Data dependent scan

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karthikskamath
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Data dependent scan

Postby karthikskamath » Wed Apr 18, 2012 8:51 am

Hello,

I have analyzed tryptic peptides of a pure protein on LTQ-Orbitrap with DDA and additionally I have added a parent ion list (generated through in-silico digestion of protein. Monoisotopic masses generated are used in inclusion list). Also I have enabled function of choosing most intense ion if the masses from parent ion inclusion list are not found. I have asked the system to perform MS/MS of +2 and +3 charged peptides.

Now my question is will same rule of charge state be considered for masses entered in parent ion inclusion list? Although my peptide is of length ~5 amino acids and most likely has a chance of being singly charged, I have disabled function of taking MS/MS of +1 charged stuffs to avoid collection of MS/MS infromation of single charged background ions.

Can I trick the method saying that, perform MS/MS of +1 charged ions if only the parent masses fall under the inclusion list?

Derek
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Postby Derek » Wed Apr 18, 2012 11:06 am

Unfortunately you are unable to trick the MS system to do what you want. If you enable charge exclusion, the FT preprocesses the MS1 spectrum, filtering it so only correctly assigned peaks make it through. It is those peaks that are then subsequently used for any inclusion list checks in DDA Top-N selection.

I don't know how much information you will get from a 5 amino acid peptide. Even if you selected it as a +1, your fragmentation would be very poor. Is it absolutely necessary to observe this portion of your protein? If so, maybe using multiple proteases would help you get the sequence coverage.

karthikskamath
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Postby karthikskamath » Wed Apr 18, 2012 7:54 pm

Thanks Derek!

Indeed yes. This peptide is of very much interest to me. I am checking N-ter acetylation on this peptide. As matter of fact I could get back this tryptic peptide but it was 6 amino acid length (through one missed cleavage, trypsin cleaved after adjacent K), though the ms/ms seemed to be poor. Now I am intending to use other peptidase to have better quality peptide.


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