Biochem 523b: Advanced Physical Methods: Mass Spectrometry, X-ray Crystallography and NMR

1. Biochem 523b: Advanced Physical Methods: Mass Spectrometry, X-ray Crystallography and NMR • Biological Mass Spectrometry (Lajoie) • (3 +1 lectures) • 2. X-ray Crystallography (Ling) • (3 +1 lectures) • 3. NMR (Shaw) • (3 +1 lectures)

2. Biochem 523b: Advanced Physical Methods: Mass Spectrometry, X-ray Crystallography and NMR First lecture: January 11(room DSB 3008) 2:30-5:30pm Last Lecture: March 29Final Exam: TBA, Mid-AprilReference material: Course notes and journal articles. Evaluation: Presentations (2) 30 marks Assignments (3) 30 marks Final exam 40 marks Students will give a 20 min presentation in two of the three topics discussed in the course.There will be an assignment for each topic. The final exam will be a 3hr exam with questions in each section. Note: Course notes from Biochem 440 and 465a will be available for review

3. Biochem 523b: Advanced Physical Methods: Mass Spectrometry, X-ray Crystallography and NMR A. Mass Spectrometry Lecture 1 Introduction Definitions Basic concepts Mass Spectrometer Ionization MALDI ESI Multiply charged ions and deconvolution MS/MS sequencing Lecture 2 Mass analyzers

4. Biochem 523b: Advanced Physical Methods: Mass Spectrometry, X-ray Crystallography and NMR Lecture 3 Quantitation General Principles ICAT, iTRAQ, SILAC,etc. PTMs Phosphorylation Glycosylation Lecture 4 Short presentations by students Non-Covalent studies Protein Folding HTP Proteomics Metabolomics Other PTMs Etc

5. Mass Spectrometry • An instrument that measures the masses of individual molecules • that have been converted into ions, i.e., molecules that • have been electrically charged. Measure mass of ions not of neutral • molecules. • Since molecules are so small, it is not convenient to measure • their masses is kilograms, or grams, or pounds. • In fact, the mass of a single hydrogen atom is approximately • 1.6726 X 10-24 grams. • The convenient unit of mass is often referred to by chemists and • biochemists as the dalton (Da) and is defined as follows: 1 Da=(1/12) • of the mass of a single atom of the isotope of carbon-12(12C). • This follows the accepted convention of defining the 12C isotope as • having exactly 12 mass units 12C= 12.00000. • Dalton (Da) is also known as unified atomic mass unit (u or amu) 1 Da = 1 u = 1.66055 x 10-27 Kg

6. Why Mass Spectrometry? Highly selective: Can monitor a certain analyte with minimum interference by other species in the sample The high selectivity also to distinguish several species at the same time for measure multiple species at the same time with high resolution for each as opposed to average signal for many spectroscopic techniques such as UV absorbtion, fluorescence, etc. Selectivity can be increased by coupling with separation technique such as LC or GC MS is highly sensitive or has very low limit of detection picomole (10-12 mol) to zeptomole (10-21 mol). Much more sensitive than NMR Concentration in NMR is typically mM MS can handle nM or better

7. Use of Mass Spectrometry • Identify structures of biomolecules such as proteins, • carbohydrates, nucleic acids and steroids • Sequence biopolymers: proteins and oligosaccharides • Determine how drugs are used by the body (metabolites) • Perform forensic analyses: confirmation and quantitation • of drugs of abuse • Analyze for environmental pollutants • Determine the age and origins of specimens in geochemistry • and archaeology • Identify and quantitate compounds of complex organic • mixtures

8. Applications in Biochemistry • Characterization of biomolecules including proteins and their modifications (phospho, glyco) • Sequence determination of proteins (peptides), polysaccahrides, lipids • Protein–Ligands Interactions • Protein abundance • Protein-Protein interactions (network) • Stoichiometry of complexes (quaternary structure, metal, etc.) • Cellular localization (organellar proteomics) • Protein dynamics and folding • Proteomics, Glycomics, Lipidomics, Metabolomics (HTP of above)

9. The Mass Spectrometer High Vacuum Sample Ion Source Mass Analyzer Detector Data System Creates ions in the gas phase Separates ions in space or time according to mass to charge ratio m/z Collects ions and amplifies signals Stores and analyzes data Controls the mass spectrometer Electron Impact (EI) Chemical Ionization (CI) Fast atom bombardment (FAB) Magnetic sector Time-of-flight (TOF) Quadrupole (Q) Hybrid : Q-TOF Linear Ion Trap FT ICR Orbitrap MALDI Electrospray

10. Why high vacuum in mass analyzer? High vacuum = low pressure High vacuum is necessary to minimize collisions with other gazeous molecules. Collisions would produce deviation of the trajectory and ions would loose their charges on walls of instrument. Ion-molecules could produced unwanted reactions and increase complexity of spectrum (controlled collisions can be useful and will be discussed later. The average distance a particle can travel before colliding is called the mean free path L: L = kT/ 2 ps • k = Boltzmann constant, T is temperature in K • p = pressure in Pa and s is collision cross-section (m2) • = p d2 where d is sum of radii of the stationary molecule and colliding molecule d = r1 + r2 r1 r2 K = 1.38 x 10-21 J K-1, T ~ 300 K, s ~ 45 x 10-20 m2 L (cm) = 0.66/ p (Pa) or = 4.95/ p (milliTorr)

11. Why high vacuum in mass analyzer?... The mean free path L must be larger than the dimension of the mass analyzer In a typical mass spec the mean free path should be at least 1 meter and hence the maximum pressure should be no more than 66 nbar However we need L = 10 to 100 times the free ion path to reduce The probability of ion/neutral collision to 10% or better to 1% or less Typical vacuum in MS systems is 10-5to a10-10 Torr. For air molecule at 10-7 Torr, L is ~1 km Note: Measurement of cross section can yield information on the conformation of molecules in the gas phase

12. Useful Definitions and Units Prefix for SI units 10-1 deci 10-3 milli 10-6 micro 10-9 nano 10-12 pico 10-15 femto 10-18 atto 10-21 zepto Quantities Charge of electron e 1.60219 x 10-19 C Mass of the electron me 9.10953 x 10-31 kg Mass of the proton mp 1.67265 x 10-27 kg Mass of the neutron mn 1.67495 x 10-27 kg Unified of atomic mass u 2.99793 x 10-27 kg Avogrado constant NA 6.02205 x 1023 mol-1 Pressure Energy 1 pascal (Pa) = 1 Newton (N) m-2 1 bar = 10 6 dyn cm-2 =105 Pa 1 millibar (mbar) = 10-3 bar = 102 Pa 1 atmosphere (Atm) = 1.1013 bar = 101 308 Pa 1 Torr = 1 mmHg = 1.33 mbar = 133.3 Pa 1 psi = 1 pound per square inch = 0.07 atm 1 cal = 4.184 J eV = 1.602 x 10-19 J

13. Mass Spectrum 2 dimensional representation of signal intensity (y axis) vs m/z (x axis) The intensity reflects the abundance of ionic species 100 Most intense peak is called the base peak and is most often normalized to 100% relative Intensity. Plot centroid peak 50 intensity 50 100 150 200 m/z m/z is dimensionless, z = is an integer, 1 or more

14. Back to Basics… Chemical Composition of Living Matter 27 of 92 natural elements are essential.   Elements in biomolecules (organic matter): H, C, N, O, P, S These elements represent approximately 92% of dry weight. Organic Matter Organized in "building blocks" amino acids polypeptides ( proteins) monosaccharides starch, glycogen nucleic acids DNA, RNA

15. Mass (Weights) of Atoms and Molecules Element Nominal Exact Percent Average mass mass abundance mass C 12 12.00000 98.90% 13 13.00335 1.10% 12.0115 H 1 1.00783 99.986% 2 2.01410 0.015% 1.00797 O 16 15.99491 99.762% 17 16.9991 0.038% 18 17.9992 0.2% 15.9994 N 14 14.00307 99.63% 15 15.00011 0.37% 14.0067 S 32 31.9721 95.02% 32.066 33    32.9714 0.75% 34 33.9678 4.21% 36 35.9671 0.02% P 31 30.9737 100% 30.9737

16. Calculation of Atomic and Molecular mass Nominal Mass: To calculate the approximate mass of a molecule Use the mass of the element present eg CO2 12 + (2x16)= 44; not precise but sufficient in many applications Isotopic mass: is calculated from the exact mass of the isotopes. It is close but not equal to the nominal mass. The monoisotopic mass of a molecule is the addition of the exact mass of the most abundant isotopes for each atom Present. For CO2 12.00000 u + (2 x 15.994915) u = 43.989830 (u or Da) Exact ionic mass: Depends on how the ions are formed. For CO2+. 12.0000000u + 2 x (15.994915) – 0.000548 u (mass of e) = 43.989282. For ESI or MALDI in positive ion mode, we will add the mass of one or more proton. Relative Atomic Mass (average mass): calculated from the weighted average of naturally occurring isotopes of an element. The relative molecular Mass Mr is calculated from the relative atomic masses of the elements in the empirical formula. Eg CO2 12.0108 + 2 x 15.9994 = 43.9988

17. Mass spectrum A mass spectrum is a graph of ion intensity as a function of mass-to-charge ratio. Mass spectra are often depicted as simple histograms as shown Most abundant =100%

18. Mass Spectrometry + (CH3) 3N-CH2-CH2-OH 104 (Choline ion) relative Intensity (number of ions counted) 5 C (12) = 60 14 H (1) = 14 1 O (16) = 16 1 N (14) = 14 --------- 104 150 100 50 m/z Low resolution mass spectrum

19. Formation of Ions by Electron Ionization Removal of 1 electron

20. Mass or Molecular Weight of Molecules Ethyl acetate C4H8O2 4 C12 4 x 12.0000 48.0000 8 H1 8 x 1.00783 8.06264 2 O16 2 x 15.9949 31.98982 Nominal Mass: 48 + 8 + 32 = 88 Monoisotopic Mass: 88.0546 Average Mass: 48.046 + 8.06376 + 31.988 = 88.10856

21. Mass Spectrum of Ethyl Acetate by Electron Impact (EI) Harsh ionization causes fragmentation m/z = 43 m/z = 15 m/z = 88 43.02 (100%) Monoisotopic peaks Base peak %relative intensity 88.05 (10%) 15 (1%) 44.02 (2.2%) 89.05 (0.44%) 20 40 80 m/z

22.   Approximation of Isotopic Distribution • Ethyl acetate C4H8O2 • 1st PEAK (100%intensity) • 4 C12 4 x 12.0000 48.0000 • 8 H1 8 x 1.0078 8.0624 • 2 O16 2 x 15.99949 31.9898 • 88.0522 • Second peak (4.56 % intensity) • 3 C12 3 x 12.0000 36.0000 • 1 C13 1 x 13.000333 13.0335 • 8 H1 8 x 1.0078 8.0624 • 2 O16 2 x 15.99949 31.9898 • (1.1% x 4 = 4.4%) 89.055 • 4 C12 4 x 12.0000 48.0000 • 7 H1 7 x 1.0078 7.0546 • 1 H2 1 x 2.0140 2.0140 • 2 O16 2 x 15.99949 31.9898 • (0.020 x 8 = 0.16%) 89.0584

23. Amino Acids (20) Intact nominal mass Exact Mass of Amino Acid Residues in Proteins Note: Leu (L) = Ile (I) = 113.08410

24. Amino Acids and Proteins Have Mass (or Weight) Ala-Ser-Phe (ASF) Nominal (MW 89 + 106 + 165 - (2 x 18)) = 323 or C15H21N3O5 monoisotopic mass: 71.03711 + 87.03203 +147.0684 + 18.0105 (H2O) = 323.1481 Mr average mass 323.3490

25. Mass accuracy and resolution Mass accuracy: the difference between measured and accurate mass and calculated exact mass. Mass accuracy can be stated as absolute units of u (or mmu) or as relative mass accuracy in ppm (most common): (Experimental – Calculated) (106) = ppm Calculated (0.0406) (106) (3708.99 -3708.9494) (106) = 11 ppm = 3708.9494 3708.9494 Resolution:good mass accuracy can only be obtained from sharp peaks that are evenly shaped signals that are well separated form each other

26. Resolution and mass accuracy Resolution (R) is a measure of separation between two adjacent peaks (masses). Dm is the smallest mass difference at which two masses can be resolved. R = m/Dm Resolving power (R) is also a performance characteristics of MS instruments, that is its ability to distinguish between two Ions that differ only slightly in their m/z rario • There are a number of ways to describe resolution(R): • Peak width at 10% valley for two overlapping peaks (2x 5%) • Peak width at 5% maximum for a single peak • Peak full width at half maximum (FWHM) (most common) • ie in Da at 50% of the intensity Since resolution is also related to peak width, resolution will also affect mass accuracy. On most instruments higher resolution means lower sensitivity.

27. Resolution and mass accuracy Two overlapping peaks Single peak 2 peaks at 10% valley Full Width at Half Maximum (FWHM)

28. Consequences of resolution on mass accuracy 1u 1 u 1u 1 u 0.1u 50 51 1000 1001 500 501 Signals at m/z 50, 500 and 100 at R = 500. At m/z 100 the peak maxima are shifted towards each other due to superimposing of the peaks.

29. Importance of Resolution

30. Glucagon: Monoisotopic and Average Mass As the mass increase the monoisotopic peak is less and less evident First peak C153 H225N42 O50S100% Second Peak: 12C-13C 153 x 1.1% 170% 1H-2H(D) 225 x 0.02% 4.5% 14N- 15N 42 x 0.37% 15.5% 190% Monoisotopic mass: 3,482.61 Average mass: 3,484.75

31. * Note: Peaks of highest intensity is 1 Da higher than monoisotopic for each ~1500 Da (ie for mass ~3000 the highest peak is 2 Da higher than the monoisotopic peak

32. Resolution and mass accuracy… Mass accuracy: ppm = 106 /R = 106Dm/M Example: Measure a mass at 1,000 +/- 0.5Da Mass accuracy = 106 (0.5)/ 1,000 = 500 ppm Resolution R = M/Dm = 1,000/0.5 = 2,000 • Higher resolution gives higher mass accuracy • For a given resolution mass accuracy decrease with higher m/z • m/z Dm (+/-) Resolution ppm (+/-) Mass range • 1,000 0.05 20,000 50 999.95-1000.05 • 2,000 0.05 40,000 25 1999.95-2000.05 • 10,000 0.5 20,000 50 9999.5-10,000.5 • 10,000 0.05 200,000 5 9999.95-10,000.05 • 10,000 0.005 2,000,000 0.5 9999.995-10,000.005

33. Characteristics of Mass Spectrometers - Sensitivity: expressed in lowest detection limit eg picomolar (10-12 mole), now subfemtomolar (< 10-15) - Mass range eg 50-4000 - Mass accuracy expressed in u or ppm (best 1- 5 ppm) -Resolving power: ability to separate two peaks (masses) For R = 20,000 can see two masses at 100.000 and 100.005 - dynamic range: ability to observe two peaks at very different intensities eg 1000:1 (103)- best 104 (LTQ-FTMS) -others: cost, ease of operation, etc.

34. Characteristics of Some Mass Spectrometers - Sensitivity for tryptic peptides MALDI–TOF/R 10-100 x 10-15mole Q-TOF2 50-200 x 10-15mole • Resolution • MALDI -TOF/R 10,000 at mass 2,000 • Q-TOF2 10-15,0000 • FTMS 100,000-3,000,000 - Mass Accuracy  MALDI-TOF/R: external calibration +/- 50 ppm internal calibration +/- 20 ppm Q-TOF2: external calibration +/- 50 ppm   FTMS +/- 1-10 ppm

35. MALDI-TOF Q-TOF Ion Trap FTICR (9.4T) Sensitivity Highest High Medium High Mass Narrow High Poor Highest Accuracy range Sequencing Difficult Yes Yes Yes (MS/MS) Throughput High Med Med Med Ease of Easiest Med Med Hardest operation Cost 300K650K300K1.0M Newest: MALDI -TOF/TOF; MALDI- Qq-TOF; FTICR MS 12 Tesla MALDI-ion trap/quadrupole; ESI Quad/Trap/TOF, Orbitrap

36. Matrix Assisted Laser Desorption/Ionization(MADLI) 1. Matrix containing analytes (eg proteins) absorbs UV (or IR) energy from a pulse laser (10 nanoseconds) 2. The matrix ionizes and dissociates; it undergoes a phase change to a supercompressed gas; it then transfers it charges to the analyte molecules 3. Matrix expands at supersonic velocity; additional analytes are formed in the gas phase; the resulting ions are entrained in the expanding plume 4. The analyte ions are accelerated by a voltage pulse and analyze in the mass spectrometer

37. Matrix Assisted Laser Desorption/Ionization Sample is co-crystallized with matrix (solid) Formation of singly charged ions Koichi Tanaka, Nobel Prize 2002

38. MALDI-TOF/R MS of Peptides from a Tryptic Digest Peptides from trypsin self-digestion internal calibrants Mass “Fingerprint” of a Pure Protein

39. Protein Identification with MALDI-TOF/R 1. Cut spots from 2D Gel, destained, reduce disulfide bonds, alkylate with iodoacetamide and trypsin digestion of each spot (medium to high silver stained spot) 2. Extract peptides and purify by ZipTip, containing reverse phase or by capillary HPLC. 3. Mix with matrix and analyze by MALDI-TOF/R 4. Compare observed masses with masses in databases obtained from virtual tryptic digest of all proteins (mass fingerprinting) 5. Confidence for hits depends on coverage: minimum 5 masses(should get >30% coverage)

40. Proteomic Analysis with MALDI: Mass Fingerprinting

41. 1000 1500 2000 “Bottom-up Approach” Peptides to proteins Mass(m/z) Extract peptides; mass analyze Pick spots on a gel Protein(s) in solution Digest – site specific protease Databasesearch or sequence

42. Difficulties With Mass Fingerprints Many tryptic peptides have similar masses resulting in numerous false positives. Mass accuracy is critical !! Mass from 1000.30-1000.70

43. Typical Problems • No MS signals!! Insufficient sample (poor digestion, poor extraction) Contaminants that affect ionization: SDS, acrylamide, salts, detergents, PEG 2. Protein contamination Keratins, peptides from trypsin self-digestion, bacterial proteins, etc.. 3. Detect the most abundant proteins only 4. Masses affected by PTMs, adducts, etc, wrong assignment

44. Electrospray Ionization –MS ESI MS

45. Formation of Charged Droplets and Mutilply Charged Ions Formation of multiply charged ions

46. Mass Spectrum of a Multiply Charged Protein

47. Raw Data Spectrum for Myoglobin (Denaturing conditions) +18 Maximum number of charges is dependent on number of basic residues (Lys, Arg, His) +17 +16 +15 +14 +13 +12 +11 +10 +9

48. Deconvoluted Spectrum for Myoglobin myo 12 (0.467) M1 [Ev-145428,It12] (Gs,0.700,650:1950,1.00,L33,R33); Sb (25,10.00 ); Cm (5:20) TOF MS ES+ 2.30e5 100 A: 16951.50±0.02 % 0 mass 14000 14500 15000 15500 16000 16500 17000 17500 18000 18500 19000 19500 A 16951.85 Myoglobin: Deconvoluted Spectrum Expected MW = 16951.49 Da R = 8000 17049.71 17567.41

49. MS of Glu-fibrinopeptide +2 M= (785.85 x 2) -2H = 1569.79 +3

50. MS of Glu-fibrinopeptide: doubly charged ion 785.85 786.36 M= (785.85 x 2) -2H = 1569.79 0.5 Da Monoisotopic 786.86 0.5 Da 787.38 0.5 Da