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What is mass spectrometry.pdf

29 Mar 2023
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What is mass spectrometry.pdf

  1. What is mass spectrometry? • Analytical method which measures the mass/charge ratio and intensity of ions (and fragments) in the gas phase • Precise (<1 part per million mass accuracy) • Sensitive (<10-15 mol) • Fast (µ sec) • Unlimited mass range • >6 orders of magnitude dynamic range • No special reagents needed (low cost/sample) • Generic • Sample must be salt-free and relatively pure • Hyphenation usually needed eg. GC-MS, LC-MS
  2. All mass spectrometers consist of 3 basic components: ION SOURCE - gas phase ions are produced MASS FILTER - ions of different m/z are separated either in space or in time DETECTOR - ions generate an electrical signal
  3. All mass spectrometers consist of 3 basic components: ION SOURCE - gas phase ions are produced MASS FILTER - ions of different m/z are separated either in space or in time DETECTOR - ions generate an electrical signal • Positively charged droplets form at the spray tip • Droplets are drawn into a heated capillary in a stream of nitrogen • Desolvation occurs via a series of Coulomb explosions • Gas phase ions are produced Electrospray
  4. All mass spectrometers consist of 3 basic components: ION SOURCE - gas phase ions are produced MASS FILTER - ions of different m/z are separated either in space or in time DETECTOR - ions generate an electrical signal Time of Flight
  5. All mass spectrometers consist of 3 basic components: ION SOURCE - gas phase ions are produced MASS FILTER - ions of different m/z are separated either in space or in time DETECTOR - ions generate an electrical signal Micro Channel Plate
  6. MS workflow Sample prep (slow) data acquisition (fast) data analysis (slow)
  7. Tryptic digest MSMS workflow Sample prep Data acquisition Data analysis Data dependent peptide fragmentation. Less than 1 second per peptide fragment spectrum. Data acquired over 16 min HPLC run. (automated) Compound extraction, charge deconvolution and mgf. file generation (automated, 30 sec) 2. SDS-PAGE (60 min) 4. Trypsin digestion (16 hours) 5. In-line reversed phase HPLC (automated, 16 min) 1. Protein purification (days) Submission to batch queue and 3-phase Mascot search (automated, 3 min) Manual data evaluation, annotation and input into Scarab (2-3 min/ sample) E-mail notification 3. Reduction-alkylation (90 min)
  8. Protein intact mass workflow Sample prep Data acquisition Data analysis Data acquired over 2 min HPLC run. (automated) Compound extraction, charge deconvolution (automated, 30 sec) 2. In-line reversed phase desalting (automated, 2 min) 1. Protein purification (days) Manual data evaluation Comparison between observed and expected mass from sequence Identification of mass discrepancies PTMs (Unimod) Truncations (PAWS)
  9. Protein native MS workflow Sample prep Data acquisition Data analysis Manual direct infusion via syringe pump (20 min) Manual data analysis 1. Monomer mass 2. Non-covalent interactions 3. Charge assignment 4. Multimeric state 5. Charge distribution analysis (conformational states) 6. Charge radius (hours) 2. Off-line desalting via size exclusion spin column x 3 (30 min) 1. Protein purification (days)
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