Karteikarten
Proteomics II
Karten in diesem Stapel (13)
- 1A protein is broken into peptides. You measure - your mass spectrometer has a mass tolerance of 1 Da. You find 500 hits, which it too much. What is your solution?Use a machine with a higher precision (e.g. mass tolerance of 0.00001 Da) --> machine more accurate and reduces number of hits.
- 2Is it adequate to use one peptide for identification of a protein with many repeats?No, this will not be adequate. Instead, you need more peptides to identify such a protein.
- 3What is comparative genomics?A way to find proteins which have become more abundant after a certain treatment.
- 4What is the principle behind SILAC?Amino acid (e.g. K) replaced with an isotope version --> cells are grown --> medium replaced with heavy medium --> proteins now labeled with heavy K --> MS --> see if protein was already there at time of switching or had to be synthesized.
- 5What else can SILAC be used for (relative)?SILAC can be used to measure ratios(!) between samples (different labels) by using different isotopes/different amino acids.
- 6What else can SILAC be used for (absolute)?You can take a standard (precisely determined by chemical means) to give you an idea on how many of the original peptides were present.
- 7Advantages of SILAC- Estimate relative protein levels between samples
- Can be used on complex mixtures of proteins
- Peptides can be sequenced directly if tandem MS-MS is used
- Complete in vivo labeling
- No addition of tag --> no altered chromotography - 8Disadvantages of SILAC- Expensive
- Labeling is tricky for whole organisms - 9What is the procedure for Label Free Quantification (LFQ)?- at least 3 tech. replicates of complex protein mixture --> compare with biological variant again using tech. replicates --> stat. analysis to identify proteins and calculate intensity-based absolute quantification (iBAQ) ratio by ranking t-test results --> how reliable is output? --> plot: total intensity vs. relative protein abundance ratio for each protein
- 10Advantages of LFQ?- Can be applied to all types of samples
- Estimate relative protein levels between samples with good accuracy
- Can be used on complex protein mix.
- Proteins can be sequenced directly if tandem MS-MS is used - 11Disadvantages of LFQ?- Requires multiple replicates so more materials
- Requires more MS/MS runs in case of complete proteomes
- Requires well-annotated genomes - 12How can PTM be identified when the peak height is very low?Using precursor scanning, the 3rd tube is trained to distinguish/report the presence of the mass of e.g. phosphate --> also tracks to which peptide it belongs.
- 13If there are multiple S/T/Y amino acids in a row, it is almost impossible to determine on which of the AA the modification is. What is a way to do so?B-elimination --> forces S/T/Y to lose a phosphate group --> dehydrogenated AA --> different m/z value.
However, if there are 2 S/T/Y in a row it is impossible.