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10 Cards in this Set
- Front
- Back
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Gel Spot Protein ID
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How to get proteins ready for MS
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excise spot from gel
reduce disulfide bonds with DTT or other reducing agent alkylate sulfhydrils with iodoacetate Digest with trypsin (or other protease) |
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MALDI-TOF-MS
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Matrix assisted lasor desorption ionization- Time of Flight- mass spec
(MALDI) The sample is co- crystallized with a low MW uv absorbing matrix. A uv laser is fired on the crystals, the matrix absorbs the uv energy, vaporizing and ionizing the matrix and sample. (TOF) The molecules are run through a time of flight analyzer |
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Tandem Mass Spectrometers
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Allows structural analysis of ions
• MS/MS in space • MS/MS in time |
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• MS/MS in space
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– Triple quadrupole
– Q-TOF – Reflectron TOF |
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• MS/MS in time
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– Quadrupole ion trap
– Fourier transform ion cyclotron resonance |
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protein mass fingerprinting
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relies only on molecular weight determination of peptide mixture (from trypsin digest)
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Mass spectrometry is:
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an analytical technique that identifies the chemical composition of a compound or sample based on the mass-to-charge ratio of charged particles
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Reversed phase HPLC
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has a non-polar stationary phase and an aqueous, moderately polar mobile phase. Fractions are then run on tandem MS.
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Tandem MS (MS/MS)
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involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring in between the stages.
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