Mass Spectrometry 101 An Introductory Lecture On Mass Spectrometry Fundamentals .

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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.
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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

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What does a mass spectrometer do? 1. It quantifies mass superior to whatever other procedure. 2. It can give data about substance structures. What are mass estimations useful for? To recognize, confirm, and quantitate: metabolites, recombinant proteins, proteins separated from normal sources, oligonucleotides, sedate applicants, peptides, manufactured natural chemicals, polymers

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Applications of Mass Spectrometry Pharmaceutical investigation Bioavailability considers Drug digestion system concentrates on, pharmacokinetics Characterization of potential medications Drug debasement item examination Screening of medication competitors Identifying drug targets Biomolecule portrayal Proteins and peptides Oligonucleotides Environmental examination Pesticides on nourishments Soil and groundwater sullying Forensic investigation/clinical

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Ion source : makes particles How does a mass spectrometer function? Test Mass analyzer : isolates particles Mass range: presents data

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Mass Spectrometer Block Diagram High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

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Mass Spectrometer Block Diagram Turbo sub-atomic pumps High Vacuum System Ion source Mass Analyzer Data System Inlet Detector

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Sample Introduction High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector HPLC Flow infusion Sample plate

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Ion Source High Vacuum System Ion Source Mass Analyzer Data System Inlet Detector MALDI ESI FAB LSIMS EI CI

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Ion Sources make particles from test atoms (Ions are less demanding to identify than unbiased particles.) Partial vacuum Sample Inlet Nozzle (Lower Voltage) Pressure = 1 atm Inner tube diam. = 100 um MH + N 2 + MH 2 + Sample in arrangement + + N 2 gas + MH 3 + High voltage connected to metal sheath (~4 kV) Charged beads Electrospray ionization:

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MALDI: Matrix Assisted Laser Desorption Ionization Sample plate Laser h n 1. Test is blended with grid (X) and dried on plate. 2. Laser streak ionizes lattice atoms. 3. Test particles (M) are ionized by proton exchange: XH + M  MH + X. MH + Grid (0 V) +/ - 20 kV

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Mass Analyzer High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Time of flight (TOF) Quadrupole Ion Trap Magnetic Sector FTMS

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Mass analyzers isolate particles in view of their mass-to-charge proportion (m/z) Operate under high vacuum (keeps particles from chancing upon gas atoms) Actually measure mass-to-charge proportion of particles (m/z) Key details are determination , mass estimation exactness , and affectability . A few sorts exist: for bioanalysis, quadrupole, time-of-flight and particle traps are generally utilized.

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Quadrupole Mass Analyzer Uses a blend of RF and DC voltages to work as a mass channel. Has four parallel metal bars. Lets one mass take a break. Can look over all masses or sit at one settled mass.

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m1 m2 m4 m3 m2 m1 m4 m3 mass checking mode m1 m2 m4 m3 single mass transmission mode Quadrupoles have variable particle transmission modes

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Time-of-flight (TOF) Mass Analyzer Source Drift locale (flight tube) + finder + V Ions are framed in heartbeats. The float area is sans field. Measures the ideal opportunity for particles to achieve the indicator. Little particles achieve the finder before huge ones.

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Ion Trap Mass Analyzer Top View Cut away side view

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Detector High Vacuum System Ion source Mass Analyzer Data System Inlet Detector Microchannel Plate Electron Multiplier Hybrid with photomultiplier

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Ions are identified with a microchannel plate essential particle - 1000V + - e - e L - e - e - 100V D L >> D

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

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40000 30000 20000 10000 0 The mass range demonstrates the outcomes MALDI TOF range of IgG MH + Relative Abundance (M+2H) 2+ (M+3H) 3+ 50000 100000 150000 200000 Mass (m/z)

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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 proportion

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How do mass spectrometers get their names? Sorts of particle sources: Electrospray (ESI) Matrix Assisted Laser Desorption Ionization (MALDI) Types of mass analyzers: Quadrupole (Quad, Q) Ion Trap Time-of-Flight (TOF) Either source sort can work with either analyzer sort: "MALDI-TOF," "ESI-Quad." Analyzers can be consolidated to make "half breed" instruments . ESI-QQQ, MALDI QQ TOF, Q Trap

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Voyager-DE STR MALDI TOF Sample Linear Extraction plate Reflector locator matrices Timed particle finder selector Reflector Laser Camera Pumping

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QSTAR TM ESI QQ TOF or MALDI QQ TOF Sample Q0 Q1 Q2 Effective Flight Path = 2.5 m Ion Mirror (reflector)

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QTRAP: Linear Ion Trap on a Triple Quadrupole another sort of instrument… . Q0 Q1 Q2 Q3 Exit direct particle trap

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Summary: securing a mass range Ionization Mass Sorting (separating) Detection Ion Source Ion Detector Mass Analyzer Form particles (charged atoms) Sort Ions by Mass (m/z) Detect particles 100 75 Inlet • Solid • Liquid • Vapor 50 25 0 1330 1340 1350 Mass Spectrum

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How is mass characterized? Appointing numerical esteem to the inherent property of "mass" depends on utilizing carbon-12, 12 C, as a source of perspective point. One unit of mass is characterized as a Dalton (Da). One Dalton is characterized as 1/12 the mass of a solitary carbon-12 molecule. Subsequently, one 12 C molecule has a mass of 12.0000 Da.

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Isotopes + Most components have more than one stable isotope. For instance, most carbon iotas have a mass of 12 Da, however in nature, 1.1% of C particles have an additional neutron, making their mass 13 Da. + Why do we give it a second thought? Mass spectrometers can "see" isotope crests if their determination is sufficiently high. On the off chance that a MS instrument has determination sufficiently high to determine these isotopes, better mass exactness is accomplished.

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Stable isotopes of most bounteous components of peptides

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1981.84 1982.84 1983.84 Mass range of peptide with 94 C-iotas (19 amino corrosive buildups) "Monoisotopic mass" No 13 C particles (every one of the 12 C) One 13 C molecule Two 13 C particles

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4361.45 4360.45 m/z Isotope design for a bigger peptide (207 C-particles)

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Mass range of insulin 2 x 13 C 13 C 12 C : 5730.61 Insulin has 257 C-molecules. Over this mass, the monoisotopic pinnacle is too little to ever be extremely helpful, and the normal mass is typically utilized.

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Monoisotopic mass When the isotopes are unmistakably determined the monoisotopic mass is utilized as it is the most precise estimation.

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Average mass Average mass relates to the centroid of the uncertain pinnacle group When the isotopes are not determined, the centroid of the envelope compares to the weighted normal of all the isotope tops in the bunch, which is the same as the normal or substance mass.

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What if the determination is not all that great? At lower determination, the mass measured is the normal mass . Better determination Poorer determination 6130 6140 6150 6160 6170 Mass

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How is mass determination figured? M R = M/D M FWHM = D M

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Resolution =18100 8000 6000 Resolution = 14200 Counts 4000 Resolution = 4500 2000 0 2840 2845 2850 2855 Mass (m/z) Mass estimation precision relies on upon determination High determination implies better mass exactness 15 ppm mistake 24 ppm blunder 55 ppm mistake

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How would we accomplish predominant mass determination? Reflector TOF Mass Analyzer Delayed Extraction on a MALDI source

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Important execution components Mass exactness : How precise is the mass estimation? Determination : How all around isolated are the tops from each other? Affectability : How little a sum can be broke down?

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What is MSMS? MS/MS implies utilizing two mass analyzers (consolidated in one instrument) to choose an analyte (particle) from a blend, then produce sections from it to give auxiliary data. Blend of particles Single particle Fragments Ion source MS-1 MS-2

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What is MS/MS? 1 peptide chose for MS/MS Peptide blend + MS/MS + The masses of the considerable number of pieces give a MS/MS range Have just masses to begin

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Interpretation of a MSMS range to infer auxiliary data is closely resembling settling a confuse + Use the section particle masses as particular bits of the confound to sort the in place atom pull out

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Cleavages Observed in MS/MS of Peptides y n-i x n-i an i b i low vitality high vitality z n-i v n-i w n-i - HN- - CH- - CO- - NH- - CH- - CO- - NH-R i CH-R\' R" c i d i+1

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E=Glu G=Gly S=Ser F=Phe N=Asn P=Pro V=Val A=Ala R=Arg Peptide Fragmentation => E G S F G 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 =

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Protein Identification 1. Peptide Mass Finger Printing (PMF) from MS information 2. Database look utilizing piece particle masses from MS/MS information 3. Grouping Tags from MS/MS information

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Bank President Who looted the bank? Scholar What protein was detached? Issue

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Mass Spectrometrist 1. Talk with researcher who separated the protein 2. Separate protein to get peptide blend 3. Break down peptide blend by MS to acquire peptide sub-atomic masses! Cop 1. Talk with witnesses 2. Tidy for fingerprints GATHER EVIDENCE catalyst

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Police Officer Height: 5\'7" Weight: 160 lbs Gender: male Age: 35-40 Fingerprints Mass Spectrometrist Approx. atomic weigh

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