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Lecture 5

Lecture 5. Condensed phase ionisation techniques: spray methods. At the end of this lecture you should be able to:. describe the ion formation models in ESI-MS calculate molecular weights and charge states from low- and high-resolution ESI-MS spectra. Ionisation Techniques: Overview.

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Lecture 5

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  1. Lecture 5 Condensed phase ionisation techniques: spray methods

  2. At the end of this lecture you should be able to: • describe the ion formation models in ESI-MS • calculate molecular weights and charge states from low- and high-resolution ESI-MS spectra

  3. Ionisation Techniques: Overview Gas-Phase Methods • Electron Impact (EI) • Chemical Ionization (CI) Desorption Methods • Secondary Ion MS (SIMS) and Liquid SIMS • Fast Atom Bombardment (FAB) • Laser Desorption/Ionization (LDI) • Matrix-Assisted Laser Desorption/Ionization (MALDI) Spray Methods • Atmospheric Pressure Chemical Ionization (APCI) • Electrospray (ESI)

  4. Condensed phase ionisation techniques (2): spray methodsOverview • Thermospray • APCI: Atmospheric pressure chemical ionisation • APPI: Atmospheric pressure photoionisation • Electrospray ionisation

  5. Atmospheric pressure chemical ionisation (APCI) • For non-polar and thermally stable compounds < 1500 Da • Useful to combine with liquid chromatography Ions Spray needle/ Capillary Spray To mass spectrometer Flow Cone Nebuliser gas Skimmers Corona discharge Vacuum Atmospheric pressure www.chm.bris.ac.uk/ms/theory/apci-ionisation.html

  6. Electrospray Ionisation (ESI) • Nobel Prize to Fenn in 2002 • Also atmospheric pressure ionisation • Very versatile • Also works for (very) large (bio)molecules, including proteins, nucleic acids, carbohydrates • Softest ionisation technique of all Spray needle/Capillary Spray To mass spectrometer Flow 3-4 keV 2-10 ml/min Cone Skimmers Atmospheric pressure Vacuum http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html

  7. Electrospray Ionisation (ESI) • Flowrates: 2 to 10 ml/min: Best interface for LC/MS • Can be combined with almost any mass analyser Common: TOF, Ion Trap, Quadrupole, FT-ICR • Uses: Mass detection, structure elucidation, protein folding, H/D exchange, protein sequencing….

  8. Sample characteristics • Common solvents: mixtures of water with acetonitrile or methanol • Usually with added acid (acetic, formic), < 1% • Can’t tolerate (non-volatile) salt or buffers • Can do positive or negative electrospray: selected by capillary voltage

  9. What it looks like

  10. Ionisation models Spray needle tip Multiply charged droplet Ion evaporation model • Spray generates multiply charged droplets • Solvent evaporation leads to increasing charge density • When charge density too high: Coulombic explosion Rayleigh limit is reached Analyte molecule Multiply charged droplet www.chm.bris.ac.uk/ms/theory/esi-ionisation.html Charged residue model

  11. 4.24 3.65 Acidic Basic 12.5 + N O Aspartate Glutamate N O O 10.8 N Arginine O- N N O + O O- O H3N Lysine N N Charged amino acids Usually negatively charged at pH 7 O 6.00 Positively/uncharged at pH 7 N N NH Histidine Always positively charged

  12. Charges on surface of proteins Red: Negatively charged Blue: Positively charged White: No charge; hydrophobic

  13. 11+ 12+ 10+ 9+ 13+ 8+ 7+ 6+ Examples • ESI of large molecules usually produces multiply charged ions • e.g. proteins: • Each peak corresponds to the same protein, but with different number of protons attached: Observed ions are[M+nH]n+ Charge state series 750 1000 1250 1500 1750 2000 2250 2500 2750 m/z

  14. [M (n 1)H] + + n 1 + How to determine the molecular mass of a protein from an ESI-MS spectrum • Observed ions have composition [M+nH]n+ • Let m1,m2,…, mn : m/z values of the different peaks • mn = • Its neighbouring peak to the left: • mn+1 = • Solving both equations for n and M: • n = • M = n(mn – H) • e.g. m2 = 998.1 and m1 = 1060.5 • n = 16, and M = 16952 Da 848.7 808.3 893.3 771.6 738.1 942.8 707.4 998.1 1060.5 m/z n must be an integer

  15. Deconvolution of ESI mass spectra Deconvoluted spectrum Charge states M = n(mn – H) 3000 30000 Mass (Da)

  16. Quick reminder: average and monoisotopic mass

  17. The distance between isotopic peaks reveals charge state 505.3506 +1 1.00 506.3584 915.7363 915.4818 507.3566 +4 915.9765 915.2247 916.2311 916.4857 0.25 1086.5515 1086.0433 0.51 1086.0444 +2 1087.5529 Jonathan A. Karty 1088.0460

  18. a.i. 1.5 1.0 0.5 0.0 1695.7 1696.2 1696.7 m/z Charge States and distance between isotopic peaks 0.1 +10

  19. Example beyond molecular mass: Protein folding • Calbindin: Calcium binding induces protein folding: increase in charge states with higher m/z (=lower charge, more folded) No Ca2+ excess Ca2+ raw data deconvoluted

  20. Recent developments: ambient mass spectrometry:DESI and DART • DESI: Desorption electrospray ionisation • DART: Direct analysis in real time • Applicable to solids, liquids, and gases • No prior sample treatment !

  21. Ionsiation techniques: Summary Table adapted from http://www.scs.uiuc.edu/~msweb/SLM530.pdf

  22. Summary: Application ranges of various techniques masspec.scripps.edu/MSHistory/whatisms.php

  23. Self-assessment questions • Q1 Describe the two ion formation models in ESI • Q2 A positive-ion ESI spectrum shows the following adjacent signals at m/z 4348.8, 4546.5, 4762.9, 5001.0 5264.2. Calculate the molecular mass of the molecule. • Q3 The following m/z (979,1040,1109,1189,1280,1387,1512,1664) were obtained by electrospray ionisation of a protein from an aqueous solution. • Calculate the molecular mass of the protein within 10 Da. • Describe how the mass spectrum would have looked if the same protein had been ionised by MALDI. • Q4 What distance between isotopic peaks would you expect for a +12 charge state ? • Q5 The following peaks arose from the different isotopes contributing to the ESI mass spectrum resulting from the protonation of a species of relative molecular mass m to reach a charge z.:m/z=848.40, 848.45, 848.50, 848.55, 848.60, 848.65, 848.70. • What is the value z and what is the mass of the species giving rise to the peak at m/z 848.55

  24. Lecture 6 Tandem MS Peptide/protein identification by MS

  25. At the end of this session you should be able to • explain how structural information can be obtained by Tandem MS and MALDI-TOF/PSD • explain how mass spectrometry data can be used to identify known and unknown proteins

  26. Tandem MS(also termed MS2 and MSn) • Used for: • Identify and quantify compounds in complex mixtures • Structure elucidation of unknown compounds • Applied in: • Proteomics • Metabolomics • Biomarker discovery • De novo protein sequencing

  27. Mass analysis of product ions • Activation and fragmentation MS-1 MS-2 Ion source Tandem MS • Multistage technique: • mass selection of precursor ion MS/MS spectrum Normal spectrum

  28. Tandem MSFragmentation techniques • Collision-induced dissociation (CID): • most common • Possible with ESI coupled to triple-quad, Ion trap, FT-ICR, and MALDI-TOF • Electron Capture Dissociation (ECD): • only for multiply charged biopolymers • Primarily with FT-ICR • Electron-Transfer Dissociation (ETD) • Absorption of electromagnetic radiation • Not Tandem MS, but useful fragmentation technique: Post-source decay (PSD) combined with MALDI reflectron TOF

  29. i n d u c e s p o s t s o u r c e d e c a y D e t e c t o r r e f l e c t r o n Reflectron-TOF and Post-Source decay for MALDI-TOF Field-free region • (Some) parent ions fragment in field-free drift region • Parent and product ions arrive at reflectron simultaneously (same velocity) • Product ions leave Reflectron earlier (smaller Ekin)

  30. Example: MALDI-PSD TOF spectrum of a neuropeptide Parent Ion Necla Birgül, Christoph Weise, Hans-Jürgen Kreienkamp and Dietmar Richter , The EMBO Journal (1999) 18, 5892–5900

  31. Simplified schematic for protein identification from biological samples (“Proteomics”) Cell culture Tissue Biofluid Extraction Complexprotein mixture Individual or small sets of proteins Separation Peptide mass mapping Cleavage Peptide-mass fingerprints Peptides MALDI Database search Sequencing (LC-MS/MS) Database search Identified proteins Peptide sequences

  32. Peptide mass mapping/ fingerprinting • Makes use of specific cleavage agents • Chemical cleavage: e.g. CNBr • Digestion with endoproteases (proteolytic enzymes): Trypsin, pepsin, chymotrypsin etc. • See exercises

  33. Peptide mass mapping/fingerprinting Mass Spectrum  Peptides Protein Digest Abundance 600 900 1200 1500 1800 m/z Compare Protein Sequence (in database) Theoretical Mass Spectrum Peptide sequences Theoretical Digest NTFVRPFWWRPR IVTPCVAYGLGR CLVGVVNR APIAPCCR FQNIP ... QNICPRVNRIVTPCVAYGLGRAPIAPCCRALNDLRFVNTRNLRRAACRCLVGVVNRNPGLRRNPRFQNIPRDCRNTFVRPFWWRPRIQCGRIN Abundance 600 900 1200 1500 1800 m/z

  34. De novo protein discovery • Mass fingerprinting only practicable if protein is already in a database • If previously undiscovered protein: Need to sequence • Can be done by sequencing peptides

  35. Peptide sequencing:fragmentation rules R3 R2 R4 R1 • Three types of bonds along backbone • amino alkyl bond • alkyl-carbonyl bond • amide bond +NH3―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2― N-terminus C-terminus

  36. Peptide sequencing:fragmentation rules b1 a1 c1 R3 R4 R1 R2 NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H y3 z3 x3 • Each bond can be broken by fragmentation  Six possible product ions • But: peptide bond most likely to break in low energy CID (and MALDI/PSD): • Mostly b and y fragments

  37. Peptide fragments generated by low energy CID or PSD b1 b2 b3 R3 R2 R4 R1 NH2―CH―CO―NH―CH―CO―NH―CH―CO―NH―CH―CO2H N-terminus C-terminus y3 y2 y1 For example: R3 R2 R1 + b3:NH2―CH―CO―NH―CH―CO―NH―CH―C=O R4 R3 y2: +NH3―CH―CO―NH―CH―CO2H m/z values of b ions: residue masses + 1 (+H+) m/z values of y ions: residue masses +17 (OH-) + 2 (2H+) = 19

  38. Example: Peptide analysed by MALDI TOF reflectron and PSD y7 y8 y6 y5 y4 y3 y1 y2 His Thr Leu/ Ile/Asn His Leu/ Ile/Asn Phe Ala Arg Leu/Ile: 113.08 Asn: 114.04  Proposed sequence:HTH[LIN]FA[LIN]R

  39. Self-assessment questions • Q1 How are MALDI and ESI used for the identification of proteins ? • Q2 Describe how Peptide Fingerprinting works • Q3 A MALDI-PSD spectrum of a peptide shows the following y-peaks:174.8 / 288.3 / 359.0 / 506.1 / 619.6 / 756.0 / 857.6 / 995.2Find out the masses for amino acids (e.g. at Wikipedia). Calculate the differences between peaks in this spectrum and suggest possible sequences for this peptide

  40. Exercises • Exercise 1: Protein cleavage/digestion 1. Go to http://www.expasy.ch/tools/peptidecutter/ 2. In the box, enter ALBU_HUMAN (this is the swissprot name of human serum albumin) - you can also choose a different protein if you like. Sequences and swissprot codes can for example be found in the swissprot database (at www.expasy.ch). 3. Scroll down, and tick the box “only the following selection ofenzymes and chemicals”,and then select one chemical or enzyme, which you want to use for cleaving the albumin protein, from the list 4. Scroll back up, and click “Perform” 5. Inspect the output. How many times is albumin cleaved by your chosen cleavage agent ? Find out what the specificity of your cleavage agent is.

  41. Exercise 2: Calculation of molecular masses of proteins and peptides 1. Copy a peptide fragment from the output of Exercise 1 (or make one up yourself), go to http://www.expasy.ch/tools/protparam.html and paste your sequence into the appropriate box (the large one). 2. Click “Compute Parameters”. 3. Inspect the output. What is the molecular formula of the peptide ? How many positively and negatively charged side-chains does your peptide have ? What charge would it have at pH 7 ?

  42. Exercise 3. Average and monoisotopic masses 1. Copy the molecular formula (or the one-letter code sequence) of the peptide from exercise 2, and go to http://education.expasy.org/student_projects/isotopident/htdocs/ 2. Paste the formula or sequence in the appropriate box. Make sure that you have selected the correct “Type of composition” (e.g. “chemical formula”) in the respective pulldown menu. 3. Click “Submit query”. 4. Inspect the output. How many isotopic peaks are there? Which is the most abundant peak ? What are the monoisotopic and the average masses ?

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