Dear all,
I have been working on the localization of phosphosites on my protein of interest using a variety of approaches (PhosTag SDS-PAGE, S-to-A mutations etc.) and, among others, I submitted excised CBB-stained SDS-PAGE gel bands to our MS core.
After tryptic digestion the core identified a single phosphosite in the LSAASSASSLASAGSAEGVGGAPTPK peptide (which is where we expected it to be) and shared the attached MS2 spectra comparing the putative phosphopeptide fragment pattern (top) with the non-phosphorylated one (bottom, in MS1 you can see the two +2 peaks 40 Da apart). MaxQuant localizes the phospho on a serine among the several ones present in this stretch, roughly with the same occupancy for each site (which is fine).
My question is about the masses of the fragments observed. I understand that there is no clean +80 shift anywhere, but MaxQuant annotates (unfortunately I don't have a high-res annotated spectrum right now) a series of ys (y13, y14, y15, y16, the peaks from 1109 to 1338) as 'starred', which I suppose means that they are carrying the PTM. All these seem to have a water loss shift (-18) and I understand that sometimes we observe a P+H2O loss (-98), but is there any literature you can refer me to regarding phosphoserine water losses and/or phosphopeptide scoring when a clean-cut 80-Da shift is not observed? In other words, when describing these result, should I simply say that the water losses are indicative of the presence of a +P in that region, especially since there is no corresponding '+18' peak (which in our case would be the phospho +98, lost during ionization, I guess)?
Here's the MaxQuant scoring, in case you're curious, and thank you so much to all of you who will be kind enough to weigh in!
LS(0.133)AAS(0.133)S(0.133)AS(0.133)S(0.133)LAS(0.133)AGS(0.134)AEGVGGAPT(0.068)PK
Hi,
If I remember correctly, a clean 80 Da loss is usually observed for tyrosine-phosphorylated species.
You are probably looking at the presence of a dehydroalanine in your peptide of interest that was formed upon the loss of H3PO4 from a serine in MS/MS. Usually, this loss is (also) observed in MS1.
Based on the fact that you observe a single loss of water up and until the y19 ion, I'd say the phosphorylation is on/distributed over the first four serines (from the N-terminus) in the peptide.
Hope this helps.
Dear Eef Dirksen,
thanks a lot, this clears up the picture quite a bit. I kind of see y19, but not y17/y18 in the phosphopeptide (y19 also has ~1 Da of discrepancy with the predicted mass, but I don't know what to do with that). Also not sure what to make of the 1559 and 2166 peaks (both of them, and also the putative y19, are not annotated in MaxQuant).
I took some time to dig into these MS2 spectra since I posted this question and I happen to have one more doubt on which I could use some advice/validation. If, by following the logic of the 98 Da loss (H3PO4), I ask any online MS2 fragment calculator to give me the masses for the bs and ys in the case of a S12 and an S15 phosphorylation (giving -18 Da as the PTM-induced mass shift), only S15 – and not S12 – reproduces the pattern of peaks that we observe on the spectrum (in particular y13 and y14 discriminate between the two phosphosites and we only see the peaks for a pS15, see attached). Without knowing the scoring I would feel comfortable calling the phosphosite on S15, but, while my interpretation nominally agrees with that of the program, MaxQuant barely assigns it to S15, giving it an occupancy score just above the others, thus suggesting a more complex picture than what I see. Is this a quirk of the MaxQuant scoring algorithm and I shouldn't worry too much about the occupancy absolute values or am I missing something in the interpretation of the MS2?
Hi,
You are right and I should correct my earlier statement: the phosphorylation is indeed likely to be on Ser15, even though the y12 ion seems to be missing in the MS/MS data.
Perhaps the latter gap results in a better MaxQuant score for the Ser12 variant, as the y15 for that peptide is present (but is also an expected fragment ion of the Ser15-phosphorylated peptide).
- Badges
- Science topic