Dear Parimal,

looking through your data I was surprised of the low intensity of the reporter ions in the reporter ion region of m/z 114 - 121 and the high background noise. Using TMT only, we normally see high intensities of the reporter ions and not much background in the lower m/z region.

It looks like your fragmentation of the reporter ion was not that succesfully as it should be.

What kind of samples are you measuring? In the slides it seems that you are monitoring singly charged molecules only due to your mass range in MS2 spectra in relation to your precursor. 3 of the 4 slides show only singly charged molecules. Are you actually observing peptides?

Hi Stephan,

Thanks for your reply. You have keen eyes :)

I do get the peptide IDs in these experiments, but not from HCD spectra. Also, I do not expect to get peptide IDs from the HCD spectra., I am using CID/HCD combination on a LTQ Orbitrap XL instrument. I first fragment the precursor using CID and scan the fragment ions using the ion trap. The same precursor is fragmented using HCD, and the fragment ion spectrum obtained using the Orbitrap. I hope that the CID spectra will give me the fragment ions for peptide ID and HCD spectra will give me the reporter ion intensities.

I merge the two spectra from one precursor together before the database search.

Another observation, I should have mentioned is that these peak clusters are present early in the LC gradient ( low organic ~5%). I do sometimes see them later in the gradient.

I think you might want to optimize HCD collision energy. I found for ur instrument, 35 to 38 seems ideal. Not sure about yours.

Hi Parimal,

I am familiar with the combined CID/HCD workflow on the Orbitrap XL instrument.

Their could be many reasons for the peak clusters (contamination due to wide isolation window, NCE settings, and so on) . Would you be so kind to upload the instrument settings from the XCalibur view to have a better overview for the instrument and method parameters?

What kind of software do you use for the database searching? In Proteome Discoverer there is no need to merge CID/HCD spectra for ID and quantification.

By the way, did you already contacted your local Thermo application specialist or their support service for this finding?

Some peaks at around 141 and M-120 are correct and here is the explanation for the peaks. These have not impact on quantitation or identification

The cluster of peaks at 141-149 belong to the reagent. They are the NMP reporter ion with the CO still attached (before the neutral loss that results in the reporter ions at 113-121). You do not see 8 ions in the group 141-149 because the way they are isotope coded means that some are still the same mass. This is outlined as follows, where -> means "gives rise to" following the loss of the coded CO:

141 -> 113 reporter

143 -> 114 & 115 reporters

145 -> 116 & 117 reporters

147 -> 118 & 119 reporters

149 -> 121 reporter

Because some of the ions in the 141-149 cluster are derived from more than one iTRAQ tag you cannot use these ions for quantitation (...well...you could use the 141 & 149 ions as these are unique).

This cluster is the same chemical species as the residual 145 ion seen in iTRAQ 4-plex. In that case, all the ions are isobaric. With the 8-plex, however, the CO is not always used in the coding, so they turn out as different masses

You should optimize CE and these should reduce.

Thanks Matthias. This is really helpful. Am I over fragmenting the labeled peptides?

I would rather think of too low collision energy for the reporter. You may want to combine actually the fragmentation of the peptide bone and the fragmentation of the reporter signal for quant in one peaklist.

Please review publications from Karl Mechtler here. E.g.

Peptide Labeling with Isobaric Tags Yields Higher Identification Rates Using iTRAQ 4-Plex Compared to TMT 6-Plex and iTRAQ 8-Plex on LTQ Orbitrap

and

Improved Precision of iTRAQ and TMT Quantification by an Axial

Extraction Field in an Orbitrap HCD Cell

Thanks Matthias. I really appreciate your help.