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. 2016 Jan 7;3:70. doi: 10.3389/fchem.2015.00070

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Copyright © 2016 Nuriel, Whitehouse, Ma, Mercer, Brown and Gross.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Optimization of the ANSID protocol. Base peak chromatograms (BPCs) showing the results of optimization experiments for (A) the amine-blocking step, (B) the nitro group reducing step, and (C) the amine-specific tagging/capture step. In Panel (A), a pure synthetic peptide, ALAAG(Y-NO₂)DVEK (spectrum a), derived from histone H1.2, was modified by either acetylation with acetic anhydride (spectrum b) or reductive dimethylation with formaldehyde and dimethylamine borane (spectrum c). When the unmodified peptide (m/z 576.8) was modified by acetylation, it became undetectable by MS (presumably due to loss of the essential positive charge), whereas reductive dimethylation resulted in the near-complete modification of the peptide to its dimethylated form (m/z 604.8) and did not interfere with its discovery by MS. In Panel (B), the nitrotyrosine group on the dimethylated histone H1.2-derived nitrated peptide (spectrum a) was reduced to aminotyrosine with either sodium dithionite (spectrum b) or hemin-agarose beads and dithiothreitol (spectrum c). While reduction with sodium dithionite caused the formation of a plus 80-dalton side product (m/z 629.8) in addition to the reduced dimethylated peptide (m/z 589.8), reduction with hemin-agarose beads and dithiothreitol resulted in near-complete reduction of nitrotyrosine without the significant formation of side-products. In Panel (C), the ANSID was performed on nitrated BSA spiked into untreated rat brain homogenate at a concentration of 100 μg NO₂BSA per mg total protein, using either amine-specific tagging with Sulfo-NHS-SS-biotin and then affinity capture with streptavidin-agarose beads (spectrum a) or direct amine-specific capture with aldehyde-agarose beads (sectrum b). While the Sulfo-NHS-SS-biotin-mediated ANSID, which was performed on 20 mg total protein, resulted in the identification of only one nitrated BSA peptide, the aldehyde-agarose-mediated ANSID, which was performed on only 1 mg of total protein, resulted in the identification of eight nitrated BSA peptides.