Reversible protein phosphorylation is a central signaling mechanism in eukaryotic cells. While the identification of canonical phosphorylation sites using mass-spectrometry (MS) based proteomics has become routine, annotation of non-canonical phosphorylation has remained a challenge. Here, we report a tailored pyrophosphoproteomics workflow to detect and reliably assign protein pyrophosphorylation in two human cell lines, providing the first direct evidence of endogenous protein pyrophosphorylation. Detection of protein pyrophosphorylation was reproducible, specific and consistent with previous biochemical evidence relating the installation of the modification to inositol pyrophosphates (PP-InsPs). We manually validated 148 pyrophosphosites across 71 human proteins, the most heavily pyrophosphorylated of which were the nucleolar proteins NOLC1 and TCOF1. A predictive workflow based on the MS data set was established to recognize putative pyrophosphorylation sequences, and UBF1, a nucleolar protein incompatible with the proteomics method, was biochemically shown to undergo pyrophosphorylation. When the biosynthesis of PP-InsPs was perturbed in a model cell line, proteins expressed in this background exhibited lower levels of pyrophosphorylation. Disruption of PP-InsP biosynthesis also significantly reduced rDNA transcription, potentially by lowering pyrophosphorylation on regulatory proteins NOLC1, TCOF1, and UBF1. Overall, protein pyrophosphorylation emerges as an archetype of non-canonical phosphorylation, and should be considered in future phosphoproteomic analyses.
An important step in elucidating the function of protein posttranslational modifications, such as pyrophosphorylation, is gaining access to site-specifically modified samples for biochemical characterization.
The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.
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