Lanthanides (Lns) have been shown recently to be essential cofactors in certain enzymes in methylotrophic bacteria. Here we identify in the model methylotroph, Methylobacterium extorquens, a highly selective LnIII-binding protein, which we name lanmodulin (LanM). LanM possesses four metal-binding EF hand motifs, commonly associated with CaII-binding proteins. In contrast to other EF hand-containing proteins, however, LanM undergoes a large conformational change from a largely disordered state to a compact, ordered state in response to picomolar concentrations of all LnIII (Ln = La–Lu, Y), whereas it only responds to CaII at near-millimolar concentrations. Mutagenesis of conserved proline residues present in LanM’s EF hands, not encountered in CaII-binding EF hands, to alanine pushes CaII responsiveness into the micromolar concentration range while retaining picomolar LnIII affinity, suggesting that these unique proline residues play a key role in ensuring metal selectivity in vivo. Identification and characterization of LanM provides insights into how biology selectively recognizes low-abundance LnIII over higher-abundance CaII, pointing toward biotechnologies for detecting, sequestering, and separating these technologically important elements.

Proteoglycans are complex glycoconjugates that regulate critical biological pathways in all higher organisms. Bikunin, the simplest proteoglycan having a single glycosaminoglycan chain, is a serine protease inhibitor used to treat acute pancreatitis. Unlike the template driven synthesis of nucleic acids and proteins, Golgi synthesized glycosaminoglycans are not believed to have predictable or deterministic sequence. Bikunin peptidoglycosaminoglycans were prepared and fractionated to obtain a collection of size similar and charge similar chains. Fourier transform mass spectral analysis identified a small number of parent molecular-ions corresponding to mono-compositional peptidoglycosaminoglycans. Fragmentation using collision induced dissociation surprisingly afforded a single sequence for each mono-compositional parent-ion, unequivocally demonstrating the presence of a defined sequence. The common biosynthetic pathway for all proteoglycans suggests that even more structurally complex proteoglycans, such as heparan sulfate, may have defined sequences, requiring a readjustment of our understanding of information storage in complex glycans.

Structural characterization of glycosaminoglycans (GAGs) has been a challenge in the field of mass spectrometry, and the application of electron detachment dissociation (EDD) Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has shown great promise to GAG oligosaccharide characterization in a single tandem mass spectrometry experiment. In this work, we apply the technique of negative electron transfer dissociation (NETD) to GAGs on a commercial ion trap mass spectrometer. NETD of GAGs, using fluoranthene or xenon as the reagent gas, produces fragmentation very similar to previously observed EDD fragmentation. Using fluoranthene or xenon, both glycosidic and cross-ring cleavages are observed, as well as even-and odd-electron products. The loss of SO 3 can be minimized and an increase in cross-ring cleavages is observed if a negativelycharged carboxylate is present during NETD, which can be controlled by the charge state or the addition of sodium. NETD effectively dissociates GAGs up to eight saccharides in length, but the low resolution of the ion trap makes assigning product ions difficult. Similar to EDD, NETD is also able to distinguish the epimers iduronic acid from glucuronic acid in heparan sulfate tetrasaccharides and suggests that a radical intermediate plays an important role in distinguishing these epimers. These results demonstrate that NETD is effective at characterizing GAG oligosaccharides in a single tandem mass spectrometry experiment on a widely available mass spectrometry platform.