peptide LC/MS/MS

[littlewenwen]

Hi, Friends,

I have two questions for PROTEIN/PEPTIDE LC/MS/MS:

1. To check for b or y ions in MS/MS, the selected parent ions must be SINGLY charged?

2. What is the most probable charge state of the daughter ions if the parent ions is singly charged?

[Triple Quad]

Hi, Friends,

I have two questions for PROTEIN/PEPTIDE LC/MS/MS:

1. To check for b or y ions in MS/MS, the selected parent ions must be SINGLY charged?

2. What is the most probable charge state of the daughter ions if the parent ions is singly charged?

Q1. No the parent doesn't necessarily need to be singly charged to produce y or b series ions.

If you have a M+2H parent ion the you may have (y+2H)/2 ions or (b+2H)/2 ions or you can have singly charged y and b series ions from a multiply charged parent of precursor ion.

Q2. If the parent is singly charged then the product ions will be singly charged also.

Do you have a chart of the possible b1, b2, y1, and y2 ions handy to you?
manually just check to see if you have any ions that are on the chart to ID b and y series ions. Or use something like MS-Product at the Protein Prospector site to see what b and y series ions your peptides can have.

[littlewenwen]

Thank you for reply.

Do you suggest that:

1. If the parent ion is SINGLY charged, the daughter ion can ONLY be singly charged?

2. If the parent ion is multiply charged, the daughter ion can be either SINGLY or MULTIPLY charged?

[Kostas Petritis]

That is correct:

Singly charged ions give only single charged fragments

Mutliple charged ions can give single charge fragments or fragments up to the charge of the peptide. I guess they could be some exceptions but these should be really rare...

[littlewenwen]

Thank you.

I guess the usual way people is doing is to select SINGLY charged parent ions to do tandem mass which is easier to analysis. Is this correct?

If it is correct, is there any trick to make the SINGLY charged parent ions be the predominnant peak in LCMS rather than those multiply charged parent ions? By changing pH?

[Kostas Petritis]

No that is not correct, at least in the case of proteomic experiments. After tryptic digestion of proteins, the majority of the peptides will be +2 followed by +3 charge states.

You can indeed decrease the charge state of your peptides by shifting the pH to higher values but in detriment of your sensitivity. If that is not a concern to you, you can try it. Remember though that the lower the sensitivity of your molecular ion that you select for fragmentation, the less ions you will get during fragmentation...

Actually, double charge ions are quite easy to interpret, triple charged ions are more challenging, same thing for higher charge states...

[littlewenwen]

Thank you.

I saw you were also answering my another question about deconvolution. I am kinda surprised that no software is available (I used google, but couldn't find one) for such a seeming easy task (i.e., deconvolution of multiply charged ions, i don't care about deisotope). But i did see in some papers that people is talking about their "deconvoluted" ESI spectra which are apprently not in high resolution. But they didn't mention how they did the deconvolution.

I think maybe the better way to get the M.W. is by using MALDI (Because I also want to see what is the impurities in my sample, the RAW ESI spectra without deconvolution can not tell you what the impurities could be there).

[Kostas Petritis]

They do deconvolution with the help of MS/MS spectra. For example if they see in the MS/MS spectra ions with higher mass than the parent ion, it is evidently >+1 charge. If they see ions with >2 times the MW of the parent ion then it is>+2 charge. They can then test several assumptions and select the charge state that will give higher Sequest or other bioinformatic tools for their peptide...

The raw spectra it wil show you all/most impurities. Use the strength of the LCQ and try to fragment each one of your full spectra ions and try to interpret the results...