1. Mass spec lecture

2. What kind of info can mass spec give you? Molecular weight Elemental composition (low MW with high resolution instrument) Structural info (hard ionization or CID)

3. How does it work? Gas-phase ions are separated according to mass/charge ratio and sequentially detected

4. Parts of a Mass Spec Sample introduction Source (ion formation) ________________________ Mass analyzer (ion sep.)high vac Detector (electron multiplier tube)

5. Sample Introduction/Sources Volatiles Probe/electron impact (EI),Chemical ionization (CI) GC/EI,CI Involatiles Direct infusion/electrospray (ESI) HPLC/ESI Matrix Assisted Laser Adsorption (MALDI) Elemental mass spec Inductively coupled plasma (ICP)

6. EI, CI EI (hard ionization) –Gas-phase molecules enter source through heated probe or GC column –70 eV electrons bombard molecules forming M+* ions that fragment in unique reproducible way to form a collection of fragment ions –EI spectra can be matched to library stds CI (soft ionization) –Higher pressure of methane leaked into the source (mtorr) –Reagent ions transfer proton to analyte

8. CI/ ion-molecule reaction 2CH 4 + e-  CH 5 + and C 2 H 5 + CH 5 + + M  MH + + CH 4 The excess energy in MH + is the difference in proton affinities between methane and M, usually not enough to give extensive fragmentation

9. Electrospray 5 kV voltage on a needle Nebulization gas Produces gas-phase protonated analytes Little to no fragmentation Multiple charging 10  M angiotensin at 5  l/min direct infusion, MW 1269

10. ] 200400600800100012001400160018002000 m/z 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 433.3 649.2 1296.6 671.1 682.1 647.5 1340.6 784.4 534.4 371.1 1362.5 1028.4 214.0 1127.3 1440.1810.81743.0928.61525.61820.41989.01607.1 1160.0

11. MALDI Matrix -UV absorber, ex. picolinic acid, cinnimic acid Singlely charged ions Need mass analyzer with a large m/z range – TOF Laser pulse as opposed to continuous source

13. Mass Analyzers Low resolution –Quadrupole –Ion trap High resolution –TOF time of flight –Sector instruments (magnet) Ultra high resolution –ICR ion cyclotron resonance

14. Resolution R = m/z/  m/z Unit resolution for quad and trap TOF up to 15000 FT-ICR over 30000 –MALDI, Resolve 13 C isotope for a protein that weighs 30000 –Resolve charge states 29 and 30 for a protein that weighs 30000

15. High vs low Res ESI Q-TOF, ICR –complete separation of the isotope peaks of a +3 charge state peptide –Ion abundances are predictable –Interferences can be recognized and sometimes eliminated Ion trap, Quad –Unit resolution

16. MVVTLIHPIAMDDGLR

17. Exact Mass Determination Need Mass Spectrometer with a high mass accuracy – 5 ppm (sector or TOF) C 9 H 15 NO 4, FM 201.1001 (mono-isotopic) Mass accuracy = {(Mass Error)/FM}*10 6 Mass Error = (5 ppm)(201.1001)/10 6 =  0.0010 amu

18. Mass accuracy Mass Error = (5 ppm)(201.1001)/10 6 =  0.0010 amu 201.0991 to 201.1011 (only 1 possibility) Sector instruments, TOF mass analyzers How many possibilities with MA = 50 ppm? with 100 ppm?

19. Quadrupole Mass Ion Filter

20. Ion Trap

21. Time of Flight -TOF

22. Where: m i = mass of analyte ion z i = charge on analyte ion E = extraction field t i = time-of-flight of ion l s = length of the source l d = length of the field-free drift region e = electronic charge (1.6022x10-19 C)

23. TOF with reflectron http://www.rmjordan.com/tt1.html

24. Sector instruments http://www.chem.harvard.edu/mass/tutorials/magnetmovie.html

25. FT-ICRMS http://www.colorado.edu/chemistry/chem5 181/MS_FT-ICR_Huffman_Abraham.pdf

26. CID or MS-MS –MS-MS sequencing the peptides or oligonucleotides structural characterization of drugs and metabolites Assay development, sensitivity enhancement

27. HPLC-MS-MS time response chromatogram m/z

28. hybrids Ex.Q-TOF –Trap has excellent sensitivity (can store essentially all ions), mass selectivity (can store ions of a particular m/z ratio –TOF is a high resolution mass analyzer Triple quadruple –Neutral loss scan

29. HPLC-MS Reverse-phase HPLC –Separation of involatiles (peptides) –The lower the flow the greater the sensitivity –Column ID (300  m – 50  m)

30. Proteomics using LC-MS Protein identification, characterization, quantification Extract proteins, fractionate proteins (typically using 2D-gel electrophoresis) Digest protein(s) with a protease to produce peptide mixture (lysine, arginine) LC-MS-MS analysis Database searching identifies proteins –Mascot, Expasy (tools)

31. 051015202530354045 Time (min) 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 Relative Abundance 11.36 17.23 12.57 12.74 17.68 36.21 1.21 15.13 24.95 24.53 22.46 2.54 3.01 21.735.43 6.14 25.20 20.41 48.55 27.31 37.18 29.53 32.43 40.11 45.43 RP-HPLC Separation of a Tryptic Digest of BSA Peptide used in following illustration

32. 784.6 785.4 1568.6 795.6 1567.5 1121.4 812.5 1122.5 830.4 1280.4 1234.4 1064.41570.6 852.5 691.5 1365.5 997.4 1180.3 391.0428.9 1477.0 591.3 1706.0 1768.7 Mass Spectrum of a Tryptic Peptide from BSA (M+2H) +2 (M+H) +

34. L S Y E Y L F S G D A F L G S F L Y E Y S R MS-MS Spectrum D A F L G S + m/z 590 Residue mass S87.0 Y 163.1 E 128.1 L 113.1 F 147.1 G 57.0

36. LC-MS-MS analysis Characterization of synthetic processes Drug metabolism studies – structural elucidation of metabolites Quantification of polar molecules in biological samples – NAD

37. NAD Assay Goal to determine the relative importance of the different biosynthetic pathways. Stable isotopic incorporation

38. NAD Synthesized from Labeled Precursors NAD synthesized from a) 2 H 4 labeled nicotinic acid and nicotinamide (m/z 667) b) 13 C 5 labeled tryptophan (m/z 669) c) 13 C 6 labeled quinolinic acid (m/z 670)

39. The Experimental Strategy Culture cells in media containing isotopically labeled precursors for fixed time intervals Harvest and lyse the cells, extract the NAD, and quantitate the unlabeled and labeled NAD using reverse-phase LC-MS- MS

40. Sensitivity advantage m/z 664  m/z 542 m/z 692  m/z 562

41. Standard Curve 39 pts Detection Limit: 5 pg/  l or 35 fmol on column

42. 8.08.59.09.510.010.511.011.5 0 20 40 60 80 100 0 20 40 60 80 100 0 20 40 60 80 100 660665670675680685690695 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 664 667 692 Unlabeled NAD Internal Standard NAD synthesized from labeled nicotinamide Sample from experiment 1: Nicotinamide at 72 hrs m/z 664 m/z 692 m/z 667

43. Elemental Mass Spectrometry ICP-MS (inductively coupled plasma) SIMS-TOF (secondary ion mass spectrometry CRIMS

44. ICP-MS (vs. ICP-UV/vis) ICP (ch 10, pg 231-232) ICP-MS (ch 11) A spark ignites flowing argon forming a self-sustaining plasma (T ≈ 10000 K) Sample is aspirated/pumped into plasma forming elemental cations and some simple polyatomic ions Ions are pushed into mass analyzer by high voltage

45. Isotope ratio mass spectrometry Elemental analysis (geologists, archeologist, isotope tracer studies) High resolution sector mass analyzers Faraday cups ThermoFinnigan Neptune http://www.thermo-optek.it/GetBrochure.php?ID=43 CRI-MS (chemical reaction interface) –Converts all carbon to CO 2