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Mass Spectrometry: An enabling technology for biomedical research New Applications for Mass Spectrometry Technology Genomics (Genotype) •Genetic disease markers (e.g. SNP’s) Proteomics (Phenotype) •Protein based disease markers ‘Metabolomics’ (Chemotype) • Metabolite based disease markers •The ultimate expression of a disease These 3 application areas represent new and exciting opportunities for mass spectrometry. The 3 areas are closely related to one another and to human health. Important Recent Developments in Biological Mass Spectrometry • API Ionization-MSMS (ITD or QqQ) – Electrospray – APCI • MALDI-TOF • Qq-TOF • FT-MS with MALDI and ESI Fastest Growing Applications • Biomedical – Proteomics – Genomics – Clinical “Metabolomics” (metabolic disorders, TDM) • Pharmaceutical – Preclinical pharmacology (Drug discovery) – Combinatorial chemistry (Drug discovery) – Clinical trials (Drug development) Atmospheric Pressure Ionization (API) A A A A+ MS Before API e.g. GC/MS Liquid Introduction Mass Spectrometry A A = Analyte; A = Solvent; A+ MS After API e.g. LC/MS = Vacuum system API Analytical Domains Ionic Analyte Polarity Ion Spray (Nebulizer Assisted Electrospray) Heated Nebulizer (APCI) GC/MS Neutral 101 102 103 Molecular Weight 104 105 IonSpray™ Ion Source IonSpray = nebulizer assisted electrospray (ESI) Heated Nebulizer (APCI) Gas Curtain Interface Tandem Mass Spectrometer Analyzers The API 2000 Triple Quadrupole MSMS MSMS Ion Optics Ion source Interface Analyzer Ion creation Ion Ion Ion Ion transfer focussing selection fragmentation and desolvation • New Patented LINAC Collision Cell • New High Efficiency Vacuum System • Compact Size • Pulse Counting Detector Product ion Ion selection detection Mass Analysis Phe - Molecular Weight= 165.08 9 1 11 2 of C (12.000) of N (14.003) of H (1.0080) of O (15.995) O H2N CH C CH2 OH MSMS Ion Optics Ion source Interface Analyzer Ion creation Ion Ion Ion Ion transfer focussing selection fragmentation and desolvation • New Patented LINAC Collision Cell • New High Efficiency Vacuum System • Compact Size • Pulse Counting Detector Product ion Ion selection detection Quadrupole Mass Filter Butyl-Ester of Phe (MW= 221.14) Acquired by NL 102 Exp’t O CH C OC4H9 100 222.1 2H -Phe 0 CH 2 2H 13C -Phe 0 1 % Intensity H2N 2H -Phe 5 227.1 220 230 m/z, amu MSMS Ion Optics Ion source Interface Analyzer Ion creation Ion Ion Ion Ion transfer focussing selection fragmentation and desolvation • New Patented LINAC Collision Cell • New High Efficiency Vacuum System • Compact Size • Pulse Counting Detector Product ion Ion selection detection MSMS of Tryptophan Bu Ester Scan Types Ion Path • Q0 and LINAC Patented for use at high pressures • Detector floated several kV • Full Autotune (calibration, resolution and optimization) Ion Path - Linac • Q2 rods are tilted and separate DC potentials are applied to each pair of rods to create an axial electric field • Q2 Linac (linear accelerator) eliminates cross-talk and allows faster MS/MS scanning without sensitivity losses • Linac collision cell used at high pressures demonstrates 100% efficiencies in both product ion formation and transmission Ion Path - Linac Entrance y x x > 2Ro = y Exit y x V1 V2 • Terminating geometries of the Linac collision cell LC/MS/MS Scan Modes Triple Quadrupole Analyzer • • • • Precursor Scan (PS) Product Ion Scan (PI) Multiple Reaction Mode (MRM) Neutral Loss Scan (NL) Product Ion Scan (PI) Precursor Ions Q1 Product Ions * Q2* * * * * * Q3 * ** ** * * * * * * * * CAD GAS DETECTOR (CEM) Product Ion Scans Illustrated MS PQZ MS/MS FGC FG FGC HJC CBA ABC WXZ While Q1 scans, Q3 detects “C” ions GC G DEC KLC F C MOLECULAR MASS MOLECULAR MASS 123 1, 2, 3, 1-2, 2-3 e.g. Collisional Fragmentation F, G, C, FG, GC FGC Collisional Fragmentation MSMS of Tryptophan Bu Ester Multiple Reaction Monitoring (MRM) CAD Precursor Ion Q1 Q1 scan for a fixed mass however more than one molecule may have the same mass * Q2* * * ** * * ** ** * * * *** * * CAD GAS Q2 Collision Product Ion Q3 DETECTOR (CEM) Q3 Scan for another fixed mass as a component of the desired molecule Homocysteine by LC-MSMS NH2 HOOC SH Homocysteine Exact Mass: 135.04 Mol. Wt.: 135.18 Cardiovascular Risk Factor: mechanism currently unknown, however, believed will become as important to cardiovascular health and wellness as cholesterol Acknowledgements: Dr. Piero Rinaldo, et. al. from Clinical Chemistry, 45(1999)1517 Homocysteine: MSMS Product Ion Spectrum [M+H-HCO2H]+ [M+H]+ Homocysteine in Plasma: LC-MSMS Response (15 mM) 140+ 94+ 136+ 90+ Homocysteine in Plasma: LC-MSMS Response (0.8 mM) Homocysteine Method Comparison LC-MSMS vs. IMx 40.00 y = 0.9797x + 0.2047 LC-MSMS Conc. (mM) 35.00 2 R = 0.9767 30.00 25.00 20.00 15.00 10.00 5.00 0.00 0 5 10 15 20 IMX Conc. (µM) 25 30 35 40 Homocysteine: HPLC vs. LC-MSMS • Equipment costs are $0.14 greater per assay for LC-MSMS vs. HPLC, however… • 1 LC-MSMS replaces 5 HPLC’s • Supply costs are 35% less for LC-MSMS • Space requirements 80% less for LCMSMS • Personnel costs 29% less for LC-MSMS • Turnaround time 81% less for LC-MSMS MSMS experiments for the determination of Amino acid and Acylcarnitine “Panels” Amino acids Acylcarnitines Neutral loss of 102 & 119 Da Product Ion Scan of 85 Da O R RC-O O NH2-CH-COC4H9 O O + (CH3)3N-CH2-CH-CH2-COC4H9 (CD3 ) H-COC4H9 O NL 102 Da + NH2 = CH-R + CH2-CH = CH-COH PS of 85 NL 102, PS of 85 and MRM Experiments Run Concurrently Covering Over 50 Analyses (360 channels of information) Precursor Ion Scan (PS) OR CH 3 CH 3 N CH 3 CH 2 CH CH 2 C CAD + H 2C O Acylcarnitines Q1 OC 4H 9 * CHCHCO2H m/z 85 * * * * * * * * * * * * * * * *CAD * GAS Q2 Q3 Detector (CEM) Control Subject Profile (MSMS PS 85) Acylcarnitine Profile from a Normal Control Blood Spot 100 C2 C2 INTERNAL STANDARDS % Intensity C16 C8 50 C18:1 C4 C3 C16 C3 C4DC C5OH 270 300 330 C14:1 360 390 420 m/z, amu Acknowledgement: Dr. Don Chace 450 480 510 540 Medium Chain Acyl CoA Dehydrogenase Deficiency (MCAD) (MS/MS PS 85) Acylcarnitine Profile from a Filter Paper Blood Spot: MCAD 100 1.16 - 25.8 µM vs. 0.22 µM C8 % Intensity INTERNAL STANDARDS 50 C16 C2 C8 C10:1 C2 C4 C6 C10 C16 C18:1 C3 270 300 330 360 390 420 450 Acknowledgement: Dr. Don Chace 480 510 540 Amino acidopathies: Phenylketonuria (PKU) O O H 2N CH C OH CH2 H 2N CH C OH CH2 CO2, H2O Phenylalanine Phenylalanine Hydroxylase OH Tyrosine Neutral Loss Scans (NL) H R O H + H CAD N C H H C OC 4 H N C 9 H H -HCO 2C 4 H 9 Amino acids (Bu esters) Q1 R + Butyl formate (mass 102) * * Q2 * * * * * * * ** * * * * * * * * CAD GAS Q3 DETECTOR (CEM) Control Subject Profile (MSMS NL 102) Amino Acid Profile from a Normal Control Blood Spot 100 Pro INTERNAL STANDARDS % Intensity Ala Phe 50 Leu + Ile Phe Leu Gln Val Ala Gly Gly 140 Ser Val 160 180 Tyr Tyr Cit Met 200 220 m/z, amu Glu Asp Glu 240 260 Acknowledgement: Dr. Don Chace 280 300 Phenylketonuria (PKU) (MS/MS NL 102) Amino Acid Profile from a Filter Paper Blood Spot: PKU Phe 100 Pro % Intensity INTERNAL STANDARDS Ala 50 Phe Leu + Ile Gln Val Ala Gly Gly 140 Ser 160 Val 180 Leu Cit Met 200 220 m/z, amu Tyr Glu Asp Glu Tyr 240 Acknowledgement: Dr. Don Chace 260 280 300 Quadrupole Ion Trap Schematic Representation of Ion Trap Operation (a) Schematic representation of working points (that is, coordinates in az, qz space) in the stability diagram for several species of ions stored concurrently. The arrangement of working points with respect to mass/charge ratio is depicted by figures which differ in size. (b) Ions are shown residing near the bottom of their respective axial potential wells of depth Dz; the ladder represents the opportunity for resonant ejection of an ion species. Schematic representation of a quadrupole mass filter and an ion trap, where f o is the potential applied to opposite pairs of rods or and caps az = ax = -ay = 4zU / mw 2ro2 qz = qx = -qy = 2zV / mw 2ro2 Tandem Mass Spectrometry: Quadrupole Ion Trap Tandem-in-Time Eject > M Daughter Ion Scanning Eject < M CAD rf Injection Ionization 0 Precursor Ion Isolation Collisionally Activated Dissociation Product Ion Scanning Time (ms) MSn • since product ions are formed in the same device in which they are generated, it is possible to perform multiple stages of MS (MSn) • genealogical elucidation information facilitates structure • for protein/peptide sequencing, low mass B and Y ions can be generated, thereby negating any disadvantages due to a low mass cut-off of 1/4*parent mass Introduction • Apigenin (m/z 271) is a base component of flavonoids • Apigenin has a characteristic fragmentation spectrum • MSn of suspected flavonoids can yield the base component ion m/z 271 • Subsequent fragmentation of the m/z 271 ion can verify the compound is a flavonoid ESI-MS MH+ 100 271.2 OH 95 O 90 85 80 75 OH O Relative Abundance 70 65 OH 60 Apigenin 55 50 MW 270 45 40 35 30 25 20 15 10 5 186.7 210.7 237.0 0 100 150 200 250 274.0 300 358.4 380.3 350 m/z 422.3 400 479.1 450 524.0 548.5 500 550 600 2 MS 153.1 100 MS/MS of Apigenin 95 90 85 80 75 Relative Abundance 70 65 60 55 50 45 40 35 30 243.3 25 225.3 20 15 145.1 10 5 79.8 90.9 107.3 118.8 121.2 144.1 0 80 100 120 140 271.2 203.2 229.1 163.2 173.0 211.6 197.1 219.4 185.1 160 180 m/z 200 220 253.2 240 260 279.8 280 297.0 300 ESI-MS 579.1 Suspected Flavonoid 100 95 90 85 80 75 Relative Abundance 70 65 60 601.1 55 50 45 40 271.3 35 30 25 602.2 20 15 518.5 10 5 0 200 223.1 263.1 285.3 309.4 250 300 345.1 365.2 350 393.3 419.5 433.2 400 463.4 486.0 516.9 534.8 450 m/z 500 617.5 623.1 645.1 551.5 550 600 650 MS/MS of m/z 579 271.2 100 95 Unknown 90 85 O OH 80 CH 75 3 Relative Abundance 70 H 65 OH 60 OH 55 50 45 40 35 30 432.9 25 20 (-147) 15 10 5 0 161.6 203.2 225.2 200 270.3 272.2 250 313.2 300 337.1 417.0 366.8 381.0 350 400 m/z 436.5 450 475.0 497.0 518.3 536.5 500 561.7 550 579.3 584.2 600 MS/MS/MS 271.2 100 579>433>products 95 90 (-147) 85 (-162) 80 O 75 Relative Abundance 70 O O OH CH 3 65 OH 60 H 55 OH Unknown CH 2 H OH OH OH 50 45 40 35 30 25 20 433.0 15 415.1 10 5 215.2 228.9 270.4 271.8 0 150 200 250 313.3 300 367.1 337.1 397.1 517.2 542.5 448.6 350 m/z 400 450 500 550 600 MS4 153.1 100 95 579>433>271>products 90 85 80 75 Relative Abundance 70 65 60 55 50 45 40 35 30 243.3 25 225.3 20 15 145.1 10 5 79.8 90.9 107.3 118.8 121.2 144.1 0 80 100 120 140 271.2 203.2 229.1 163.2 173.0 211.6 197.1 219.4 185.1 160 180 m/z 200 220 253.2 240 260 279.8 280 297.0 300 MS/MS Apigenin 153.1 100 95 O OH 90 85 Relative Abundance 80 75 70 65 OH 60 O 55 50 45 OH 40 35 243.3 30 25 225.3 20 145.1 15 10 5 107.3 79.8 90.9 0 80 121.2 102.4 100 163.2 118.9 120 173.0 144.1 140 160 203.1 197.1 176.8 185.1 271.2 229.1 211.6 253.2 279.8 219.4 180 200 220 240 260 m/z 153.1 280 297.0 300 O OH 100 MS4 m/z 579 95 90 85 Relative Abundance 80 75 O 70 65 CH 2 CH 3 60 H 50 OH O OH 55 45 O O O OH OH H OH OH OH 40 35 243.3 30 25 225.3 20 145.1 15 10 163.2 5 107.3 79.8 90.9 118.9 121.2 102.4 144.1 0 80 100 120 140 160 176.8 185.1 180 197.1 211.6 253.2 279.8 219.4 200 m/z 271.2 229.1 203.1 173.0 220 240 260 280 297.0 300 Multiply Charged Ions for Biopolymer Analysis IonSpray-MS of Diluted Blood • Full scan mass spectrum of 10 µL blood diluted 500 fold 18+ 841.3 100 90 19+ 797.1 17+ 890.8 80 Ions chosen for 'SIM' 70 16+ 946.4 % Intensity 60 ß17+ 934.4 50 40 30 ß20+ ß19+ 836.1 ß16+ 992.7 ß18+ 882.5 ß15+ 15+ 1009.4 1058.8 14+ 1081.4 794.4 20 ß14+ 1134.3 13+ 1164.6 ß13+ 1221.5 10 800 900 1000 m/z, amu 1100 1200 12+ 1261.5 Hemoglobin MW Spectrum • Data transformed from m/z to Mr (Da) 100 -hemoglobin 15126.4 ß-hemoglobin 90 15867.1 80 %GHb = 50 {[ g/( +g) + [ßg/(ß+ßg )]} 70 = 50{[8.7/(100+8.7) + [8.7/(82.1+8.7)]} % Intensity 60 = 8.8% 50 40 30 20 10 Glycated- Glycated- 16029.2 15288.5 15501.7 15000 15500 16000 Mr (Da) 16500 Hemoglobin Variants • e.g. sickle cell hemoglobin shows base-line resolution -hemoglobin 15126.4 90 80 70 % Intensity 60 50 40 ß-hemoglobin SC-ß-hemoglobin 15867.1 15836.1 30 20 10 Glycated-SC- 15998.0 Glycated- 16029.2 Glycated- 15288.5 15200 15400 15600 15800 16000 Mass, amu 16200 16400 16600 Protein/Peptide Molecular Weights by Charge State “Deconvolution” Protein/Peptide Ubiquitin (bovine) Cytochrome C (bovine) Lysozyme (chicken egg) Hemoglobin-alpha chain (bovine) Hemoglobin-beta chain (bovine) Apomyoglobin (equine) B-lactoglobulin A (bovine milk) Carbonic Anhydrase (bovine erythrocytes) Bovine Serum Albumin Theoretical Experimental %Mass Avg. MW Avg. MW Accuracy 8564.9 12230.9 14306.2 15053.2 15953.3 16951.5 18363.3 29024.6 66430.3 8565.0 12231.5 14305.0 15053.7 15954.1 16951.1 18364.5 29025.2 66432.3 0.001 0.005 0.008 0.003 0.005 0.002 0.006 0.002 0.003 Average % Mass Accuracy = 0.004% Schematic of MALDI-TOF Mirror Laser Sample Tray Reflectron Flight Tube Detector Sample Preparation for MALDI-TOF Analysis MALDI-TOF Apparatus v = (2zVacc / m)1/2; t = (m / 2zVacc)1/2L; t = a (m/z)1/2 + b TOF Principles TOF MS Advantages: Drawbacks: Parallel detection of all ions Originally, low mass resolution Virtually unlimited m/z range Requires complicated electronics for spectra recording: time-to-digital converter (TDC) or transient recorder No slits or apertures, rods or magnets High mass accuracy Limited dynamic range (with TDC) Typical TOF parameters: Length ........................................................ Accelerating voltage ................................. Drift time ..................................................... Mass resolution (FWHM) .......................... 20 cm to 5 m 1 to 30 kV 5 to 200 µs up to 35,000 (Bergmann et al., 1989) Mass range ................................................. up to ~1 Megadalton Factors limiting resolution R= m/Dm=t/2Dt Dt Reflecting TOF, or “Reflectron” MALDI-TOF of Protein Digest: Adenylate Kinase 17 peptides MALDI-TOF of Protein Digest: matching peptides and mass tolerance MALDI-TOF of Protein Digest: matching peptides and mass tolerance MALDI-TOF of Protein Digest: search results MALDI-TOF of Protein Digest: sequence coverage Short Video MALDI-TOF of Oligonucleotides for SNP’s Analysis Electrostatic Ion Mirror ESI-TOF University of Manitoba Standing et al., 1993-95 Field-free drift region z y x Conducting Sheath Object plane Deflection plates Beam optics Detector Electrospray Source Modulator ~ 10 -7 Torr D Quadrupole rods ~10 -5 Torr ~10 -2 Torr Vacuum Pumps ~2.5 Torr ESI-TOF: The advantages of higher resolution ESI-TOF of Small Molecules: The advantages of higher resolution ESI-TOF of Peptides: Determination of charge state ESI-TOF of Peptides: Determination of charge state ESI-TOF of Peptides: Determination of molecular weight ESI-TOF of Peptides&Proteins: Molecular weight ranges No mass discrimination ESI-TOF of Proteins: Non-covalent Complexs clusters of catalase HP II M.W. up to 1.38 MDa The QSTAR™ Pulsar Hybrid LC/MS/MS System • Hybrid MS /MS (Quadrapole / TOF) • Most Accurate Hybrid MS/MS System • Accurate molecular weight and sequence information which can be used for data base searching • De Novo Peptide Sequencing • High Sensitivity Post Translational Modification Analyses • Interface with a choice of: LC or cap.LCNanospray MALDI plate- QSTAR: Hybrid Quadrupole TOF 770 L/s 250 L/s Modulator Focusing grid Accelerator column 4 anode detector Sample Ions q0 Q1 q2 10 mTorr 2.5 Torr Curtain Gas Conducting liner 770 L/s 10-2 Torr Effective Flight Path = 2.5 m Field Free Drift region 7x10-7 Torr Ion Mirror (reflector) QSTAR: Mass Resolution Peptide Sequencing by MS/MS N2 CAD Gas Q0 Q1 MS - Peptide Mass Fingerprint Q2 Q3 MS/MS - Peptide tag QSTAR: MS/MS Sensitivity QSTAR De Novo Peptide Sequencing with 18O labeling Shevchenko et al., 1997 De Novo Peptide Sequencing: two peptides with same m/z Peptide Sequencing: K vs Q (∆m=0.036 Da), and F vs Mox (∆m=0.033 Da) MS/MS on Large Ions: MW 4587.33 Da, charge 6+ (sample received from BioVisioN, Hannover, Germany) Product Ions of Metabolite 565 (m/z 565) 565 234 and 235 HO NH N O O O O Cl Cl N N O MH+ 565 Metabolite 565 (m/z) HO NH N O O O N N O Cl O MH+ 565 Cl HO NH -102 N H O + O O H+ O N N O Cl N O Cl N O m/z 463 m/z 234 463.1130 Accurate 463.1217 Observed (5.6 ppm) 234.1130 Accurate 234.1164 Observed (14.5 ppm) O O O N H+ m/z 235 235.1447 Accurate 235.1464 Observed (7.2 ppm) m/z 234 and 235 at 8,000 resolution m/z 234.1164 (14.5 ppm) m/z 235.1464 (7.2 ppm) BSA: isotopically resolved 47+ charge state HiResESI @ 7.0 T HighResMALDI: DNA 20-mer R50% > 70.000 HiResMALDI 4.7 T: Equine Myoglobin HiResMALDI 4.7 T: Cytochrome C R50% > 80.000 @ 4.7 T Quick Tuning of Instrumental Parameters e.g. for MS/MS Wizard to select the basic experiment Tuning of the ‚Arbitrary Waveform Generator‘ to select only a single ion for MS/MS You will have simple access to all parameters. ESI spectrum of Cytochrome C with one isotopic peak isolated from the +13 charge state of the molecular ion (ULTIMA 7 T) LINAC Collision cell Duty Cycle = 25% for the heaviest ions Duty Cycle < 5% for light ions TOF -4kV Q2 IQ3 Accelerator IQ2 Slit Delay V Trapping Extraction region 4-anode detector Releasing Duty Cycle >90% for a pre-selected ion QSTAR Pulsar: Q2 Pulsing Q2, collision cell IQ3/IRP IRD IRW Ions are Stored in Q2, then Pulsed into the TOF QSTAR Pulsar: Pulsing ON • TOF Duty Cycle increase: »from 5% or less (for low mass ions) to 100% for the ion of interest • Sensitivity Increase: »10-20x at low mass »3-5x at higher mass (>500 amu) Can Pulse MS/MS product ions as well as Precursor Ions MS/MS Pulsing: for post-translational modification (PTM) identification and for enhanced sensitivity of product ions Peptide Immonium Ions: Nanospray infusion of tryptic digest m/z 86 (Ile/Leu) m/z 175 (Arg) m/z 147 (Lys) m/z 120 (Phe) Useful for finding peptides for MS/MS in complex, dirty samples (e.g. in-gel digests) Fragment ions of m/z=829 (ALILTLVS) normal spectrum.... 10000 5000 0 100 200 300 400 500 300 400 500 ....and with trapping in Q2 m/z=86 Gain ~ 17 100k 0k 100 200 m/z Tryptic digest of myoglobin Precursor ion scan for m/z=86; with no trapping... 20 0 600 700 800 1000 … and with trapping in Q2 Gain ~ 15 500 0 900 600 700 800 m/z 900 1000 Mixture of lipids: TOF MS vs Precursor Ion scan Precursor ion scan: parallel detection of two classes of lipids: phosphatidylcholines and ceramides Precursor ion scanning for Phosphorylated Peptides 100 fmol of b-casein tryptic digest – QSTAR Nanospray *NEGATIVE ION* Full TOF MS Spectrum Specific phosphopeptide scan Phosphopeptide Pulsar on x13 gain LC-MS/MS Applications with Precursor Ion Scans Information Dependent Acquisition for LC-MS/MS IDA Principal: Survey Scan Filter/Identify Ion(s) of Interest ? Not necessarily most intense ion of interest Identify Ion(s) MSMS Ion of Interest Dynamic process repeated throughout the LC analysis Protein Identification Using Precursor Ion Scans and Information Dependent Acquisition (IDA) 0.2 pM/mL solution of BSA tryptic digest LC: Column type Aquapore Brownlee (AB) 7 micron size 1 x 100 mm; 60mL/min; 0.5% Formic acid ACN/H2O 10 mL injection PE 200 Autosampler MS: Precursor Ion scan: Shimadzu LC-10AVP m/z from 450 to 950 in 2s with step 1, collision energy 75 eV Two Dependent Product Ion scans: 1.5s acquisition, collision energy 45/30 eV Quad resolution: Low (2-3 amu) IDA of a BSA Tryptic Digest LC: C18 column (1x100mm, 7m); 10mL of 0.2pM/mL injected IDA: 2s precursor scan followed by 2x1.5s product ion scans T L E N V Product Ion Scans from IDA of BSA Tryptic Digest (1.5s acquisition time for each) E D Y V L F A F L S L L G L V O H Monoacetylmorphine Metabolism O O Deacetylation O O N O N O O O Diacetylmorphine Monoacetylmorphine MWt =327.38 Formula =C19H21NO4 MW =369.42 Formula =C21H23NO5 Deacetylation O OH O OH O H O O O Glucuronidation O-methylation OH O O O N N N H O H O H O Morphine Codeine MW=285.35 Formula =C17H19NO3 MW =299.37 Formula =C18H21NO3 Morphine 3-glucuronide MW =461.47 Formula =C23H27NO9 Glucuronidation Glucuronidation O-methylation H O O H O O O O OH O OH O N O O O OH N O O H O OH OH Morphine 6-glucuronide MW =461.47 Formula =C23H27NO9 Normorphine MW =271.32 Formula =C16H17NO3 N O OH Codeine glucuronide MW =475.50 Formula =C24H29NO9 Fragmentation Pattern of MAM Metabolites Common fragments selected for precursor ion scanning: 153.1, 155.1, 165.1, 181.1, 183.1, 191.1 and 193.1 (M+H)+ (M+H)+ (M+H+ Identification of Monoacetylmorphine (MAM) Metabolites Using Precursor Ion Scans and Information Dependent Acquisition (IDA) Urine sample from subject exposed to Monoacetylmorphine LC: Column type C18(2) Luna Phenomenex 3 micron size 2 x 100 mm; 200mL/min; 0.5% Formic acid ACN/H2O 10 mL injection PE 200 Autosampler MS: Precursor Ion scan: Product Ion scan: Quad resolution: Shimadzu LC-10AVP m/z from 200 to 500 in 2s, step 1 amu, collision energy 45 eV 1s acquisition, collision energy 35 eV Low (2-3 amu) Monoacetylmorphine Metabolites in Urine: IDA with Multiple Precursor Ion Scan Morphine (286) Codeine (300) Morphine Gluc. (462) Codeine Gluc. (476) Monoacetylmorphine (328) 286.0 Monoacetylmorphine Metabolites in Urine: IDA with TOF MS as a Survey scan m/z=278 m/z=265 Morphine Gluc. (462) Morphine (286); Codeine Gluc. (476) Monoacetylmorphine (328) Codeine ??? (300) MALDI QqTOF Orthogonal MALDI with Collisional Cooling Advantages 1. Ion source decoupled from analyzer stable calibration diversity of target materials laser fluence, pulse width not critical no “ghost” peaks of metastable ions 2. Collisional cooling of ions reduced fragmentation 3. Quasi-continuous ion beam higher repetition rate less peak saturation possibility to use TDC 4. Compatible with tandem MS control of degree of fragmentation same two-point calibration and mass accuracy in MS/MS mode Drawbacks 1. Lower sensitivity: reduced duty cycle losses at TOF entrance 2. Discrimination against heavy ions in quadrupoles and at the detector MALDI QqTOF of a peptide mixture Single MS mode Substance P Resolution ~ 10 000 Accuracy < 10 ppm Melittin 20000 Fragment of CD4 Insulin 10000 0 1000 2000 File: 007 mixture, Date: 12/5/1998 9:53 Resolution: 1.2 ns, Display bin: 65.0 ns, Starts: 1 3000 m/z 4000 5000 6000 [Courtesy of Alexander Loboda, Werner Ens and Ken Standing, University of Manitoba] Mass spectrum of substance P obtained from 70 amol sample deposited on the probe; DHB matrix; 1200 laser shots in 60 s [Courtesy of Alexander Loboda, Werner Ens and Ken Standing, University of Manitoba] Tryptic mass map of citrate synthase: single MS and MS/MS of tryptic fragment 11 [Courtesy of Alexander Loboda, Werner Ens and Ken Standing, University of Manitoba] Protein Analysis Scheme Digested Sample MALDI MS Protein Database search Peptide List de novo sequencing Match ? Off-line analysis On-line analysis Tag search Tag search (EST, PTM) (EST, PTM) de novo sequencing Yes Remove identified or predicted peptides Daughter Lists Yes Match ? No MALDI MS/MS ESI MS/MS on selected on max possible Peaks peaks TMS Analyzer Attributes MS high res. NL* PS* MRM* Product ion Cost Ion collection efficiency Duty cycle Ion Trap Yes No No No Yes Low 5% Low* Triple Quads No Yes Yes Yes Yes Medium 90+% High QSTAR (QqTOF) Yes Yes Yes No Yes High 50% High FT-ICR Yes No No No Very High Low Low *Commonly used scan modes for NBS Yes Acknowledgements • • • • • • • • • • Patricia Iliusu (PE Biosystems) Igor Chernushevich (MDS SCIEX) Bruce Thomson (MDS SCIEX) Ron Bonner (MDS SCIEX) Lorne Taylor (Ocada) Brian Musselman (Perceptive Biosystems) Larry Haf (Perceptive Biosystems) Wade Hines (Perceptive Biosystems) Don Chace (NeoGen Screening) Werner Sievers (GSG Instruments)