0
$\begingroup$

I saw this figure in this atricle:
enter image description here
It's a really nice representation of what happens when using MS/MS. However I was wondering how this works when there are more proteins present in the gel spot. How would you be able to select the parent ion(because there are multiple of them)?

$\endgroup$
2
$\begingroup$

Liquid chromatography (LC) allows the peptides from a complex sample to be fed into the mass spectrometer over time. The bottom half of the image shows an MS spectrum from a single precursor scan and the machine is programmed to select the three most intense peaks (parent ions or precursor ions) from this scan for MS/MS (MS2).

enter image description here

This process is repeated throughout the LC-MS/MS run and programs usually include exclusion criteria where a parent ion is ignored for 30 - 90 seconds once it has been scanned 1 - 2 times. Usually the machine will also ignore singly charged parent ions as many of these ions will be inorganic contaminants and you want the machine to be busy with peptides.

Efficient use of machine time is critical to getting good coverage of the proteins in the sample. Proteins that are small create fewer peptides that are amenable to sequencing so these can be missed. Also those proteins that are less abundant may produce peptides that stay below the selection criteria. Good chromatography and efficient use of machine time are critical to getting good results and even in good conditions most peptides will not be subjected to MS2 sequencing. This estimates that of 100 000 peptides with only 16% were 'sequenced'. This explains why proteomics labs are speed freaks, and has driven the development of faster machines. The ~ 1 hour yeast proteome paper demonstrates the 'work' that the newer machines can get through;

Over a 1.3 h chromatographic method .... collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 peptide spectral matches and 34,255 peptides with unique amino acid sequences (1% false discovery rate (FDR)).

$\endgroup$
  • $\begingroup$ Thankyou! One thing isn't clear for me, from the MS spectra 3 peaks were selected(for peptide fragmentation). But how are the parent peaks detected? e.g. how could the program "know" if a peak is a parent ion peak or not? Because in the spectra (bottom right) all the peaks (from three peptides) are mixed up. $\endgroup$ – KingBoomie Oct 29 '16 at 11:09
  • $\begingroup$ Precursors are selected for MS2 based on their intensity, the three in the image are the three tallest (most intense) peaks. The machine switches between MS1 where the precursors are screened and MS2 where mass filters close to the source (entrance to machine) only allow precursors within the precursor window to continue to the fragmentation and measurement sections of the machine. $\endgroup$ – Michael_A Oct 29 '16 at 11:33
  • $\begingroup$ So the precursors are selected based on their intensity and if they "match" the mass of the input (which makes sense to me because they only differ by one H atom)? @Michael_A $\endgroup$ – KingBoomie Oct 29 '16 at 11:47
  • $\begingroup$ I'm not able to find information about the determination of the input mass you mentioned. Can you please add some information about the determination of these parent ion peaks, that would be great! $\endgroup$ – KingBoomie Oct 29 '16 at 11:56
  • $\begingroup$ No shotgun sequencing relies on collecting the most abundant precursors that are in the machine at any given time. This bit is not true for shotgun sequencing; and if they "match" the mass of the input (which makes sense to me because they only differ by one H atom)? The bracketed portion is only true of singly-charged precursor ions. I said mass filters in my previous comment but it is really an m/z filter. If you can quote where I mention "determination of the input mass" as I can't see where you got that from but I clearly need to improve my answer so it's clearer. $\endgroup$ – Michael_A Oct 29 '16 at 20:27

Your Answer

By clicking “Post Your Answer”, you agree to our terms of service, privacy policy and cookie policy

Not the answer you're looking for? Browse other questions tagged or ask your own question.