Figure 4

Residue-based labeling with ²H-Leu and ¹³C-Ile. The mass spectrum for ribosomal protein S4, residues 204–224 (GEILGGMAAVEQPEKPAAQPK, z = +2). Shown in grey are experimental data points. In blue is the calculated distribution for the unlabeled species, in orange the calculated distribution for the labeled species, and in black the sum of the two. The labeled species is composed of 72% isoleucine labeled with 97% ¹³C, and 72% leucine labeled with 97% ²H₉ and 85% ²H₁ (due to hydrogen exchange). These enrichment levels were determined empirically using Envelope. In the case of the extent of amino acid enrichment, values dictated by the experimental protocol were used as a starting point. Four distinct clusters of peaks are observed in the spectrum resulting from peptides which have no labeled amino acids, a single labeled isoleucine, a single labeled leucine, or both a labeled isoleucine and a labeled leucine. Note that there is a small but significant contribution to the unlabeled peak from the fractionally labeled species (orange). This contribution is due to partial labeling with both leucine and isoleucine (72%), resulting in a statistical subset of labeled peptides that do not contain either a labeled leucine or a labeled isoleucine. The amplitude of the unlabeled species (blue) must be adjusted so that the sum of the two contributions (black) matches the experimental spectrum. The data used is available for download (Additional File 3).