邱老師說希望大家有學到東西,所以公佈題目及參考答案
(還有英文答案是偶寫的...不是邱寫的,所以中文化的英文,大家應該還是看的懂吧)
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
(就是說至少要寫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)
這題如果寫LTQ-FTMS如上,或者寫其它儀器的scan functions(儀器及scan種類)
兩者答案任寫一種.
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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