Mass Spectrometry and HPLC of peptides
The report will include:
1- Demonstration and explanation of the electrospray ionisation process using a sample peptide mixture.
2- Explanation of the principles behind reverse phase high pressure liquid chromatography.
3- Determination the mass of charge ratio (m/z) and the charge state of ionised peptides and determination of the molecular weight of the peptides by electrospray
4- Determination the mass of charge ratio (m/z), of large molecules such as proteins ionised such as lysozyme and bovine serum albumin (BSA).
5- Understanding how the quadrupole time of fight (QTOF) mass spectrometer works.
6- Separation (using chromatography) and identification (using mass spectrometry)of a protein.
7- Using ms/ms to identify a protein using Mascot for database searching (fragmentation).
The next part of this report will be answers of the following questions:
1-Document and explain the retention time elution order of the peptides from the HPLC column.
2-For HPLC why were the peptides dissolved in a solution containing 0.1% v/v formic in water acid i.e. a solution with low acetonitrile content?
3-Find out the charge state of each peptide during the chromatographic run and tabulate. Now work out the neutral mass of each peptide.
4-Why is the mass spectrometer response (i.e. signal intensity) for each peptide different during the HPLC run?
5-What is the exact mass of lysozyme and BSA determined by electrospray QTOF? How will does this compare with the theoretical mass?
6-What is the total ion chromatograph (TIC), the extracted ion chromatograph (EIC) and the base peak chromatograph (BPC)?
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