邱老师说希望大家有学到东西,所以公布题目及参考答案 (还有英文答案是偶写的...不是邱写的,所以中文化的英文,大家应该还是看的懂吧) A number of proteins that contain composite phospho-sumoyl switch sequence motif KxExxS/T, for phosphorylation and sumoylation, have recently been identified. Give critical thoughts and short answers to following questions as an exercise in designing proper mass spectrometry(MS)-based proteomic approaches to identify novel candidate proteins carrying these coordinated site-specific post translational modifications(PTM) 1.The first step in any PTM analysis is to enrich a)In brief or by schematic drawing, propose sample enrichment step(s) that would help eventual detection of the Ser/Thr phosphorylation by shotgun proteomic analysis. answer: for enrich phophopetide using SCX followed by IMAC or only by IMAC (就是说至少要写IMAC) b)Assuming you will be using an LTQ-FTMS as your principal MS instrument for data dependent LC-MS/MS analysis, which scan functions that would help ID pSer/Thr sites with confidence ? How? You can propose alternative scan functions based on other appropriate MS instrument. answer: for LTQ-FTMS instrument---data dependent "neutral loss scan" function can be used. For example, if an fragment ion has a neutral loss correspond to H3PO4 (m/z 98), will trigger instrument to acquire additional stages of mass spectrometric fragmentation(MSn) 这题如果写LTQ-FTMS如上,或者写其它仪器的scan functions(仪器及scan种类) 两者答案任写一种. alternative ---for Triple quadruple instrument "precursor ion scan" can be used. In negative mode, if an ion peak correspond to PO3- (m/z 79)is detected, then instrument will switch to postive mode in order to acquire peptide sequence of precusor ion. c)How would the above experimental design be different or similar if you are aiming for i)pTyr and not for pSer/Thr ii) targeting a specific protein of interest and not for global analysis? answer: i) for pTyr using anti-pTyr antibody to enrich. And pTyr would not give a neutral loss. ii) for targeting protein ---to purify the targeting protein (affinity purification or other way). --

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