Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity. There is an increasingly urgent need to understand the causal factors that contribute to excess lipid accumulation in the liver known as hepatosteatosis, and an equally important need to discover biomarkers and interventions for its early diagnosis and treatment. A major proportion of current and predicted global health burden stems from conditions in which hepatosteatosis is an underlying pathology 1. Defining the mechanisms that causally influence hepatosteatosis has historically proven challenging, largely owing to an ill-defined interaction between genetic and environmental factors 2. This, together with the insufficient ability for standard genome-wide association studies to capture the effect of environment on complex traits, probably explains why only a small fraction of the estimated 30% heritability for hepatosteatosis has been assigned to specific gene variants 3. Genetic reference panels (GRPs) have become a more tractable way of studying the influence of genetics and environment on complex traits, because unlike studies in humans, GRPs allow for accurate control of environment as well as access to critical metabolic tissues. Importantly, integrating intermediate phenotypes such as transcriptomics, Parker et al.

Intraoperative delineation of tumor margins is critical for effective pancreatic cancer surgery. Yet, intraoperative frozen section analysis of tumor margins is a time-consuming and often challenging procedure that can yield confounding results due to histologic heterogeneity and tissue-processing artifacts. We have previously described the development of the MasSpec Pen technology as a handheld mass spectrometry–based device for nondestructive tissue analysis. Here, we evaluated the usefulness of the MasSpec Pen for intraoperative diagnosis of pancreatic ductal adenocarcinoma based on alterations in the metabolite and lipid profiles in in vivo and ex vivo tissues. We used the MasSpec Pen to analyze 157 banked human tissues, including pancreatic ductal adenocarcinoma, pancreatic, and bile duct tissues. Classification models generated from the molecular data yielded an overall agreement with pathology of 91.5%, sensitivity of 95.5%, and specificity of 89.7% for discriminating normal pancreas from cancer. We built a second classifier to distinguish bile duct from pancreatic cancer, achieving an overall accuracy of 95%, sensitivity of 92%, and specificity of 100%. We then translated the MasSpec Pen to the operative room and predicted on in vivo and ex vivo data acquired during 18 pancreatic surgeries, achieving 93.8% overall agreement with final postoperative pathology reports. Notably, when integrating banked tissue data with intraoperative data, an improved agreement of 100% was achieved. The result obtained demonstrate that the MasSpec Pen provides high predictive performance for tissue diagnosis and compatibility for intraoperative use, suggesting that the technology may be useful to guide surgical decision-making during pancreatic cancer surgeries.

Minimally invasive robotic-assisted surgeries have been increasingly used as a first-line of treatment for patients undergoing oncologic surgeries. In-situ tissue identification is critical to guide tissue resection and assist decision-making. Traditional intraoperative histopathologic analysis of frozen tissue sections can be time-consuming and present logistical challenges which interrupt surgical workflows. We report the development and implementation of a laparoscopic, drop-in version of the MasSpec Pen device integrated into the da Vinci Xi Surgical system for in vivo tissue analysis in a robotic-assisted porcine surgery. We evaluated the performance of the drop-in MasSpec Pen during surgery by introducing the device into the animal upper gastrointestinal system and performing in vivo analyses of the stomach and liver, including charred and bloody tissues after electrocauterization. The molecular profiles obtained included ions tentatively identified as metabolites and lipids typically observed with MasSpec Pen analysis, without causing observable tissue damage. Statistical classifiers built to distinguish porcine liver and stomach tissues using the in vivo data yielded an overall tissue identification accuracy of 98% (n = 53 analyses). The results provide evidence that the drop-in MasSpec Pen developed can be used to acquire mass spectra in vivo during a robotic-assisted surgery and might be used as an in vivo tissue assessment tool to help guide surgical resections and streamline surgical workflows.

Post-translational modifications create a diverse mixture of proteoforms, leading to substantial challenges in linking proteomic information to disease. Top-down sequencing of intact proteins and multiprotein complexes offers significant advantages in proteoform analysis, but achieving complete fragmentation for such precursor ions remains challenging. Intact proteins that undergo slow-heating generally fragment via charge directed (i.e., mobile proton) or charge remote fragmentation pathways. Our efforts seek to alter this paradigm by labeling proteins with trimethyl pyrylium (TMP), which forms a stable, positively charged label at lysine residues. Fixing positive charges to the protein sequence reduces the availability of mobile protons, driving fragmentation to charge remote channels. Furthermore, we demonstrate that capping acidic side chains with carbodiimide chemistry obstructs this pathway, restoring charge-directed fragmentation and resulting in more even coverage of the protein sequence. With large amounts of fixed charge and few mobile protons, we demonstrate that it is also possible to direct fragmentation almost exclusively to lysine residues containing the charged label. Finally, our data indicate that when electrosprayed under native conditions, protein ions possess an immense capacity to accommodate excess positive charge. Molecular dynamics simulations of such ions bearing intrinsically charged labels reveal evidence of numerous charge solvation processes, including the preferential formation of helices that solvate charged labels through interactions with their macro-dipoles. Thus, these studies reveal the extent to which intact gas-phase protein ions are capable of solvating charge, and provide the most complete indication to date of the extensive physical forces opposing the goal of comprehensive top-down protein sequencing.