Why tandem mass spectrometry? by 5c4dsW9

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									台灣質譜學會93年度教育訓練課程
    -質譜分析技術


      93/4/9 (五)
 串聯質譜儀(Tandem-MS) (二) –
    TOF instrument

       廖寶琦
   成功大學環境醫學研究所
 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+
        ionization source                             C+
                    A+ , B+ , C+ , D+       I    D+
                                                                     B+
         mass analyzer I

                    A+                                         m/z
 precursor ion

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

                                                                                A+
        mass analyzer II

                                                               m/z
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, photo-
  dissociation, 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                                                                         O
                                                          OH
                                              CH2
                               C              +     CH2                                                  C                  +

Precursor             N             C
                                         NH
                                                                                                    N         C
                                                                                                                    NH2                       Product
                                                  CH                                                                            CH
   Ion                C             C
                                          N                      CH2CH2O                            C         C
                                                                                                                        N
                                                                                                                                                Ion
                NH2        NH                                                           NH2             NH

                          m/z      196                                                                  m/z   152

                               O
                                                          OH              O
                                             CH2                                                        OH                  O                                               O
                               C                    CH2                                     CH2                                                           OH
                                         NH                                                                                                  CH2
                                                                          C                       CH2
                      N             C                                                   N                               +C                          CH2                     C
                                                  CH              N              C                                                       N                                              NH
                                                                                                                    N                C                              N               C
                                                                                              CH
                      C             C                                                                                                          CH                                                CH
            +                            N                NH3    +C              C                                                                                              +C
                NH3        NH                                                           N                           C                C                    CH2CH2O   C
                                                                          NH                                                             N                                                  N
                                                                                                                         NH                                             NH
                          m/z      196
                                                                                                                                                                            m/z 135
                               O                                          O                                              O                                              O
                                                          OH                                            OH                                                OH
                                             CH2                                            CH2                                              CH2
                               C                    CH2                   C
                                                                                        N
                                                                                                  CH2               + C                  N
                                                                                                                                                   CH2                  C
                                         N                                                                                                                                              NH
                      N             C                             N              C                                               C                                              C
                                                  CH                                          CH                                               CH                                               CH
                      C             C                             C   +          C                       NCNH2                   C                        CH2CH2O           +C
                                         N                                              N                                                N                                              N
                NH2        NH2                             NH2            NH2                                           NH2                                         NH2
                           +
                          m/z      196                                                                                                                                      m/z 110

                               O
                                                          OH
                                             CH2
                               C             +      CH2
                                         NH
                      N             C
                                                  CH                            + CH2                    OH
                      C             C                                                         CH2
                                         N
                NH2        NH

                          m/z      196                                                      m/z    45
High Sensitivity and Specificity Provided
  by LC-MS/MS for N7-HEG Detection
           Without matrix       Matrix effect considered

                       4 fmol           16 fmol




LC-MS/MS



                     128 fmol          1.02 pmol
 LC-MS
High Sensitivity and Specificity Provided
  by LC-MS/MS for N7-HEG Detection
           Without matrix       Matrix effect considered

                     128 fmol          1.02 pmol



LC-MS/MS



                     128 fmol          1.02 pmol
 LC-MS
          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    scan
                                  dissociation (CID)
       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                              Single reaction monitoring




         scan         (CID)      select ion               select ion   (CID)     select ion


                                              N
                              mpre+               mpro+ + mneu                      Efficiency

Scan                             mpre+              mpro+                mneu
Product ion scan                 Fix                Vary                 Vary Q-TOF >> TSQ
Precursor ion scan               Vary               Fix                  Vary
Neutral loss scan                Vary               Vary                 Fix    Q-TOF ~ TSQ
Single reaction monitoring       Fix                Fix                  Fix
    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




                                Recording of product ions
Precursor ion   Fragmentation
selection
An example of
product ion data of
tryptic peptides
generated by
tandem time-of-
flight (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
  reflectron-less used now.
• Quadrupole-time-of-flight (Q-TOF) tandem
  mass spectrometer-gaining popularity in
  proteomics, pharmaceutical, bioanalytical,
  and environmental applications.
• Tandem time-of-flight (TOF/TOF) mass
  spectrometer-may have a great potential in
  high-throughput proteomics.

								
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