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Mass Spectrometry: Methods & Theory David Wishart University of Alberta Edmonton, AB [email protected] MS Principles • Different elements can be uniquely identified by their mass Lecture 2.1 2 MS Principles • Different compounds can be uniquely identified by their mass Butorphanol L-dopa N -CH2OH Ethanol COOH HO -CH2CH-NH2 CH3CH2OH HO HO MW = 327.1 Lecture 2.1 MW = 197.2 MW = 46.1 3 Mass Spectrometry • Analytical method to measure the molecular or atomic weight of samples Lecture 2.1 4 Mass Spectrometry • For small organic molecules the MW can be determined to within 5 ppm or 0.0005% which is sufficiently accurate to confirm the molecular formula from mass alone • For large biomolecules the MW can be determined within an accuracy of 0.01% (i.e. within 5 Da for a 50 kD protein) • Recall 1 dalton = 1 atomic mass unit (1 amu) Lecture 2.1 5 Masses in MS • Monoisotopic mass is the mass determined using the masses of the most abundant isotopes • Average mass is the abundance weighted mass of all isotopic components Lecture 2.1 6 Isotopic Distributions 1H = 99.9% 2H = 0.02% Lecture 2.1 12C = 98.9% 13C = 1.1% 35Cl = 68.1% 37Cl = 31.9% 7 Isotopic Distributions 1H 12C = 99.9% 2H = 0.02% = 98.9% 13C = 1.1% 35Cl = 68.1% 37Cl = 31.9% 100 32.1 6.6 2.1 0.06 0.00 m/z Lecture 2.1 8 Mass Calculation (Glycine) NH2—CH2—COOH Amino acid R1—NH—CH2—CO—R3 Residue Monoisotopic Mass 1H = 1.007825 12C = 12.00000 14N = 14.00307 16O = 15.99491 Lecture 2.1 Glycine Amino Acid Mass 5xH + 2xC + 2xO + 1xN = 75.032015 amu Glycine Residue Mass 3xH + 2xC + 1xO + 1xN =57.021455 amu 9 Amino Acid Residue Masses Monoisotopic Mass Glycine Alanine Serine Proline Valine Threonine Cysteine Isoleucine Leucine Asparagine Lecture 2.1 57.02147 71.03712 87.03203 97.05277 99.06842 101.04768 103.00919 113.08407 113.08407 114.04293 Aspartic acid Glutamine Lysine Glutamic acid Methionine Histidine Phenylalanine Arginine Tyrosine Tryptophan 115.02695 128.05858 128.09497 129.0426 131.04049 137.05891 147.06842 156.10112 163.06333 186.07932 10 MS History • JJ Thomson built MS prototype to measure m/z of electron, awarded Nobel Prize in 1906 • MS concept first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 • Designed to measure mass of elements (iso.) • Aston Awarded Nobel Prize in 1922 • 1920s - Electron impact ionization and magnetic sector mass analyzer introduced Lecture 2.1 11 MS History • 1948-52 - Time of Flight (TOF) mass analyzers introduced • 1955 - Quadrupole ion filters introduced by W. Paul, also invents the ion trap in 1983 (wins 1989 Nobel Prize) • 1968 - Tandem mass spectrometer appears • Mass spectrometers are now one of the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY Lecture 2.1 12 MS Principles • Find a way to “charge” an atom or molecule (ionization) • Place charged atom or molecule in a magnetic field or subject it to an electric field and measure its speed or radius of curvature relative to its mass-to-charge ratio (mass analyzer) • Detect ions using microchannel plate or photomultiplier tube Lecture 2.1 13 Mass Spec Principles Sample + _ Ionizer Lecture 2.1 Mass Analyzer Detector 14 Typical Mass Spectrometer Lecture 2.1 15 Typical Mass Spectrum aspirin Lecture 2.1 16 Typical Mass Spectrum • Characterized by sharp, narrow peaks • X-axis position indicates the m/z ratio of a given ion (for singly charged ions this corresponds to the mass of the ion) • Height of peak indicates the relative abundance of a given ion (not reliable for quantitation) • Peak intensity indicates the ion’s ability to desorb or “fly” (some fly better than others) Lecture 2.1 17 Resolution & Resolving Power • Width of peak indicates the resolution of the MS instrument • The better the resolution or resolving power, the better the instrument and the better the mass accuracy DM • Resolving power is defined as: M • M is the mass number of the observed mass (DM) is the difference between two masses that can be separated Lecture 2.1 18 Resolution in MS Lecture 2.1 19 Resolution in MS 783.455 QTOF 784.465 785.475 783.6 Lecture 2.1 20 Inside a Mass Spectrometer Lecture 2.1 21 Mass Spectrometer Schematic Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Inlet Sample Plate Target HPLC GC Solids probe Lecture 2.1 Ion Source Mass Filter MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Detector Microch plate Electron Mult. Hybrid Detec. Data System PC’s UNIX Mac 22 Different Ionization Methods • Electron Impact (EI - Hard method) – small molecules, 1-1000 Daltons, structure • Fast Atom Bombardment (FAB – Semi-hard) – peptides, sugars, up to 6000 Daltons • Electrospray Ionization (ESI - Soft) – peptides, proteins, up to 200,000 Daltons • Matrix Assisted Laser Desorption (MALDI-Soft) – peptides, proteins, DNA, up to 500 kD Lecture 2.1 23 Lecture 2.1 24 Electron Impact Ionization • Sample introduced into instrument by heating it until it evaporates • Gas phase sample is bombarded with electrons coming from rhenium or tungsten filament (energy = 70 eV) • Molecule is “shattered” into fragments (70 eV >> 5 eV bonds) • Fragments sent to mass analyzer Lecture 2.1 25 EI Fragmentation of CH3OH CH3OH CH3OH+ CH3OH CH2O=H+ CH3OH + CH2O=H+ Lecture 2.1 + H CH3 + OH CHO=H+ + H 26 Why You Can’t Use EI For Analyzing Proteins • EI shatters chemical bonds • Any given protein contains 20 different amino acids • EI would shatter the protein into not only into amino acids but also amino acid subfragments and even peptides of 2,3,4… amino acids • Result is 10,000’s of different signals from a single protein -- too complex to analyze Lecture 2.1 27 Soft Ionization • Soft ionization techniques keep the molecule of interest fully intact • Electro-spray ionization first conceived in 1960’s by Malcolm Dole but put into practice in 1980’s by John Fenn (Yale) • MALDI first introduced in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) • Made it possible to analyze large molecules via inexpensive mass analyzers such as quadrupole, ion trap and TOF Lecture 2.1 28 Lecture 2.1 29 Soft Ionization Methods 337 nm UV laser Fluid (no salt) + _ Lecture 2.1 cyano-hydroxy cinnamic acid Gold tip needle MALDI ESI 30 Electrospray (Detail) Lecture 2.1 31 Electrospray (Detail) Lecture 2.1 32 Electrospray Ionization • Sample dissolved in polar, volatile buffer (no salts) and pumped through a stainless steel capillary (70 - 150 mm) at a rate of 10100 mL/min • Strong voltage (3-4 kV) applied at tip along with flow of nebulizing gas causes the sample to “nebulize” or aerosolize • Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size (still carrying charges) Lecture 2.1 33 Electrospray Ionization 5%H2O/95%CH3CN 95%H2O/5%CH3CN 100 V 1000 V 3000 V Lecture 2.1 34 Electrospray Ionization • Can be modified to “nanospray” system with flow < 1 mL/min • Very sensitive technique, requires less than a picomole of material • Strongly affected by salts & detergents • Positive ion mode measures (M + H)+ (add formic acid to solvent) • Negative ion mode measures (M - H)- (add ammonia to solvent) Lecture 2.1 35 Positive or Negative Ion Mode? • If the sample has functional groups that readily accept H+ (such as amide and amino groups found in peptides and proteins) then positive ion detection is used • If a sample has functional groups that readily lose a proton (such as carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative ion detection is used Lecture 2.1 36 Electrospray Ionization • Samples of MW up to 1200 Da usually produce singly charged ions with observed MW equal to parent mass + H (1.008 Daltons) • Larger samples (typically peptides) yield ions with multiple charges (from 2 to 20 +) • Multiply charged species form a Gaussian distribution with those having the most charges showing up at lower m/z values Lecture 2.1 37 Multiply Charged Ions ESI spectrum of HEW Lysozyme MW = 14,305.14 Lecture 2.1 38 Peptide Masses From ESI Each peak is given by: m/z = (MW + nH+) n m/z = mass-to-charge ratio of each peak on spectrum MW = MW of parent molecule n = number of charges (integer) H+ = mass of hydrogen ion (1.008 Da) Lecture 2.1 39 Peptide Masses From ESI Charge (n) is unknown, Key is to determine MW Choose any two peaks separated by 1 charge 1431.6 = (MW + nH+) 1301.4 = (MW + [n+1]H+) [n+1] n 2 equations with 2 unknowns - solve for n first n = 1300.4/130.2 = 10 Substitute 10 into first equation - solve for MW MW = 14316 - (10x1.008) = 14305.9 Lecture 2.1 14,305.14 40 ESI Transformation • Software can be used to convert these multiplet spectra into single (zero charge) profiles which gives MW directly • This makes MS interpretation much easier and it greatly increases signal to noise • Two methods are available – Transformation (requires prior peak ID) – Maximum Entropy (no peak ID required) Lecture 2.1 41 Maximum Entropy Lecture 2.1 42 ESI and Protein Structure • ESI spectra are actually quite sensitive to the conformation of the protein • Folded, ligated or complexed proteins tend to display non-gaussian peak distributions, with few observable peaks weighted toward higher m/z values • Denatured or open form proteins/peptides which ionize easier tend to display many peaks with a classic gaussian distribution Lecture 2.1 43 ESI and Protein Conformation Native Azurin Denatured Azurin Lecture 2.1 44 Matrix-Assisted Laser Desorption Ionization 337 nm UV laser cyano-hydroxy cinnamic acid MALDI Lecture 2.1 45 MALDI • Sample is ionized by bombarding sample with laser light • Sample is mixed with a UV absorbant matrix (sinapinic acid for proteins, 4hydroxycinnaminic acid for peptides) • Light wavelength matches that of absorbance maximum of matrix so that the matrix transfers some of its energy to the analyte (leads to ion sputtering) Lecture 2.1 46 MALDI Ionization Matrix + + +-+ Laser Analyte + + ++ + --+ -+ + + + + + Lecture 2.1 • Absorption of UV radiation by chromophoric matrix and ionization of matrix • Dissociation of matrix, phase change to supercompressed gas, charge transfer to analyte molecule • Expansion of matrix at supersonic velocity, analyte trapped in expanding matrix plume (explosion/”popping”) 47 MALDI • Unlike ESI, MALDI generates spectra that have just a singly charged ion • Positive mode generates ions of M + H • Negative mode generates ions of M - H • Generally more robust that ESI (tolerates salts and nonvolatile components) • Easier to use and maintain, capable of higher throughput • Requires 10 mL of 1 pmol/mL sample Lecture 2.1 48 MALDI Sample Limits • • • • • • • • Phosphate buffer < 50 mM Ammonium bicarbonate < 30 mM Tris buffer < 100 mM Guanidine (chloride, sulfate) < 1 M Triton < 0.1% SDS < 0.01% Alkali metal salts < 1 M Glycerol < 1% Lecture 2.1 49 MALDI = SELDI 337 nm UV laser cyano-hydroxy cinnaminic acid MALDI Lecture 2.1 50 MALDI/SELDI Spectra Normal Tumor Lecture 2.1 51 Mass Spectrometer Schematic Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Inlet Sample Plate Target HPLC GC Solids probe Lecture 2.1 Ion Source Mass Filter MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Detector Microch plate Electron Mult. Hybrid Detec. Data System PC’s UNIX Mac 52 Different Mass Analyzers • Magnetic Sector Analyzer (MSA) – High resolution, exact mass, original MA • Quadrupole Analyzer (Q) – Low (1 amu) resolution, fast, cheap • Time-of-Flight Analyzer (TOF) – No upper m/z limit, high throughput • Ion Trap Mass Analyzer (QSTAR) – Good resolution, all-in-one mass analyzer • Ion Cyclotron Resonance (FT-ICR) – Highest resolution, exact mass, costly Lecture 2.1 53 Magnetic Sector Analyzer Lecture 2.1 54 Mass Spec Equation (Magnet Sector) 2 2 B r m = z 2V M = mass of ion z = charge of ion V = voltage Lecture 2.1 B = magnetic field r = radius of circle 55 Quadrupole Mass Analyzer Lecture 2.1 56 Quadrupole Mass Analyzer • A quadrupole mass filter consists of four parallel metal rods with different charges • Two opposite rods have an applied potential of (U+Vcos(wt)) and the other two rods have a potential of -(U+Vcos(wt)) • The applied voltages affect the trajectory of ions traveling down the flight path • For given dc and ac voltages, only ions of a certain mass-to-charge ratio pass through the quadrupole filter and all other ions are thrown out of their original path Lecture 2.1 57 Q-TOF Mass Analyzer NANOSPRAY TIP MCP DETECTOR PUSHER HEXAPOLE QUADRUPOLE ION SOURCE Lecture 2.1 HEXAPOLE COLLISION CELL TOF REFLECTRON SKIMMER HEXAPOLE 58 Mass Spec Equation (TOF) 2 2Vt m = z L2 m = mass of ion z = charge of ion V = voltage Lecture 2.1 L = drift tube length t = time of travel 59 Ion Trap Mass Analyzer • Ion traps are ion trapping devices that make use of a threedimensional quadrupole field to trap and massanalyze ions • invented by Wolfgang Paul (Nobel Prize1989) • Offer good mass resolving power, and even MSn capability. Lecture 2.1 60 Ion Trap Mass Analyzer Lecture 2.1 61 FT-Ion Cyclotron Analzyer Lecture 2.1 62 FT-ICR • Uses powerful magnet (5-10 Tesla) to create miniature cyclotron • Originally developed in Canada (UBC) by A.G. Marshal in 1974 • FT approach allows many ion masses to be determined simultaneously (efficient) • Has higher mass resolution than any other MS analyzer available • Will revolutionize proteomics studies Lecture 2.1 63 Mass Spectrometer Schematic Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Inlet Sample Plate Target HPLC GC Solids probe Lecture 2.1 Ion Source Mass Filter MALDI ESI IonSpray FAB LSIMS EI/CI TOF Quadrupole Ion Trap Mag. Sector FTMS Detector Microch plate Electron Mult. Hybrid Detec. Data System PC’s UNIX Mac 64 MS Detectors • Early detectors used photographic film • Today’s detectors (ion channel and electron multipliers) produce electronic signals via 2o electronic emission when struck by an ion • Timing mechanisms integrate these signals with scanning voltages to allow the instrument to report which m/z has struck the detector • Need constant and regular calibration Lecture 2.1 65 Mass Detectors Electron Multiplier (Dynode) Lecture 2.1 66 Different Types of MS • ESI-QTOF – Electrospray ionization source + quadrupole mass filter + time-of-flight mass analyzer • MALDI-QTOF – Matrix-assisted laser desorption ionization + quadrupole + time-of-flight mass analyzer Lecture 2.1 67 Different Types of MS • GC-MS - Gas Chromatography MS – separates volatile compounds in gas column and ID’s by mass • LC-MS - Liquid Chromatography MS – separates delicate compounds in HPLC column and ID’s by mass • MS-MS - Tandem Mass Spectrometry – separates compound fragments by magnetic field and ID’s by mass Lecture 2.1 68 Tandem Mass Spectrometer NANOSPRAY TIP MCP DETECTOR PUSHER HEXAPOLE QUADRUPOLE ION SOURCE Lecture 2.1 HEXAPOLE COLLISION CELL TOF REFLECTRON SKIMMER HEXAPOLE 69 Tandem Mass Spectrometry • Purpose is to fragment ions from parent ion to provide structural information about a molecule • Also allows separation and identification of compounds in complex mixtures • Uses two or more mass analyzers/filters separated by a collision cell filled with Argon or Xenon • Collision cell is where selected ions are sent for further fragmentation Lecture 2.1 70 Tandem Mass Spectrometry • Different MS-MS configurations – – – – Quadrupole-quadrupole (low energy) Magnetic sector-quadrupole (high) Quadrupole-time-of-flight (low energy) Time-of-flight-time-of-flight (low energy) • Fragmentation experiments may also be performed on single analyzer instruments such as ion trap instruments and TOF instruments equipped with post-source decay Lecture 2.1 71 Different MS-MS Modes • Product or Daughter Ion Scanning – first analyzer selects ion for further fragmentation – most often used for peptide sequencing • Precursor or Parent Ion Scanning – no first filtering, used for glycosylation studies • Neutral Loss Scanning – selects for ions of one chemical type (COOH, OH) • Selected/Multiple Reaction Monitoring – selects for known, well characterized ions only Lecture 2.1 72 MS-MS & Proteomics Lecture 2.1 73 Proteomics Applications • • • • • • • • • Protein sample identification/confirmation Protein sample purity determination Detection of post-translational modifications Detection of amino acid substitutions Determination of disulfide bonds (# & status) De novo peptide sequencing Mass fingerprint identification of proteins Monitoring protein folding (H/D exchange) Monitoring protein-ligand complexes/struct. Lecture 2.1 74 Conclusions • Mass spectrometers exist in many different configurations to allow different problems to be solved • All mass spectrometers have a common architecture and relatively similar operating principles • Understanding the applications and limitations of MS in proteomics will help in understanding and meeting the bioinformatics needs in proteomics Lecture 2.1 75