Mass Spectrometry 101 An Introductory Lecture On Mass Spectrometry Fundamentals Presented to the Sandler Mass Spectrometry Users’ Group University of California San Francisco April 11, 2003
What does a mass spectrometer do? 1. It measures mass better than any other technique. 2. It can give information about chemical structures. What are mass measurements good for? To identify, verify, and quantitate: metabolites, recombinant proteins, proteins isolated from natural sources, oligonucleotides, drug candidates, peptides, synthetic organic chemicals, polymers
Applications of Mass Spectrometry • Pharmaceutical analysis • Bioavailability studies • Drug metabolism studies, pharmacokinetics • Characterization of potential drugs • Drug degradation product analysis • Screening of drug candidates • Identifying drug targets • Biomolecule characterization • Proteins and peptides • Oligonucleotides • Environmental analysis • Pesticides on foods • Soil and groundwater contamination • Forensic analysis/clinical
Ion source:makes ions How does a mass spectrometer work? Sample Mass analyzer: separates ions Mass spectrum:presents information
Mass Spectrometer Block Diagram High Vacuum System Ion source Mass Analyzer Data System Inlet Detector
Mass Spectrometer Block Diagram Turbo molecular pumps High Vacuum System Ion source Mass Analyzer Data System Inlet Detector
Sample Introduction High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector HPLC Flow injection Sample plate
Ion Source High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector MALDI ESI FAB LSIMS EI CI
Ion Sources make ions from sample molecules(Ions are easier to detect than neutral molecules.) Partialvacuum Sample Inlet Nozzle (Lower Voltage) Pressure = 1 atmInner tube diam. = 100 um MH+ N2 + + + + + + + + + + + + MH2+ + + + + + + + + + + + + Sample in solution + + + + + + + + + + + + + + + + + N2 gas + + + + + MH3+ High voltage applied to metal sheath (~4 kV) Charged droplets Electrospray ionization:
MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser hn • 1. Sample is mixed with matrix (X) and dried on plate. • 2. Laser flash ionizes matrix molecules. • 3. Sample molecules (M) are ionized by proton transfer: XH+ + M MH+ + X. MH+ Grid (0 V) +/- 20 kV
Mass Analyzer High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS
Mass analyzers separate ions based on their mass-to-charge ratio (m/z) • Operate under high vacuum (keeps ions from bumping into gas molecules) • Actually measure mass-to-charge ratio of ions (m/z) • Key specifications are resolution, mass measurement accuracy, and sensitivity. • Several kinds exist: for bioanalysis, quadrupole,time-of-flight and ion traps are most used.
Quadrupole Mass Analyzer Uses a combination of RF and DC voltages to operate as a mass filter. • Has four parallel metal rods. • Lets one mass pass through at a time. • Can scan through all masses or sit at one fixed mass.
m1 m2 m4 m3 m2 m1 m4 m3 mass scanning mode m1 m2 m2 m2 m2 m2 m4 m3 single mass transmission mode Quadrupoles have variable ion transmission modes
Time-of-flight (TOF) Mass Analyzer Source Drift region (flight tube) + + detector + + V • Ions are formed in pulses. • The drift region is field free. • Measures the time for ions to reach the detector. • Small ions reach the detector before large ones.
Ion Trap Mass Analyzer Top View Cut away side view
Detector High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Microchannel Plate Electron Multiplier Hybrid with photomultiplier
Ions are detected with a microchannel plate primary ion - 1000V + - e - e L - e - e - 100V D L >> D
Data System High Vacuum System Ion source Mass Analyzer Data System Inlet Detector PC Sun SPARK Station DEC Station
40000 30000 20000 10000 0 The mass spectrum shows the results MALDI TOF spectrum of IgG MH+ Relative Abundance (M+2H)2+ (M+3H)3+ 50000 100000 150000 200000 Mass (m/z)
ESI Spectrum of Trypsinogen (MW 23983) M + 15 H+ 1599.8 M + 16 H+ M + 14 H+ 1499.9 1714.1 M + 13 H+ 1845.9 1411.9 1999.6 2181.6 m/z Mass-to-charge ratio
How do mass spectrometers get their names? • Types of ion sources: • Electrospray (ESI) • Matrix Assisted Laser Desorption Ionization (MALDI) • Types of mass analyzers: • Quadrupole (Quad, Q) • Ion Trap • Time-of-Flight (TOF) • Either source type can work with either analyzer type: “MALDI-TOF,” “ESI-Quad.” • Analyzers can be combined to create “hybrid” instruments. ESI-QQQ, MALDI QQ TOF, Q Trap
Voyager-DE STR MALDI TOF Sample Linear Extraction plate Reflector detector grids Timed ion detector selector Reflector Laser Camera Pumping Pumping
QSTARTM ESI QQ TOF or MALDI QQ TOF Sample Q0 Q1 Q2 Effective Flight Path = 2.5 m Ion Mirror (reflector)
QTRAP: Linear Ion Trap on a Triple Quadrupole A new type of instrument…. Q0 Q1 Q2 Q3 Exit linear ion trap
Summary: acquiring a mass spectrum Ionization Mass Sorting (filtering) Detection Ion Source Ion Detector Mass Analyzer • Form ions • (charged molecules) Sort Ions by Mass (m/z) • Detect ions 100 75 Inlet • Solid • Liquid • Vapor 50 25 0 1330 1340 1350 Mass Spectrum
How is mass defined? Assigning numerical value to the intrinsic property of “mass” is based on using carbon-12, 12C, as a reference point. One unit of mass is defined as a Dalton (Da). One Dalton is defined as 1/12 the mass of a single carbon-12 atom. Thus, one 12C atom has a mass of 12.0000 Da.
Isotopes +Most elements have more than one stable isotope. For example, most carbon atoms have a mass of 12 Da, but in nature, 1.1% of C atoms have an extra neutron, making their mass 13 Da. +Why do we care? Mass spectrometers can “see” isotope peaks if their resolution is high enough. If an MS instrument has resolution high enough to resolve these isotopes, better mass accuracy is achieved.
1981.84 1982.84 1983.84 Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) “Monoisotopic mass” No 13C atoms (all 12C) One 13C atom Two 13C atoms
4361.45 4360.45 m/z Isotope pattern for a larger peptide (207 C-atoms)
Mass spectrum of insulin 2 x 13C 13C 12C: 5730.61 Insulin has 257 C-atoms. Above this mass, the monoisotopic peak is too small to be very useful, and the average mass is usually used.
Monoisotopic mass When the isotopes are clearly resolved the monoisotopic mass is used as it is the most accurate measurement.
Average mass Average mass corresponds to the centroid of the unresolved peak cluster When the isotopes are not resolved, the centroid of the envelope corresponds to the weighted average of all the the isotope peaks in the cluster, which is the same as the average or chemical mass.
What if the resolution is not so good? At lower resolution, the mass measured is the average mass. Better resolution Poorer resolution 6130 6140 6150 6160 6170 Mass
How is mass resolution calculated? M R = M/DM FWHM = DM
Resolution =18100 8000 6000 Resolution = 14200 Counts 4000 Resolution = 4500 2000 0 2840 2845 2850 2855 Mass (m/z) Mass measurement accuracy depends on resolution High resolution means better mass accuracy 15 ppm error 24 ppm error 55 ppm error
How do we achieve superior mass resolution? Reflector TOF Mass Analyzer Delayed Extraction on a MALDI source
Important performance factors • Mass accuracy: How accurate is the mass measurement? • Resolution: How well separated are the peaks from each other? • Sensitivity: How small an amount can be analyzed?
What is MSMS? MS/MS means using two mass analyzers (combined in one instrument) to select an analyte (ion) from a mixture, then generate fragments from it to give structural information. Mixture of ions Single ion Fragments Ion source MS-1 MS-2
What is MS/MS? 1 peptide selected for MS/MS Peptide mixture + + MS/MS + + + The masses of all the pieces give an MS/MS spectrum Have only masses to start
Interpretation of an MSMS spectrum to derive structural information is analogous to solving a puzzle + + + + + Use the fragment ion masses as specific pieces of the puzzle to help piece the intact molecule back together
Cleavages Observed in MS/MS of Peptides yn-i xn-i ai bi low energy high energy zn-i vn-i wn-i -HN--CH--CO--NH--CH--CO--NH- Ri CH-R’ R” ci di+1
E=Glu G=Gly S=Ser F=Phe N=Asn P=Pro V=Val A=Ala R=Arg Peptide Fragmentation => E G S F F G E E N P N V A R 175.10 246.14 345.21 459.25 556.30 670.35 799.39 928.43 985.45 1132.52 1279.59 1366.62 1423.64 1552.69 = = =
Protein Identification 1. Peptide Mass Finger Printing (PMF) from MS data 2. Database search using fragment ion masses from MS/MS data 3. Sequence Tags from MS/MS data
Bank President Who robbed the bank? Biologist What protein was isolated? PROBLEM
Mass Spectrometrist 1. Interview biologist who isolated the protein 2. Cleave protein to obtain peptide mixture 3. Analyze peptide mixture by MS to obtain peptide molecular masses! Police Officer 1. Interview witnesses 2. Dust for fingerprints GATHER EVIDENCE enzyme
Police Officer Height: 5’7” Weight: 160 lbs Gender: male Age: 35-40 Fingerprints Mass Spectrometrist Approx. molecular weight: 30,000 Origin: bovine liver Peptide mass list from MS analysis: 975.4832, 1112.5368, 632.3147, 803.4134, 764.3892 DATABASE SEARCH search search DATABASE OF KNOWN FELONS PEPTIDE MASS DATABASE OF KNOWN PROTEINS
Police Officer Identifies the robber Anthony J. Felon Mass Spectrometrist Identifies the protein bovine carbonic anhydrase DATABASE SEARCH RESULTS
Peptide mass fingerprint of Spot A Gel coordinates: 16kDa, 4.2 (mwt, pI)