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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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