Nerve growth factor-beta (NGF) is essential for correct development of the nervous system. NGF exists both in a mature and a pro-form (proNGF). The two forms have opposing effects on neurons, in which NGF induces proliferation, while proNGF induces apoptosis via binding to a receptor complex of the common neurotrophine receptor (p75NTR) and Sortilin. Overexpression of both proNGF and Sortilin has been associated with several neurodegenerative diseases. Insights into the conformational differences between proNGF and NGF are central to a better understanding of the opposing mechanisms of action of NGF and proNGF on neurons. However, while the structure of NGF has been determined by X-ray crystallography, structural details for proNGF remain elusive. Here, using a sensitive MS-based analytical method to measure the hydrogen/deuterium exchange of proteins in solution, we analyzed the conformational properties of proNGF and NGF. We detected the presence of a localized higher-order structure in the pro-part of proNGF. Furthermore, by comparing the hydrogen/deuterium exchange in the mature part of NGF and proNGF, we found that the presence of the pro-part in proNGF causes a structural stabilization of three loop regions in the mature part, possibly through a direct molecular interaction. Moreover, using tandem MS analyses, we identified two N-linked and two O-linked glycosylations in the pro-part of proNGF. These results advance our knowledge of the conformational properties of proNGF and NGF and help provide a rationale for the diverse biological effects of NGF and proNGF at the molecular level.