tandem Mass Spectrometer

Also referred as QqTOF, Q: first mass analyzer;

q: collision cell; TOF: second mass analyzer.

Parallel detection feature of TOF provides highsensitivity in full-scan mode.

Most Q-TOF MS have reflectrons that provide

high resolution and therefore high mass

accuracy.

Ideal for obtaining product ion scan data.


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First commercial Q-TOF mass spectrometer

was introduced ~ 1996.

Originally designed forESI source, Q-TOF MSare also used with MALDI source now.

Originally targeted forpeptide analysis, Q-TOF

MS are also applied to the analysis ofsmall

molecules such as pharmaceuticals andenvironmental toxicants.


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(From Chernushevich et al., JMS, 2001, 36, 849)

Schematic Diagram of QqTOF MS

Si l MS O ti i Q TOF MS


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Single MS Operation in QqTOF MSRF-only mode

MS/MS O ti i Q TOF MS


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MS/MS Operation in QqTOF MSRF-only mode

Precursor ion selection

Product ions recording


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can be Viewed as:

the replacement of the third quadrupole (Q3) in

a triple quadrupole by a TOF mass

spectrometer,

or

the addition of a mass-resolving quadrupole

and collision cell to an ESI-TOF.


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(From Hoffmann, Principle of Mass Spectrometry, 1998)

Principle ofOrthogonal ESI-TOF

ESI


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Important Considerations in

Q-TOF Operations

Orthogonalacceleration into TOF

Collisional cooling by RF-only quadrupoles

TOF chamber shielding

Pulsing rate of ion modulator and duty cycle

C lli i l C li i Q TOF MS


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Collisional Cooling in QqTOF MS

collisional cooling, reducing both

energy spread and beam diameter

RF-only mode TOF is more sensitive to

quality of incoming ion beam


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TOF chamber shielding in QqTOF MS

ground

voltage

Shielding is important for TOF

chamber to create a field-free

region floated at high potential


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Pulsing Rate of Ion Modulator in QqTOF MS

Pulsing too fast: overlap with slowest ion from

previous pulse

Pulsing too slow: low duty cycle

Typical pulsing rate is 5-10 kHz with 5-30% duty cycle


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Extraction Pulsing Rate, Duty Cycle, and Gating pulse

forDuty Cycle Enhancement @ a specific m/z region

Using gat ing p ulse, ~ 100% duty cyc le can be achieved @ a specif ic m /z region ,

At the expense of low du ty cyc les in o ther m/z regions


Can Q TOF be used for various MS/MS modes as provided


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scan(CID)select ion

Product ion scan

scan (CID) select ion

Precursor ion scan

scan(CID)scan

Neutral loss scan

select ionselect ion (CID)

Selected reaction monitoring

mpre+ mpro+ + mneuScan mpre

+ mpro+ mneu

Product ion scan Fix Vary Vary

Precursor ion scan Vary Fix Vary

Neutral loss scan Vary Vary Fix

Selected reaction monitorg Fix Fix Fix

N

Can Q-TOF be used for various MS/MS modes as provided

by triple quadrupole? (yes) How about efficiencies? (depends)

Efficiency

Q-TOF >> TSQ

Q-TOF ~ TSQ


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Gating pulse forDuty Cycle Enhancement @ a

specific product ion is useful in precursor ion scan

Using g at ing pu lse, ~ 100% duty cyc le can be achieved @ a specif ic prod uct ion



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LC-Q-TOF-MS with an ESI source

Liquid

Chromatography

Information-dependent LC-MS-MS of tryptic digest using a Q-TOF MS


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Information dependent LC MS MS of tryptic digest using a Q TOF MS


Information-dependent LC/MS/MS with precursor ion scan as a survey scan


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Information-dependent LC/MS/MS with precursor ion scan as a survey scan

(From Chernushevich et al.,

JMS, 2001, 36, 849)

7 fragments

with m/z

values 153.1,

155.1, 165.1,

181.1, 183.1,

191.1, and

193.1 were

selected for

multiple

precursor ionscanning


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Q-TOF-MS with an MALDI source

Ions forget their initial conditions in the

ionization region through collisional cooling

Larger laser influence can be used

to increase overall sensitivity


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Mass Calibration and Accuracy:

MALDI-Q-TOF vs . MALDI-TOF

In MALDI-Q-TOF, ion source is decoupled from

TOF using collisional cooling provided by

quadrupoles. As long as the points are not too

close together, a simple two-point calibration is

usually accurate over a wide mass range.

In MALDI-TOF, flight time can be affected by ion

source conditions such as surface distortion(sample morphology) and laser fluence, requiring

a multipoint calibration (when delayed extraction

is used) and internal standards for best mass

accuracy.


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Examples of Q-TOF MS Applications

Exact mass measurement of precursor and

product ions for structural confirmation and

identification of unknown compounds

Analysis of complex glycans


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Exact mass measurement of

precursor and product ions by Q-TOF MS

(From Miao et al., RCMS, 2003, 17, 149)

Exact mass measurement of precursor and product ions


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for structural confirmation and identification by Q-TOF

(From Clauwaert et al., RCMS, 2003, 17, 1443)



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for structural confirmation and identification by Q-TOF

(From Clauwaert et al., RCMS, 2003, 17, 1443)

A l i f l l b Q TOF MS


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Analysis of complex glycans by Q-TOF MS(From Harvey et al., RCMS, 2000, 14, 2135)


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Tandem time-of-flight (TOF/TOF)

mass spectrometer

First mass analyzer: TOF; second massanalyzer : TOF. TOF provides high sensitivityin full-scan mode.

Second TOF utilizes a reflectron that provideshigh resolution and therefore high massaccuracy.

TOF/TOF is currently used only for MALDIsource.

Ideal for obtaining product ion scan data oftryptic peptides forproteomics research.


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Diagram of a Tandem time-of-flight

(TOF/TOF)mass spectrometer


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Diagram of a Tandem time-of-flight

(TOF/TOF)mass spectrometer

Precursor ion

selection

FragmentationRecording of product ions


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(From Go et al., Anal Chem, 2003, 75, 2504)

An example of

product ion data of

tryptic peptidesgenerated by

tandem time-of-

flight (TOF/TOF)

mass spectrometry


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Time-of-Flight (TOF)-Based

Tandem Mass Spectrometers

Post source decay (PSD) analysis using a

reflectronless used now. Quadrupole-time-of-flight (Q-TOF)tandem

mass spectrometergaining popularity inproteomics, pharmaceutical, bioanalytical,

and environmental applications.

Tandem time-of-flight (TOF/TOF)massspectrometermay have a great potential inhigh-throughput proteomics.

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