2.Starting from the list of phosphoproteins thus identified, you would then scan their sequences for the presence of potential sumoylation site adjacent to identified phopsho-sites that would conform to a phospho-sumoyl motif. Ideally, you would hope to identify both modifications either on the same or different pepetides. a)Would the same tryptic phosphopeptides be produced if it is also sumoylated? Why? answer: not same tryptic peptide will be produced.Sumoylation occurs at lysine(k) of protein. If the phosphorylation and sumoylation both happened at same peptide, modifying lysine (sumoylation) will disrupt tryptic digest. And peptide will be longer than only phosphoryaltion. b)Even if the phospho-sumoylated proteins have been enriched out in the first place, you are still unlikely to detect the sumoylated peptides(with or without pSer/Thr) by your typical LC-MS/MS run why? How would it be different if i) it is in yeast system versus mammalian cells? ii) you are looking for ubiqutination and not sumoylation? Answer: why sumoylated peptides can’t detect in typical LC-MS/MS run---- Tryptic sumoylated peptides still too large to be detected in normal LC-MS/MS. Additionally, compare to phosphopetides sumoylated peptides are fewer, and have less chance to be selected for MS/MS analysis. i)after trypsin digest, the remnant sequence in yeast will be smaller than in mammalian.(參照下表) ii) tryptic ubiqutinated peptide, contain diglycine modification KxExxS/T \ (R)GG ()表示被切掉 species mass remnant sequence sumo yeast 484.5 (R)EQIGG sumo-1 human 2137.3 (K)ELGM...QTGG sumo-2,3 human 3551.7 (R)FDGQ...QTGG ubiquitin (R)GG c)if instead of starting from enriching for phosphoproteins, you choose to enrich for and to identify sumoylated proteins by using cell line expressing His6-tagged SUMO, how may quantitative labeling by SILAC help identifying true hit versus false positive ? Would you have a better chance of detecting both modifications by this alternative approach? Answer: SILAC labeling, peptide signal will appear a heavy and light m/z pair. If signal comes alone, it will be false postive. Yes, we have better chance of detecting both modification. (this assume the products of immunoaffinity purification are all sumoylated protein, and it will be easily for MS/MS analysis to find phospho site) d)If sumoylation take place only after phosphorylation of the phospho-sumoyl motif and the phospho would be taken off by phosphatase after sumoylation , how can this dynamic turnover be demonstrated by proteomic analysis? 這題是自由發揮,主要寫到利用定量及不同的時間點去偵測.大概就可以有基本分 --

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