ELECTRONIC Supplemental INFORMATION FOR

ELECTRONIC SupplementAL INFORMATION FOR

Short communication

investigation of fragmentation of tryptophan nitrogen radical cation

Andrii Piatkivskyi1, Marshall Happ1, Justin Kai-Chi Lau2,3, K. W. Michael Siu2,3, Alan C. Hopkinson2 and Victor Ryzhov1

1 Department of Chemistry and Biochemistry, and Center for Biochemical and Biophysical Studies, Northern Illinois University, DeKalb, IL, 60115 (USA).

2 Department of Chemistry and Centre for Research in Mass Spectrometry, York University, 4700 Keele Street, Toronto, M3J 1P3 Ontario (Canada).

3 Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Avenue, N9B 3P4 Ontario (Canada)

Figure S1. Detailed mechanisms showing the fragmentation pathways of tryptophan N-indole radical cation(NH3+-TrpN). The enthalpies at 0 K (Ho0) and free energies at 298 K (Go298, in parenthesis), both in kJ mol-1, are relative toNH3+-TrpN and were calculated at the B3LYP/6-311++G(d,p) level.

Figure S2.Fragmentation spectra of m/z 131 carried out as: a) MS4 of m/z 234 ion (NH3+TrpN-NO), b) MS3 of [CuII(terpy)3MI]2+.

Figure S3. Ion-molecule reactions of di-tert-butyl nitroxidewith: a) 4-hydroxypyridinium ion (m/z 96); b) 3-methyleneindoliumion (m/z 130) obtained via CID of  radical cation of Trp; showing formation of a product at m/z 146. (at pulse duration 350 s; reaction time 1000 ms)

Figure S4. Fragmentation spectrum of D3N+-TrpN•. (Spectrum on the inset: the presence of m/z 131 - 134 ions experimentally suggests involvement of 6 hydrogens in the fragmentation: 3 from N-terminus, 1 from C-terminus, and 2 from the indole ring).

H/D Exchange Spectrum

The moderate abundance of the ion at m/z 134 caused by loss of CNH2D shows that there is scrambling prior to dissociation. Previous work [26] has shown that in the fragmentation of protonated tryptophan aromatic hydrogens at positions 2 and 4 scramble. If the scrambling noted here involves both these hydrogens then an ion at 135 (loss of CNH3) should be observed and this is present but only in very low abundance. Consequently the scrambling appears to be less extensive than in the protonated tryptophan.

Formation of the m/z 132 ion in the first step in the mechanism in Figure 1 would require loss of CND3 and this is not possible unless the α-hydrogen exchanges. Here we assume that it is only the aromatic hydrogens at positions 2 and 4 that exchange. We consider three possibilities.

No scrambling. If there is no scrambling then CNHD2 would be lost and the initial product would be the ion at m/z 133. Subsequent loss of D and H would give ions at 131 and 132, with the former being more prevalent if it is the lower energy isomer 3MI•+ that undergoes the second dissociation
One hydrogen scrambles. Either CNHD2 (60%) or CNH2D (40%) would be lost. If this were the situation then the relative abundances of the ions at m/z 133 and m/z 134 would be 3:2. Subsequent losses of H and D would give ions at m/z 131, 132 and 133.
Two hydrogens scramble. Possible losses are CNHD2 (40%), CNH2D (53.3%) and CNH3 (6.7%) giving ions at 133, 134 and 135 respectively. The spectrum shows ion m/z 135 but only in very low abundance. From this it appears that the majority of the ions at m/z 134 comes from an ion in which only one aromatic hydrogen has been involved in scrambling.