Ms/Ms Peptide Identification - Please Clarify "Monoisotopic Vs Average" Parameter

Question

[Bio_X2Y]

Both Mascot and X!Tandem offer a parameter for "monoisotopic vs average" (X!Tandem calls it "spectrum, fragment mass type"). Even after reading the documentation, I'm unclear on the meaning of this parameter - perhaps someone can clarify?

In general, I understand the difference between MS1 and MS2 scans, and I understand the existence of isotope envelopes at both MS1 and MS2 levels. I'm also clear on the point that the first peak (all-12C) of an isotope envelope is the monoisotopic peak, which may or may not be the most intense peak.

My best guess regarding the parameter (although I'm not at all confident): both engines assume that you have de-isotoped your MS2 spectra before submission, and that this parameter is asking the user to clarify whether the deisotoping procedure retained the monoisotopic peak or the most intense peak.

Thanks for your time.

• X!Tandem parameter explanation
• Mascot parameter explanation

UPDATE: I have cross-posted this to the spctools-discuss mailing list since those folks are more focused on proteomics.

Answer 1

[John Cottrell]

Average is becoming obsolete. It is used when the isotopic envelope has not been resolved, so that the whole distribution has been centroided. This value may be close to the m/z of the most intense isotope peak, but it is not the same thing.

Monoisotopic is, as you say, the mass of the 12C isotope peak. De-isotoping the peak list, so as to elmiminate all but the 12C peaks, will often give better results, but it is not a requirement. If the peak list is not de-isotoped, the 13C and higher peaks simply fail to get matches. MS/MS fragments tend to be relatively low in mass, so the 12C peak is often the most intense or second most intense peak for each fragment

[Bio_X2Y]

Thanks John. I'm still unsure, however, what the parameter means in the context of Mascot/X!Tandem/etc, i.e. what are you "telling" Mascot when you choose "Monoisotopic". e.g. are you indicating that you are providing de-isotoped MS2 spectra where the monoisotopic means have been retained and the others discarded?

[Craig]

When you choose monoisotopic, you are telling the search algorithm that the input data contains monoisotopic experimental peaks so it should use monoisotopic theoretical peaks when it is doing comparisons. It doesn't necessarily mean the input data is de-isotoped.