Mass Spectrometry: Methods & Theory
Slide 2Proteomics Tools Molecular Biology Tools Separation & Display Tools Protein Identification Tools Protein Structure Tools
Slide 3Mass Spectrometry Needs Ionization - how the protein is infused into the MS machine Separation - Mass and Charge is resolved Activation - protein are broken into littler parts (peptides/AAs) Mass Determination - m/z proportions are resolved for the ionized protein sections/peptides
Slide 4Protein Identification 2D-GE + MALDI-MS Peptide Mass Fingerprinting (PMF) 2D-GE + MS Peptide Sequencing/Fragment Ion Searching Multidimensional LC + MS-MS ICAT Methods (isotope naming) MudPIT (Multidimensional Protein Ident. Tech.) 1D-GE + LC + MS-MS De Novo Peptide Sequencing
Slide 5Mass Spectrometry (MS) Introduce test to the instrument Generate particles in the gas stage Separate particles on the premise of contrasts in m/z with a mass analyzer Detect particles
Slide 6Ionization strategy MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif. How does a mass spectrometer function? Make particles Separate particles Detect particles Mass range Database investigation
Slide 7Artificially trypsinated Fragmented utilizing trypsin Artificial spectra fabricated Spot expelled from gel Generalized Protein Identification by MS Spectrum of pieces created MATCH Library Database of arrangements (i.e. SwissProt)
Slide 8Methods for protein distinguishing proof
Slide 9MS Principles Different components can be extraordinarily recognized by their mass
Slide 10N - CH 2 - OH COOH HO - CH 2 CH-NH 2 HO MS Principles Different mixes can be interestingly recognized by their mass Butorphanol L-dopa Ethanol CH 3 CH 2 OH MW = 327.1 MW = 197.2 MW = 46.1
Slide 11Mass Spectrometry Analytical technique to quantify the sub-atomic or nuclear weight of tests
Slide 12Weighing proteins A mass spectrometer makes charged (particles) from atoms. Basic route is to include or take away a particles: Na Cl + e - Na Cl - Na Cl Na Cl + e - It then examines those particles to give data about the atomic weight of the compound and its substance structure.
Slide 13Mass Spectrometry For little natural atoms the MW can be resolved to inside 5 ppm or 0.0005% which is adequately exact to affirm the sub-atomic equation from mass alone For substantial biomolecules the MW can be resolved inside a precision of 0.01% (i.e. inside 5 Da for a 50 kD protein) Recall 1 dalton = 1 nuclear mass unit (1 amu)
Slide 14MS History JJ Thomson manufactured MS model to quantify m/z of electron, granted Nobel Prize in 1906 MS idea first put into practice by Francis Aston, a physicist working in Cambridge England in 1919 Designed to gauge mass of components Aston Awarded Nobel Prize in 1922
Slide 15MS History 1948-52 - Time of Flight (TOF) mass analyzers presented 1955 - Quadrupole particle channels presented by W. Paul, additionally imagines the particle trap in 1983 (wins 1989 Nobel Prize) 1968 - Tandem mass spectrometer seems Mass spectrometers are currently a standout amongst the MOST POWERFUL ANALYTIC TOOLS IN CHEMISTRY
Slide 16MS Principles Find an approach to "charge" an iota or atom ( ionization ) Place charged molecule or particle in an attractive field or subject it to an electric field and measure its speed or span of arch in respect to its mass-to-charge proportion ( mass analyzer ) Detect particles utilizing microchannel plate or photomultiplier tube
Slide 17Mass Spec Principles Sample + _ Detector Ionizer Mass Analyzer
Slide 18Ionization strategy MALDI Electrospray (Proteins must be charged and dry) Mass analyzer MALDI-TOF MW Triple Quadrapole AA seq MALDI-QqTOF AA seq and MW QqTOF AA seq and protein modif . How does a mass spectrometer function? Make particles Separate particles Detect particles Mass range Database investigation
Slide 19Mass spectrometers Time of flight (TOF) (MALDI) Measures the time required for particles to fly down the length of a chamber. Frequently joined with (MALDI-TOF) Detections from various laser blasts are found the middle value of. Different laser Tandem MS-MS/MS -detachment and recognizable proof of mixes in complex blends -instigate fracture and mass dissect the section particles. - Uses at least two mass analyzers/channels isolated by a collision cell loaded with Argon or Xenon Different MS-MS arrangements Quadrupole-quadrupole (low vitality ) Magnetic area quadrupole ( high ) Quadrupole-time-of-flight ( low vitality ) Time-of-flight-time-of-flight ( low vitality )
Slide 20Typical Mass Spectrometer
Slide 21LC/LC-MS/MS-Tandem LC, Tandem MS
Slide 22Typical Mass Spectrum Characterized by sharp, limit crests X-hub position demonstrates the m/z proportion of a given particle (for independently charged particles this compares to the mass of the particle) Height of pinnacle shows the relative plenitude of a given particle (not dependable for quantitation) Peak force demonstrates the particle's capacity to desorb or "fly" (some fly superior to others)
Slide 23All proteins are sorted in view of a mass to charge proportion (m/z) m/z proportion: Molecular weight partitioned by the charge on this protein
Slide 24Typical Mass Spectrum Relative Abundance ibuprofen 120 m/z-for independently charged particle this is the mass
Slide 25D M Resolution & Resolving Power Width of pinnacle shows the determination of the MS instrument The better the determination or determining power, the better the instrument and the better the mass precision Resolving force is characterized as: M is the mass number of the watched mass ( D M) is the contrast between two masses that can be isolated
Slide 26Resolution in MS
Slide 27Resolution in MS 783.455 QTOF 784.465 785.475 783.6
Slide 28Turbo pumps Diffusion pumps Rough pumps Rotary pumps High Vacuum System Ion Source Mass Filter Inlet Data System Detector Sample Plate Target HPLC GC Solids test TOF Quadrupole Ion Trap Mag. Segment FTMS Microch plate Electron Mult. Cross breed Detec. PC's UNIX Mac MALDI ESI IonSpray FAB LSIMS EI/CI Mass Spectrometer Schematic
Slide 29Different Ionization Methods Electron Impact ( EI - Hard strategy) little particles, 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
Slide 30Electron Impact Ionization Sample brought into instrument by warming it until it vanishes Gas stage test is shelled with electrons originating from rhenium or tungsten fiber (vitality = 70 eV) Molecule is "broke" into pieces (70 eV >> 5 eV securities) Fragments sent to mass analyzer
Slide 32EI Fragmentation of CH 3 OH CH 3 OH CH 3 OH + CH 3 OH CH 2 O=H + H CH 3 OH + CH 3 + OH CH 2 O=H + H CHO=H + Why wouldn't Electron Impact be appropriate for investigating proteins?
Slide 33Why You Can't Use EI For Analyzing Proteins EI smashs concoction bonds Any given protein contains 20 diverse amino acids EI would smash the protein into amino acids as well as amino corrosive sub-pieces and even peptides of 2,3,4… amino acids Result is 10,000's of various signs from a solitary protein - excessively perplexing, making it impossible to examine
Slide 34Soft Ionization Methods 337 nm UV laser Fluid (no salt) + _ Gold tip needle cyano-hydroxy cinnamic corrosive MALDI ESI
Slide 35Soft Ionization Soft ionization procedures keep the atom of intrigue completely in place Electro-splash ionization initially considered in 1960's by Malcolm Dole however put into practice in 1980's by John Fenn (Yale) MALDI initially presented in 1985 by Franz Hillenkamp and Michael Karas (Frankfurt) Made it conceivable to break down huge particles by means of cheap mass analyzers, for example, quadrupole, particle trap and TOF
Slide 37Ionization techniques Electrospray mass spectrometry (ESI-MS) Liquid containing analyte is constrained through a steel hairlike at high voltage to electrostatically scatter analyte. Charge bestowed from quickly dissipating fluid. Framework helped laser desorption ionization (MALDI) Analyte (protein) is blended with substantial abundance of lattice (little natural atom) Irradiated with short heartbeat of laser light. Wavelength of laser is the same as absorbance max of framework.
Slide 38Electrospray Ionization Sample broke up in polar, unpredictable support (no salts) and pumped through a stainless steel hairlike (70 - 150 m) at a rate of 10-100 m L/min Strong voltage (3-4 kV) connected at tip alongside stream of nebulizing gas causes the specimen to "nebulize" or aerosolize Aerosol is coordinated through districts of higher vacuum until beads vanish to close nuclear size (as yet conveying charges)
Slide 39Electrospray (Detail)
Slide 40Electrospray Ionization Can be altered to " nanospray " framework with stream < 1 m L/min Very delicate method, requires not exactly a picomole of material Strongly influenced by salts & cleansers Positive particle mode measures (M + H) + (add formic corrosive to dissolvable) Negative particle mode measures (M - H) - (add alkali to dissolvable)
Slide 41Positive or Negative Ion Mode? In the event that the example has practical gatherings that promptly acknowledge H+, (for example, amide and amino gatherings found in peptides and proteins) then positive particle recognition is utilized PROTEINS If a specimen has utilitarian gatherings that promptly lose a proton, (for example, carboxylic acids and hydroxyls as found in nucleic acids and sugars) then negative particle identification is utilized DNA
Slide 42Matrix-Assisted Laser Desorption Ionization 337 nm UV laser cyano-hydroxy cinnamic corrosive MALDI
Slide 43MALDI Sample is ionized by shelling test with laser light Sample is blended with an UV absorbant lattice (sinapinic corrosive for proteins, 4-hydrox
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