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_DominikNotes/2022-04-12_anja_stefanski.md

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+

_DominikNotes/notes_proteomics.md

+
+# proteomics notes
+
+> adapted from: https://training.galaxyproject.org/training-material/topics/proteomics/slides/introduction
+
+- [glossary](#glossary)
+- [sample-prep](#sample-prep)
+- [LC](#lc)
+- [LC-MS/MS - liquid chromatography tandem mass spectrometry](#lc-msms---liquid-chromatography-tandem-mass-spectrometry)
+- [data analysis](#data-analysis)
+
+## 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
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_DominikNotes/notes_software.md

+
+# Notes on proteomics software
+
+- [MaxQuant](#maxquant)
+  - [installation](#installation)
+  - [tutorial](#tutorial)
+  - [galaxy](#galaxy)
+- [rawrr package](#rawrr-package)
+- [pyteomics](#pyteomics)
+
+## MaxQuant
+
+### installation
+
+### tutorial
+
+- hot to run maxquant on linux: https://youtu.be/KHdvO1M85VM
+- requires windows...
+
+### galaxy 
+- https://training.galaxyproject.org/training-material/topics/proteomics/tutorials/maxquant-label-free/tutorial.html
+
+
+## rawrr package
+
+- The rawrr R Package: Direct Access to Orbitrap Data and Beyond
+- https://pubs.acs.org/doi/10.1021/acs.jproteome.0c00866
+
+
+## pyteomics 
+
+- https://github.com/levitsky/pyteomics
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