Quizzes & Puzzles6 mins ago
Tandem Mass Spec
I basically understand how tandem mass spec works but I am having trouble getting my head around it in a practical sense, I have been given this scenario and have done a lot of reading but still dont quite know- Describe how you might use tandem mass spec to detect and then locate a phosphorylated amino acid in a tryptic peptide?
Answers
1 to 6 of 6
Best Answer
No best answer has yet been selected by amywy88. Once a best answer has been selected, it will be shown here.
For more on marking an answer as the "Best Answer", please visit our FAQ.
Precursor or parent ion scanning is very useful in the mixtures which fragment to produce common fragment ions. Tryptic mixtures containing glycosylated peptides is included in this category, so yes, if you want an increased detection rate, you should use it.
As a rule of thumb when using MS-MS, the first analyser allows transmission of all the sample ions, whilst the second detects specific fragment ions.
The technique used will vary from lab to lab and furthermore, can vary between equipment. It really does depend on the protocol in place at your lab, so I can only give you a general idea. In my laboratories, we analyse the course trypsin digested mixture in MS mode to create a peptide map. The information gained may be sufficient from this first stage. If not, the next stage is to switch the Q-TOFMS to MS/MS mode. The protonated ions of the digest fragments are selected and sent through into the collision cell. The sample ions then collide with argon ions introduced into the chamber which result in the sample ions fragmenting. The exact moment of the argon injection into the chamber needs to be carefully calculated depending on the expected ions present and the lab staff are superb at working this out. The fragmented ions present are then analysed by the second analyser.
What you have at the end is a MS/MS spectrum showing all the fragment ions. An ion spectrum is then produced for each of the components present in the digest. The spectra are carefully analysed and the sequences compared to that in a known database. In general MS/MS is excellent at demonstrating phosphorylation, acylation and glycosylation in the form of post-translational modifications or otherwise.
As a rule of thumb when using MS-MS, the first analyser allows transmission of all the sample ions, whilst the second detects specific fragment ions.
The technique used will vary from lab to lab and furthermore, can vary between equipment. It really does depend on the protocol in place at your lab, so I can only give you a general idea. In my laboratories, we analyse the course trypsin digested mixture in MS mode to create a peptide map. The information gained may be sufficient from this first stage. If not, the next stage is to switch the Q-TOFMS to MS/MS mode. The protonated ions of the digest fragments are selected and sent through into the collision cell. The sample ions then collide with argon ions introduced into the chamber which result in the sample ions fragmenting. The exact moment of the argon injection into the chamber needs to be carefully calculated depending on the expected ions present and the lab staff are superb at working this out. The fragmented ions present are then analysed by the second analyser.
What you have at the end is a MS/MS spectrum showing all the fragment ions. An ion spectrum is then produced for each of the components present in the digest. The spectra are carefully analysed and the sequences compared to that in a known database. In general MS/MS is excellent at demonstrating phosphorylation, acylation and glycosylation in the form of post-translational modifications or otherwise.
So in the first analyser the m/z of the trypitc peptide is measured and produces a spectrum and then the tryptic peptide fragments in the collision cell, and if i use a neutral loss scan, only ions that have lost a certain amount of neutrons from losing the phosphorylation of the amino acid enter the 2nd analyser which is then produced on a spectrum? Have I got the right idea? Because you can program the mass spec to only allow through the collision cell ions that lose a certain amount of neutrons.....
Is there any fragmentation in the first analyser?
And if it is termed as a tryptic peptide does that mean that the peptide has already been digested by trypsin so its already fragmented?
mass spec is not my favourite subject!
Is there any fragmentation in the first analyser?
And if it is termed as a tryptic peptide does that mean that the peptide has already been digested by trypsin so its already fragmented?
mass spec is not my favourite subject!
Yes, you've got the right idea. In constant neutral loss scanning both analysers either scan or collect data across the whole m/z range. However, the analysers are offset, which in practice means that the second analyser allows only through those ions which differ by a certain number of mass units from the ions sent through the first analyser. As you say, this feature can be programmed into the instrument.
The kinetic energy of ions is converted to vibrational energy in the collision cell and the ions fragment. There is no fragmentation in the first analyser. The second analyser establishes the m/z values of the resultant fragment ions arriving from the collision cell.
Trypsin cleaves proteins by severing selectively at the C-terminal side of lysine and arginine residues (except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis). The consequence of this cleavage is the production of peptide fragments between many hundred to many thousand Da molecular masses. This fragment mixture, often called a "peptide fingerprint" is specific to any given protein. Just don't confuse this fragmentation with the ion fragmentation that occurs in the collision cell!
Believe me, I'm no expert in MS either, but as a biochemist I have to have a reasonable working knowledge of most of the techniques. We have a dedicated team in the Mass Spectrometry building at my own university who are the real experts in this field. All the same, we had a new MS facility built alongside my own laboratories a couple of years ago and I've not found fault with my own technicians that man the facility.
The kinetic energy of ions is converted to vibrational energy in the collision cell and the ions fragment. There is no fragmentation in the first analyser. The second analyser establishes the m/z values of the resultant fragment ions arriving from the collision cell.
Trypsin cleaves proteins by severing selectively at the C-terminal side of lysine and arginine residues (except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis). The consequence of this cleavage is the production of peptide fragments between many hundred to many thousand Da molecular masses. This fragment mixture, often called a "peptide fingerprint" is specific to any given protein. Just don't confuse this fragmentation with the ion fragmentation that occurs in the collision cell!
Believe me, I'm no expert in MS either, but as a biochemist I have to have a reasonable working knowledge of most of the techniques. We have a dedicated team in the Mass Spectrometry building at my own university who are the real experts in this field. All the same, we had a new MS facility built alongside my own laboratories a couple of years ago and I've not found fault with my own technicians that man the facility.
1 to 6 of 6
Latest Posts
ChatterBank12 mins ago
Quizzes & Puzzles36 mins ago
Quizzes & Puzzles39 mins ago
Crosswords72 mins ago