With the rapid rise of therapeutic antibodies and antibody-drug conjugates, significant investments have been made in developing workflows that utilize mass spectrometry to detect these intact molecules, the large fragments generated by their selective digestion, and the peptides generated by traditional proteomics workflows. The resultant data is used to gain insight into a wide range of parameters, including primary sequence, disulfide bonding, glycosylation patterns, biotransformation, and more. However, many of the technologies utilized to couple these workflows to mass spectrometers have significant limitations that force nonoptimal modifications to upstream sample preparation steps, limit the throughput of high-volume workflows, and prevent the harmonization of diverse experiments onto a single hardware platform. Here, we describe a new analytical platform that enables direct and high-throughput coupling to electrospray ionization mass spectrometry. The SampleStream platform is compatible with both native and denaturing electrospray, operates with a throughput of up to 15 s/sample, provides extensive concentration of dilute samples, and affords similar sensitivity to comparable liquid chromatographic methods.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17β-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.

Background/aimsThis study aimed to establish a wide-field optical coherence tomography (OCT) deviation map obtained from swept-source OCT (SS-OCT) scans. Moreover, it also aimed to compare the diagnostic ability of this wide-field deviation map with that of the peripapillary and macular deviation maps currently being used for the detection of early glaucoma (EG).MethodsFour hundred eyes, including 200 healthy eyes and 200 eyes with EG were enrolled in this retrospective observational study. Patients underwent a comprehensive ocular examination, including wide-field SS-OCT (DRI-OCT Triton; Topcon, Tokyo, Japan). The individual wide-field scan was converted into a uniform template using the fovea and optic disc centres as fixed landmarks. Subsequently, the wide-field deviation map was obtained via the comparison between individual wide-field data and a normative wide-field database that had been created by combining images of healthy eyes into a uniform template in a previous study. The ability of the new wide-field deviation map to distinguish between EG and healthy eyes was assessed by comparing it with conventional deviation maps based on the area under the receiver operating characteristic curve (AUC).ResultsThe wide-field deviation map obtained using the normative wide-field database showed the highest diagnostic ability for the diagnosis of EG (AUC=0.980 and 961 for colour-coded pixels presenting <5% and <1%, respectively) among various deviation maps. Its AUC was significantly superior to that of most conventional deviation maps (p<0.05). The wide-field deviation map demonstrated early structural glaucomatous damage well over a wider area.ConclusionThe wide-field SS-OCT deviation map exhibited good performance for distinguishing between eyes with EG and healthy eyes. The visualisation of the wider damaged area on the wide-field deviation map could be useful for the diagnosis of EG in clinical settings.