Introduction to proteomics
- 2. Why proteomics? ● Most of the stuff are done by proteins ● Valuable publicly available resources ● Post translational modifications may influence epigenetics ● Many recent applications of machine learning in ○ Preprocessing raw files ○ Analysis ○ Interpreting ● Proteogenomics ○ Proteoepigenomics?
- 3. How does a peptide look like
- 4. How does a peptide look like
- 5. How is proteomics data generated? Image from: http://www.premierbiosoft.com/tech_notes/mass-spectrometry.html
- 6. Using trypsin to limit possibilities Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline.
- 7. 1st dimension of data: time
- 8. Other dimensions Slide from Andrei Drabovich
- 9. Possible precursor fragmentation Slide from Thomas Kislinger
- 10. In summary Slide from Thomas Kislinger
- 11. How are proteomics experiments designed? A. Labeling Cysteine after obtaining peptide mixture B. Labeling with heavy amino acids while growing cells or animal C. Reaction with iTRAQ tags Slide from Thomas Kislinger
- 12. What are the features for each peptide? 1. Time of elution 2. m/z and intensity of precursor ion (trypsinated peptides) 3. m/z and intensity of fragmented ions (mono-oligo peptides) from each precursor ion
- 13. How to analyze the data? 1. Possible expectations from a FASTA file 2. Publicly available data from known proteins 3. De novo sequencing: a. What are the possible peptides for a given precursion ion m/z b. Which of these peptides are better supported with m/z of obtained b/y ions?
- 14. How to analyze the data? Zolg, Daniel P., et al. "Building ProteomeTools based on a complete synthetic human proteome." Nature methods 14.3 (2017): 259.
- 15. How to infer post-translational modifications Chick, Joel M., et al. "A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides." Nature biotechnology 33.7 (2015): 743.
- 17. Publicly available cancer proteomics resources ● The Clinical Proteomic Tumor Analysis Consortium (CPTAC) ○ Breast cancer ○ Ovarian cancer ○ Colorectal cancer ○ Rectal adenocarcinoma ○ Skin carcinoma ○ Oral squamous cell carcinoma ○ Treated and resistant triple negative breast cancer xenografts
Editor's Notes
- #6: Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline.
- #8: SCX: Strong cation exchange C18: Hydrophobic column HPLC: High pressure liquid chromatography Acetonitrile: ACN a polar aprotic solvent Blue: reverse phase gradient Green: salt pulse and reverse phase
- #9: SCX: Strong cation exchange C18: Hydrophobic column HPLC: High pressure liquid chromatography Acetonitrile: ACN a polar aprotic solvent Blue: reverse phase gradient Green: salt pulse and reverse phase
- #12: ICAT - Isotope coded affinity tags SILAC - Stable isotope labeling by amino acids in cell culture iTRAQ - Isotope tags for relative and absolute quantification