Large molecules
23 important questions on Large molecules
What are special features of biopharmaceuticals?
- relatively few toxic side effects
- typically of macromolecular nature
- potential to evoke immunogenic reactions.
Commercially interesting (the hit rate for biopharmaceuticals is quite a big higher than for small molecules)
What are the disadvantages of ligand binding assays?
- there is a small linear calibration range
- only one analyte per assay method
- no correction for experimental variability (by internal standard)
- Relatively poor precision and accuracy (you don't know if the antibody recognises other things as well)
- comparison of results between labs is difficult
What are the disadvantages of using LC-MS for large molecules?
- the sensitivity is often insufficient. large molecules can have multiple charge states which makes it difficult to measure them in small amounts. Furthermore, only one of the charge states can be used for a quantitative method. For normal LC-MS molecules can be measured until a mass of 2000.
- decreased precision: the abundance of charge states may vary from sample to sample.
- it is complicated and expensive compared to ligand binding assays.
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What is the normal procedure of LC-MS/MS for large molecules?
- You extract the protein from it.
- You digest it
- You extract the peptide
- You measure it with LC-MS
What is the purpose of protein extraction?
- decrease the complexity of the sample
- concentrate the analyte
How does it work (immunocapture)?
Then you have a washing phase in which all the not attached stuff is washed away.
Then the elution takes place. This can been done for example very easily with magnetic beads if the antibodies are attached to magnetic beads, then with a magnet the elution can take place very easily.
What are other extraction techniques beside immunocapture?
- This is relatively selective
- However, It might be that there is an intermediate clean-up necessary.
- There is a low risk of interference and intermediate sensitivity.
- This is more generic.
What percentage of analyte can be foudn back after protein extraction?
What enzyme is mostly used for digestion?
What are alternatives for trypsin?
- lys-c: this one cleaves on the carboxyl side of lysine
- Gluc-c: this one cleaves on the carboxyl side of glutamic acid and at a lower rate aspartic acid.
However these alternatives are much more expensive than trypsin.
What are the requirements for peptide selection?
- stability: the peptide should preferably contain no unstable amino acids such as methionine, cysteine, tryptophan (oxidation) or asparagine or glutamine (deamidation)
What are the requirements for the peptide that is selected?
- mass spectrometry: the peptide should have a favourable ionization and fragmentation characteristics.
How are peptides extracted?
If protein extraction is performed before digestion, the digest is relatively clean and peptide extraction often is not needed.
If protein extraction is not performed, the digest is highly complex and peptide extraction is needed to improve selectivity.
For what reason a high concentration of NaCl is used for washing? (with the nanobody in plasma)
The compound of interest can then be eluted with aqeous ammonia.
How does the ionization takes place with large molecules?
How does fragmentation occur with large molecules?
What does an internal standard correct for with large molecules?
- extraction behaviour (if co-extracted)
- digestion efficiency (if co-digested)
- derivatization yield
- transfer and injection volumes
- chromatographic performance
- detection
What are the possibilities for an internal standard for macromolecules?
- stable-isotope form of protein
- structural analogue of protein
- stable-isotope form of peptide
- structural analogue of peptide
- stable-isotope form of extended peptide
- peptide derivatized with stable-isotope labeled reagent.
What are the advantages and disadvantages of stable-isotope labeled protein?
- optimal correction for all extraction steps (including immunoaffinity extraction)
- optimal correction for digestion
- yields stable-isotope form of signature peptide, thus optimal correction for peptide extraction and MS detection
disadvantages
- not readily available
- expensive
So what are the conclusions about internal standards?
- stable isotope labeled forms are optimal
- 18O-labeled forms can be easily prepared
- if digestion is properly controlled, protein internal standards do not seem to be necessary.
Why a high resolution mass spectrometry is used for intact protein analysis?
here
- the selection of precursor ion occurs by a quadrupole (unit mass resolution)
- there is a second quadrupole for the collision-induced dissociation
- The selection of the fragment ion occurs by TOF. This means that all ions have the same kinetic energy and the ion velocity and time to reach the detector depends on the mass to charge ratio.
What is the resolving power?
How the mass can be calculated?
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