(Introduction to Mass Spectrometry)

(Tandem Mass Spectrometry, MS/MS)

liaopc@mail.ncku.edu.tw

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

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 mass spectrometry experiments (tandem-in-time) placed one behind the other. Acronym: MS/MS; MS3; MSn

An Example of Tandem Mass Spectrometry Experiment: Product Ion Scan

analytes A, B, C, D A+ C+ I D+ B+ mass analyzer I m/z A2+ I A4+ A3
+

ionization source A+ , B+ , C+ , D+

precursor ion

A+

fragmentation product ions A1+ , A2+ , A3+ , A4+

A1+ A+ m/z

mass analyzer II

Activation (Dissociation, Fragmentation) Methods in MS/MS

Metastable decay
Collision-induced dissociation (CID), or collisionally activated dissociation (CAD). High energy vs. low energy CID. Surface induced dissociation, photodissociation, electron excitation.

Rationales for (Why) Performing Tandem Mass Spectrometry

To obtain chemical structure-related information. For example, differentiation of isobar compounds or isomers.
To increase selectivity and S/N ratio in quantitative measurement. For example, metabolite quatitation in complex biological matrices.

MS/MS by fragmentation enhances selectivity

isobar

MW: 278 Da for both

distinct fragments

Fragment Ions Derived from N7-HEG by CID-MS/MS experiment

O CH2 C NH
+

OH CH2 N

O C C C NH NH2
+

Precursor Ion

N C NH2 NH

C C

CH N CH2CH2O C NH2 N

CH

Product Ion

m/z
O

196
OH CH2 CH N NH3 O CH2 C C C NH N CH2 CH

m/z
CH2 OH

152

O CH2

C N
+

OH CH2 N

O C C NH CH

C C NH

NH

+C
N C NH C C

C NH3

+C

CH N CH2CH2O C NH

+C

m/z
O

196
O CH2 C N CH C NH2
+

m/z 135
CH2 OH CH2 OH CH2 CH C NH2 N NCNH2 NH2 C N O CH2 OH CH2 CH CH2CH2O NH2 O C C NH CH

C N C NH2 NH2 C C

+C
C

+C

m/z
O

196

m/z 110
OH CH2 CH

CH2 C N C NH2 NH C C NH
+

+ CH2
CH2

OH

m/z

196

m/z

45

High Sensitivity and Specificity Provided by LC-MS/MS for N7-HEG Detection

Without matrix
4 fmol

Matrix effect considered

16 fmol

LC-MS/MS

LC-MS

128 fmol

1.02 pmol

High Sensitivity and Specificity Provided by LC-MS/MS for N7-HEG Detection

Without matrix
128 fmol

Matrix effect considered

1.02 pmol

LC-MS/MS

LC-MS

128 fmol

1.02 pmol

Common Applications of Tandem Mass Spectrometry

Examination of ion-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 novo sequencing, and post-translational modification analysis in proteomics study

Peptide Fragmentation Pattern Obtained by Product Ion Scan of Peptide Ion

772.913

Full MS scan
Mass analyzer

scan

Product ion scan (product of 772.9)

select ion

collision-induced dissociation (CID)

scan

Time-of-Flight (TOF)-Based Tandem Mass Spectrometers

Post source decay (PSD) analysis using a reflectron Quadrupole-time-of-flight (Q-TOF) tandem mass spectrometer
Tandem time-of-flight (TOF/TOF) mass spectrometer

(From Suizdak, Mass Spectrometry for Biotechnology, 1996)

x10

High Resolution!
x10

Effect of high resolution on resolving isotopic peaks

High resolution/ resolved isotopic peaks: Use monoisotopic mass

Low resolution/ unresolved isotopic peaks: Use average mass

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 of MALDI 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.

Post Source Decay (PSD) Analysis Using a Reflectron

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

Post Source Decay (PSD) Analysis of a Charged Peptide Using a Reflectron

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 high sensitivity 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.

Quadrupole-Time-of-Flight (Q-TOF) Tandem Mass Spectrometer

First commercial Q-TOF mass spectrometer was introduced ~ 1996. Originally designed for ESI source, Q-TOF MS are also used with MALDI source now. Originally targeted for peptide analysis, Q-TOF MS are also applied to the analysis of small molecules such as pharmaceuticals and environmental toxicants.

Schematic Diagram of QqTOF MS

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

Single MS Operation in QqTOF MS

RF-only mode

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

MS/MS Operation in QqTOF MS

RF-only mode Product ions recording

Precursor ion selection

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

Quadrupole-Time-of-Flight (Q-TOF) Tandem Mass Spectrometer 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.

Principle of Orthogonal ESI-TOF

ESI

(From Hoffmann, Principle of Mass Spectrometry, 1998)

Important Considerations in Q-TOF Operations

Orthogonal acceleration into TOF

Collisional cooling by RF-only quadrupoles

TOF chamber shielding Pulsing rate of ion modulator and duty cycle

Collisional Cooling in QqTOF MS

RF-only mode TOF is more sensitive to quality of incoming ion beam

collisional cooling, reducing both energy spread and beam diameter

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

TOF chamber shielding in QqTOF MS

ground voltage

Shielding is important for TOF chamber to create a field-free region floated at high potential
(From Chernushevich et al., JMS, 2001, 36, 849)

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

Extraction Pulsing Rate, Duty Cycle, and Gating pulse for Duty Cycle Enhancement @ a specific m/z region

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

Using gating pulse, ~ 100% duty cycle can be achieved @ a specific m/z region, At the expense of low duty cycles in other m/z regions

Can Q-TOF be used for various MS/MS modes as provided by triple quadrupole? (yes) How about efficiencies? (depends)
Product ion scan

Neutral loss scan

select ion

(CID)

scan

scan

(CID)

scan

Precursor ion scan

Selected reaction monitoring

scan

(CID)

select ion

select ion

(CID)

select ion

mpre+
Scan
Product ion scan Precursor ion scan Neutral loss scan Selected reaction monitorg

mpro+ + mneu
mpro+
Vary Fix Vary Fix

Efficiency
mneu
Vary Q-TOF >> TSQ Vary Fix Fix

mpre+
Fix Vary Vary Fix

Q-TOF ~ TSQ

Gating pulse for Duty Cycle Enhancement @ a specific product ion is useful in precursor ion scan

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

Using gating pulse, ~ 100% duty cycle can be achieved @ a specific product ion

LC-Q-TOF-MS with an ESI source

Liquid Chromatography

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

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

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

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 ion scanning

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

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

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.

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

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 for structural confirmation and identification by Q-TOF

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

Exact mass measurement of precursor and product ions for structural confirmation and identification by Q-TOF

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

Analysis of complex glycans by Q-TOF MS

(From Harvey et al., RCMS, 2000, 14, 2135)

Tandem time-of-flight (TOF/TOF) mass spectrometer

First mass analyzer: TOF; second mass analyzer : TOF. TOF provides high sensitivity in full-scan mode. Second TOF utilizes a reflectron that provides high resolution and therefore high mass accuracy. TOF/TOF is currently used only for MALDI source. Ideal for obtaining product ion scan data of tryptic peptides for proteomics research.

Diagram of a Tandem time-of-flight (TOF/TOF) mass spectrometer

Diagram of a Tandem time-of-flight (TOF/TOF) mass spectrometer

Precursor ion selection

Fragmentation

Recording of product ions

An example of product ion data of tryptic peptides generated by tandem time-offlight (TOF/TOF) mass spectrometry

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

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 in proteomics, pharmaceutical, bioanalytical, and environmental applications. Tandem time-of-flight (TOF/TOF) mass spectrometermay have a great potential in high-throughput proteomics.