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notes_proteomics.md 2.59 KiB

proteomics notes

adapted from: https://training.galaxyproject.org/training-material/topics/proteomics/slides/introduction

glossary

  • LFQ = label-free quantification

  • explorative / shotgun proteomics (untargeted)

    • DDA: data dependent acquisition
    • DIA: data independent acquisition

sample-prep

  1. protein extraction
  2. reduction (of disulfide bridges) and alkylation (of cysteins)
    • ensures that tryptic peptides are separated from each other and allows their mass based identification
  3. tryptic digestion: Trypsin cleaves the amino acid sequences C-terminal of arginin and lysine
  4. desalting: clean up step to protect the instrument from contamination and clogging

LC

  • separates peptide mixture according to hydrophobicity
  • reduces sample complexity and gives the mass spectrometer time for the measurement
  • acidic LC buffer charges the peptides positively at their N-terminus and the basic lysin or arginin amino acids on the C-terminus
  • electrospray ionization:
    • LC column is directly connected with ion source needle
    • high voltage and heat are applied to evaporate the ionized peptides into the gas phase
    • mass spectrometer mass analyzer separates peptides based on their mass-to-charge ratio

LC-MS/MS - liquid chromatography tandem mass spectrometry

  • While sample elutes from the LC column, thousands of mass spectra are acquired
  • First, a mass spectrum of all peptides: MS1 spectra
  • From MS1 spectra the N most abundant peptide peaks are determined. These topN peptides get fragmented. N is typically between 3 and 20
    • filter unit of the mass spectrometer (a quadrupole) allows only these peptides to pass
    • One after the other is selected in the filter unit and then fragmented by collision with neutral gas molecules. This fragmentation breaks the peptide bonds and generates peptide fragments
    • The peptide fragments are measured again via the mass analyzer and detector: MS2 spectra.
    • After all topN peptides were fragmented and measured, another full MS1 mass spectra is acquired.
    • MS1 and MS2 spectra are acquired in this way during the elution of the sample from the LC.

data analysis

  1. peptides are identified via their MS2 fragmentation spectra
  2. From these peptide identities the corresponding proteins are assembled
  3. The MS1 spectra are used for peptide quantification
  4. Peptide quantities are summarized into protein quantities