Large-Scale Differentiation and Site Specific Discrimination of Hydroxyproline Isomers by Electron Transfer/Higher-Energy Collision Dissociation (EThcD) Mass Spectrometry

3- and 4-Hydroxyprolines (HyP) are regioisomers that play different roles in various species and organs. Despite their distinct functions inside cells, they are generally considered indistinguishable using mass spectrometry due to their identical masses. Here, we demonstrate, for the first time, that characteristic w ions can be produced by electron-transfer/higher energy collision dissociation (EThcD) dual fragmentation technique to confidently discriminate 3-HyP/4-HyP isomers. An integrated and high throughput strategy was developed which combined online LC separation with EThcD for large-scale differentiation of 3-HyP/4-HyP in complex samples. An automated algorithm was developed for charge state dependent characterization of 3-HyP/4-HyP isomers. Using this combined discrimination approach, we identified 108 3-HyP sites and 530 4-HyP sites from decellularized pancreas, allowing more than 5-fold increase of both 3-HyP and 4-HyP identifications compared to previous reports. This approach outperformed ETD and HCD in the analysis of HyP-containing peptides with unique capacity to generate w ions for HyP discrimination, improved fragmentation of precursor ions, as well as unambiguous localization of modifications. A high content of 3-HyP was observed in the C-terminal (GPP)_n domain of human CO1A1, which was previously only identified in vertebrate fibrillar collagens from tendon. Unexpectedly, some unusual HyP sites at Xaa position in Gly-HyP-Ala, Gly-HyP-Val, Gly-HyP-Gln, Gly-HyP-Ser, and Gly-HyP-Arg were also confirmed to be 3-hydroxylated, whose functions and enzymes are yet to be discovered. Overall, this novel discrimination strategy can be readily implemented into de novo sequencing or other proteomic search engines

DataSheet1_Validating expression of beta cell maturation-associated genes in human pancreas development.PDF

The identification of genes associated with human pancreatic beta cell maturation could stimulate a better understanding of normal human islet development and function, be informative for improving stem cell-derived islet (SC-islet) differentiation, and facilitate the sorting of more mature beta cells from a pool of differentiated cells. While several candidate factors to mark beta cell maturation have been identified, much of the data supporting these markers come from animal models or differentiated SC-islets. One such marker is Urocortin-3 (UCN3). In this study, we provide evidence that UCN3 is expressed in human fetal islets well before the acquisition of functional maturation. When SC-islets expressing significant levels of UCN3 were generated, the cells did not exhibit glucose-stimulated insulin secretion, indicating that UCN3 expression is not correlated with functional maturation in these cells. We utilized our tissue bank and SC-islet resources to test an array of other candidate maturation-associated genes, and identified CHGB, G6PC2, FAM159B, GLUT1, IAPP and ENTPD3 as markers with expression patterns that correlate developmentally with the onset of functional maturation in human beta cells. We also find that human beta cell expression of ERO1LB, HDAC9, KLF9, and ZNT8 does not change between fetal and adult stages.</p