Percentage of the total signal intensity attributed to phosphopeptides (blue bars; 11 unique peptide ions) and non-phosphorylated peptides (grey bars; 25 unique peptide ions) following SAX fractionation at (A) pH 6.0, (B) pH 6.8 and (C) pH 8.0, with later fractions consisting of up to 100% phosphopeptide ion signal. Representative pHis-containing myoglobin peptide (LFTGHPETLEK; solid line), and its non-phosphorylated counterpart (LFTGHPETLEK; dashed line), quantified across all 16 SAX fractions; SAX was performed at pH 6.0 (red), pH 6.8 (blue) or pH 8.0 (green). % of the total peak area of each individual peptide across the gradient is plotted for each fraction. In order to assess pHis stability and effects of SAX separation in a complex mixture (black), SAX was also repeated at pH 6.8 with phosphorylated myoglobin spiked into a human cell lysate prior to digestion. Data for the five pHis-containing myoglobin peptides is shown in Fig. EV3. 