The posttranslational modification of deimination refers to conversion of protein-bound arginine into citrulline. It is frequently detected by monoxime treatment (acid phase 2,3-butanedione and antipyrine reaction) resulting in an adduct formation. We present evidence that deiminated proteins are prone to aggregate formation upon prolonged monoxime exposure. Moreover, the efficiency of adduct formation is nonlinear and dramatically reduced with increasing polypeptide complexity [complete with smaller but progressively decreasing with higher (poly)peptides]. This nonlinearity results in vastly noncomparable detection among different methods such as immunohistochemistry, Western blot, and mass spectrometry. Mass ... More
The posttranslational modification of deimination refers to conversion of protein-bound arginine into citrulline. It is frequently detected by monoxime treatment (acid phase 2,3-butanedione and antipyrine reaction) resulting in an adduct formation. We present evidence that deiminated proteins are prone to aggregate formation upon prolonged monoxime exposure. Moreover, the efficiency of adduct formation is nonlinear and dramatically reduced with increasing polypeptide complexity [complete with smaller but progressively decreasing with higher (poly)peptides]. This nonlinearity results in vastly noncomparable detection among different methods such as immunohistochemistry, Western blot, and mass spectrometry. Mass spectrometric detection, based on mass addition of +238 on citrulline moiety with monoxime and +1 change due to deimination without monoxime treatment, corroborates serious limitations in monoxime adduct formation on proteins. Methodological limitations may also interfere with the identification of deiminated proteins as well as their modification sites and, as a consequence, the understanding of the biological role of deimination. We present here methods that alter the sequence of digestion and the combination of chromatographic techniques that collectively reduce complexity and help capture the deiminated peptides. Thin-layer chromatographic methods, together with different enzymatic digestions, can also be potentially routinely used for detection of changes in deimination sites within a given protein in different states of a cell or tissue.
