Abstract There is considerable interest in developing antibodies as modulators of signaling pathways. One of the most important signaling pathways in higher eukaryotes is the phosphoinositide 3-kinase (PI3K) pathway, which plays fundamental roles in growth, metabolism and immunity. The class IB PI3K, PI3K γ , is a heterodimeric complex composed of a catalytic p110 γ subunit bound to a p101 or p84 regulatory subunit. PI3K γ is a critical component in multiple immune signaling processes and is dependent on activation by Ras and GPCRs to mediate its cellular roles. Here we describe the rapid and efficient characterization of multiple PI3K γ single chain camelid nanobodies using hydrogen deuterium exchange mass spectrometry (HDX-MS) for structural and biochemical studies. This allowed us to identify nanobodies that stimulated lipid kinase activity, blocked Ras activation and specifically inhibited p101-mediated GPCR activation. Overall, this reveals novel insight into PI3K γ regulation and identifies sites that may be exploited for therapeutic development. Highlights – HDX-MS rapidly identifies epitopes of camelid single-chain nanobodies raised against Class IB PI3K complexes, p110 γ /p101 and p110 γ /p84 – A nanobody targeting p101 improves local resolution in EM studies with p110 γ /p101 facilitating structural characterization of the complex – Nanobodies that bind at the interfaces with the lipidated activators Ras and G βγ can prevent activation of p110 γ /p101 and p110 γ /p84

HDX-MS optimized approach to characterize nanobodies as tools for biochemical and structural studies of class IB phosphoinositide 3-kinases