Mass Spectrometry of Putrescine, Spermidine, and Spermine Covalently Attached to Francisella tularensis Universal Stress Protein and Bovine Albumin

Figure 3

MS/MS spectrum for peptide DAFLGSFLYE(184.18)Y with spermine covalently bound to Glu 356 (E356) of bovine serum albumin, in a reaction catalyzed by EDC. The parent ion mass in charge state +3 is 503.61 m/z. (a) A y1–y7 ion sequence for the peptide. The mass difference between y1 and y2 ions is 313.25 Da, consistent with glutamate-spermine. There is also a short b-ion sequence, DAF, which completes the identification of all residues in the peptide except for b4, leucine. (b) The major unassigned peaks in the spectrum define a y-ion sequence that has undergone a neutral loss of 74 Da from spermine, leaving an added mass of 110 Da. The labeled fragments, , are marked with an asterisk to denote the neutral loss. The mass of (421.25) is consistent with glutamate (148.06 for the C-terminus of a y-series) plus tyrosine (163.06) plus 110 Da. This neutral loss was not anticipated, so the search engine was not configured to identify these fragments. Their presence was only revealed by manual evaluation. A nonsequence fragment at 269.23 Da is consistent with a signature ion that is unique for the glutamate-spermine construct. Most of the remaining, unassigned fragments correspond to internal fragments or to loss of amine, water, or CO from assigned fragments.

(a)

(b)