Charge state dependent top-down characterisation using electron transfer dissociation.
The dissociation of protein ions (5-30  kDa) as a function of charge state has been explored in order to suggest the optimal charge state range for top-down sequencing. Proteins were generated under denaturing conditions and their charge states were modified via ion/ion proton transfer reactions prior to dissociation. Electron transfer dissociation (ETD) data suggested optimal sequence coverage for charge states in the m/z range from 700 to 950 while limited sequence coverage was noted when the precursor m/z was above 1000. Sequence coverage from ETD data was found to be dependent on protein size, with smaller proteins having better sequence coverage. An observed depletion in sequence-related information was mainly attributed to limited instrument (ion trap) performance (m/z range and resolution). For a combined ETD/collision-induced dissociation (CID) approach it is difficult to propose an optimal m/z range since good sequence coverage for CID is at intermediate charge states and the optimal m/z range increases with protein size. When only one charge state can be analysed in a combined ETD/CID approach, a range around 950  m/z is suggested as a starting point. Alternatively, two charge states should be explored, each optimal for either ETD or CID. Overall, these suggestions should be useful to achieve enhanced characterisation of smaller proteins/large protein fragments (generated from denaturing solutions) in minimal analysis times.
Ruđer Bošković Institute, HR-10002, Zagreb, Croatia. marko@irb.hr
Marko
M
Rožman
Simon J
SJ
Gaskell
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004583:Electrons; D007477:Ions / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry*
D007477:Ions; D011506:Proteins