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

Mass spectrum: presents information How does a mass spectrometer work? Sample Ion source: makes ions 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 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.) Electrospray ionization: High voltage applied to metal sheath (~4 kV) Sample Inlet Nozzle (Lower Voltage) Charged droplets + MH+ MH3+ MH2+ Pressure = 1 atm Inner tube diam. = 100 um Sample in solution N2 N2 gas Partial vacuum

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.

Quadrupoles have variable ion transmission modes mass scanning mode m1 m3 m4 m2 single mass transmission mode m2 m3 m1 m4

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 Microchannel Plate Electron Multiplier Hybrid with photomultiplier

Ions are detected with a microchannel plate + e - primary ion L D 1000V 100V L >> D

Data System High Vacuum System Ion source Mass Analyzer Data System Inlet Detector PC Sun SPARK Station DEC Station

The mass spectrum shows the results MALDI TOF spectrum of IgG MH+ 10000 20000 30000 40000 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 This instrument has the basic footprint of a triple quadrupole instrument - it’s the same as our small benchtop API2000. The modifications made were the addition of a gridded exit lens and the auxiliary AC- applied dipolar. Also the Q3 is now gold-coated stainless steel rods with two collar lenses, and this is to improve the resolution and peak shape when in LIT mode. Gold-coated rods have less pits on the surface than the stainless steel rods- less imperfect fields means better resolution / sensitivity performance. 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 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.

Stable isotopes of most abundant elements of peptides

Mass spectrum of peptide with 94 C-atoms (19 amino acid residues) “Monoisotopic mass” No 13C atoms (all 12C) 1981.84 1982.84 1983.84 One 13C atom Two 13C atoms

Isotope pattern for a larger peptide (207 C-atoms) m/z 4360.45 4361.45

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? R = M/DM FWHM = DM

High resolution means better mass accuracy Mass measurement accuracy depends on resolution High resolution means better mass accuracy 2000 4000 6000 8000 Counts 2840 2845 2850 2855 Mass (m/z) Resolution = 14200 Resolution = 4500 Resolution =18100 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

The masses of all the pieces give an MS/MS spectrum What is MS/MS? Peptide mixture 1 peptide selected for MS/MS + 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 bi yn-i zn-i low energy ai xn-i vn-i wn-i -HN--CH--CO--NH--CH--CO--NH- Ri CH-R’ R” high energy ci di+1

Peptide Fragmentation 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

What protein was isolated? PROBLEM Bank President Who robbed the bank? Biologist What protein was isolated?

GATHER EVIDENCE Mass Spectrometrist Police Officer 1. Interview witnesses 2. Dust for fingerprints 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! enzyme

Approx. molecular weight: 30,000 DATABASE SEARCH 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 Police Officer Height: 5’7” Weight: 160 lbs Gender: male Age: 35-40 Fingerprints search search DATABASE OF KNOWN FELONS PEPTIDE MASS DATABASE OF KNOWN PROTEINS

DATABASE SEARCH RESULTS Police Officer Identifies the robber Anthony J. Felon Mass Spectrometrist Identifies the protein bovine carbonic anhydrase

Peptide mass fingerprint of Spot A Gel coordinates: 16kDa, 4.2 (mwt, pI)

Mass accuracy tolerance = 15 ppm This means that the mass is within 0.015 Da at m/z 1000

MS/MS spectrum for tryptic peptide MH+ = 1025.5, EAFQLFDR, from Spot A. An MS-Tag search using the fragment ions from this spectrum confirmed the identity of Spot A as myosin light chain.

Sequence Tags from Peptide Fragmentation by MS/MS One sequence tag includes four components: peptide molecular weight (MW) partial sequence (region 2) molecular wt before partial sequence (region 1) molecular wts after partial sequence (region 3) A V I/L T Peptide measured molecular wt = 1927.2 1108.13 Partial Sequence - A-V-I/L-T- 381.1 region 1 region 2 region 3 1546.11

Sequence TAG Example from MS/MS Spectrum Peptide MW = 1345.70 y 1 9 8 7 6 5 4 3 2 b I / L a . - H O ( ) 200 400 600 800 1000 1200 m/z, amu 100 300 500 700 Sequence Tag (739.34)SVS(I/L)(1120.60) 739.34 1120.60 [M+2H]2+ S V I/L

Sequence Tag search identifies 1 hit (carbonic anhydrase)

Acknowledgements We thank the Applied Biosystems Mass Spectrometry Applications Laboratory for allowing the use of some of their slides for this presentation.