Recently I am facing a low labeling efficiency problem in iTRAQ experiment. The key point is, when I went to check the MS2 spectra of the non-labled peptides PD1.4 gived, I found most of them have a clear and accurate reporter ions in the low m/z range but the relative intensity is low. Do you think it is the low relative intensity of the reporter ions in a MS2 spectrum caused the spectrum being identified as a non-labled peptides? But what can cause a low relative intensity of the reporter ions in a spectrum? Thank you very much
If the parent is abundant in the MS1 spectra and the reporters are not in the MS2 you might not have enough collision energy or collision gas. There can be an issue of where the charge is located on the parent. If it's on the peptide, you don't see the reporter. If it's on the reporter, you don't see the peptide. However, this is peptide specific. If you are fragmenting a multi-charged peptide space-charging generally pushes the charges onto both the tag and the peptide. There is also the problem on not having enough tag for the amount of protein that you have in the sample. Finally, don't assume that the software gives you the right answer. It only gets the right sequence about 35% of the time on human samples, more on simpler proteomes. Try to confirm the uncharged sequence by hand. You almost never detect a non-ion in MS, although you can detect radical ions that appear to have a zero charge mass.
Firstly, you are confusing peptide identification and ITRAQ quantitation. Identification is done based on MS1 mass and MS2 fragments and does not require reporters.If a peptide is non-labelled you will see it in MS1 mass.
Secondly, as the person wrote above:
1) check collision gas
2) make sure that you are using HCD (not CID) if you have an IT/OT instrument
3) make sure you use stepped CE, i.e. low collision energy fro fragments and high for reporters
4) see what the settings are for SN in PD
You cannot identify a peptide as non-labelled (MS1 mass) but still get reporter ions (MS2 mass). Although you can, but it would be obviously wrong as mentioned in some comments above and explained below. In brief, case 1 is of identification being incorrect due to mis-assigning a labeled peptide to an incorrect unlabeled sequence of same isobaric mass, provided the reporter peaks are true. Second case could be- that the peptide is really unlabeled, but the reporters you see are just chemical noise or artefacts. If these reporters are above some threshold noise level (check S/N - signal noise ratio), then reporters may be fine and case 1 is what you observe here. If precursor is observable, at least one of reporters should have decent relative intensity as sum of these reporters (different reporter samples) results in total precursor we see in MS1. It may be also be observed (ID and Quant disagreement) due to a modified peptide which the search engine (probably sequest) assigns to either a wrong peptide or unmodified part of same peptide, being the best possible match. Also, it's possible that the peptide sequence that generated the spectrum is not in the database for whatever reason, leading to a faulty second best match turning up as the best hit.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides