16573318
Description
Flashcards by Dominique M, updated more than 1 year ago
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Created by Dominique M
about 5 years ago
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| Question | Answer |
| A 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. |
| Is 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. |
| What is comparative genomics? | A way to find proteins which have become more abundant after a certain treatment. |
| What 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. |
| What 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. |
| What 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. |
| Advantages 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 |
| Disadvantages of SILAC | - Expensive - Labeling is tricky for whole organisms |
| What 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 |
| Advantages 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 |
| Disadvantages of LFQ? | - Requires multiple replicates so more materials - Requires more MS/MS runs in case of complete proteomes - Requires well-annotated genomes |
| How 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. |
| If 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. |
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