Spatially Resolved Top-Down Proteomics of Tissue Sections Based on a Microfluidic Nanodroplet Sample Preparation Platform.

Conventional proteomic approaches measure the averaged signal from mixed cell populations or bulk tissues, leading to the dilution of signals arising from subpopulations of cells that might serve as important biomarkers. Recent developments in bottom-up proteomics have enabled spatial mapping of cellular heterogeneity in tissue microenvironments. However, bottom-up proteomics cannot unambiguously define and quantify proteoforms, which are intact (i.e., functional) forms of proteins capturing genetic variations, alternatively spliced transcripts and posttranslational modifications. Herein, we described a spatially resolved top-down proteomics (TDP) platform for proteoform identification and quantitation directly from tissue sections. The spatial TDP platform consisted of a nanodroplet processing in one pot for trace samples-based sample preparation system and an laser capture microdissection-based cell isolation system. We improved the nanodroplet processing in one pot for trace samples sample preparation by adding benzonase in the extraction buffer to enhance the coverage of nucleus proteins. Using ∼200 cultured cells as test samples, this approach increased total proteoform identifications from 493 to 700; with newly identified proteoforms primarily corresponding to nuclear proteins. To demonstrate the spatial TDP platform in tissue samples, we analyzed laser capture microdissection-isolated tissue voxels from rat brain cortex and hypothalamus regions. We quantified 509 proteoforms within the union of top-down mass spectrometry-based proteoform identification and characterization and TDPortal identifications to match with features from protein mass extractor. Several proteoforms corresponding to the same gene exhibited mixed abundance profiles between two tissue regions, suggesting potential posttranslational modification-specific spatial distributions. The spatial TDP workflow has prospects for biomarker discovery at proteoform level from small tissue sections.

TopPICR: A Companion R Package for Top-Down Proteomics Data Analysis.

Top-down proteomics is the analysis of proteins in their intact form without proteolysis, thus preserving valuable information about post-translational modifications, isoforms, and proteolytic processing. However, it is still a developing field due to limitations in the instrumentation, difficulties with the interpretation of complex mass spectra, and a lack of well-established quantification approaches. TopPIC is one of the popular tools for proteoform identification. We extended its capabilities into label-free proteoform quantification by developing a companion R package (TopPICR). Key steps in the TopPICR pipeline include filtering identifications, inferring a minimal set of protein accessions explaining the observed sequences, aligning retention times, recalibrating measured masses, clustering features across data sets, and finally compiling feature intensities using the match-between-runs approach. The output of the pipeline is an MSnSet object which makes downstream data analysis seamlessly compatible with packages from the Bioconductor project. It also provides the capability for visualizing proteoforms within the context of the parent protein sequence. The functionality of TopPICR is demonstrated on top-down LC-MS/MS data sets of 10 human-in-mouse xenografts of luminal and basal breast tumor samples.

Evaluating Linear Ion Trap for MS3-Based Multiplexed Single-Cell Proteomics.

There is a growing demand to develop high-throughput and high-sensitivity mass spectrometry methods for single-cell proteomics. The commonly used isobaric labeling-based multiplexed single-cell proteomics approach suffers from distorted protein quantification due to co-isolated interfering ions during MS/MS fragmentation, also known as ratio compression. We reasoned that the use of MS3-based quantification could mitigate ratio compression and provide better quantification. However, previous studies indicated reduced proteome coverages in the MS3 method, likely due to long duty cycle time and ion losses during multilevel ion selection and fragmentation. Herein, we described an improved MS acquisition method for MS3-based single-cell proteomics by employing a linear ion trap to measure reporter ions. We demonstrated that linear ion trap can increase the proteome coverages for single-cell-level peptides with even higher gain obtained via the MS3 method. The optimized real-time search MS3 method was further applied to study the immune activation of single macrophages. Among a total of 126 single cells studied, over 1200 and 1000 proteins were quantifiable when at least 50 and 75% nonmissing data were required, respectively. Our evaluation also revealed several limitations of the low-resolution ion trap detector for multiplexed single-cell proteomics and suggested experimental solutions to minimize their impacts on single-cell analysis.

Ruptured Splenic Artery Pseudoaneurysm Causing Hemorrhage Into a Pancreatic Pseudocyst.

ABSTRACT: This case report describes fatal exsanguination due to splenic artery hemorrhage into a pancreatic pseudocyst with cystogastrostomy in a 46-year-old woman. The decedent had a complicated medical history including necrotizing pancreatitis, giant pseudocyst formation after cystogastrostomy procedure, and coiling of a hemorrhagic splenic artery. While hospitalized, she underwent embolization of a ruptured splenic artery pseudoaneurysm. Weeks later, she went into hemorrhagic shock and was ultimately pronounced at the hospital. Doctors suspected an upper gastrointestinal (GI) bleed as the cause of death; however, the patient was too unstable to undergo interventional radiology at the time.At autopsy, the pancreas was hemorrhagic and included a 15 × 15 × 15-cm pseudocyst, which contained a metallic stent from a cystogastrostomy. This case describes a unique co-occurrence of numerous common complications of chronic pancreatitis. There are multiple ways by which pancreatitis can cause upper and lower GI bleeds. In this case, the presence of a cystogastrostomy stent allowed for a ruptured pseudoaneurysm to hemorrhage through the pancreatic pseudocyst and into the stomach and duodenum, mimicking the presentation of a more common upper GI bleed. The pseudocyst then ruptured causing abdominal hemorrhage. The passage of hemorrhage through a cystogastrostomy stent is not described in other literature.

Enhancing Top-Down Proteomics of Brain Tissue with FAIMS.

Proteomic investigations of Alzheimer's and Parkinson's disease have provided valuable insights into neurodegenerative disorders. Thus far, these investigations have largely been restricted to bottom-up approaches, hindering the degree to which one can characterize a protein's "intact" state. Top-down proteomics (TDP) overcomes this limitation; however, it is typically limited to observing only the most abundant proteoforms and of a relatively small size. Therefore, fractionation techniques are commonly used to reduce sample complexity. Here, we investigate gas-phase fractionation through high-field asymmetric waveform ion mobility spectrometry (FAIMS) within TDP. Utilizing a high complexity sample derived from Alzheimer's disease (AD) brain tissue, we describe how the addition of FAIMS to TDP can robustly improve the depth of proteome coverage. For example, implementation of FAIMS with external compensation voltage (CV) stepping at -50, -40, and -30 CV could more than double the mean number of non-redundant proteoforms, genes, and proteome sequence coverage compared to without FAIMS. We also found that FAIMS can influence the transmission of proteoforms and their charge envelopes based on their size. Importantly, FAIMS enabled the identification of intact amyloid beta (Aß) proteoforms, including the aggregation-prone Aß1-42 variant which is strongly linked to AD. Raw data and associated files have been deposited to the ProteomeXchange Consortium via the MassIVE data repository with data set identifier PXD023607.

Traceless Click-Assisted Native Chemical Ligation Enabled by Protecting Dibenzocyclooctyne from Acid-Mediated Rearrangement with Copper(I).

The scope of proteins accessible to total chemical synthesis via native chemical ligation (NCL) is often limited by slow ligation kinetics. Here we describe Click-Assisted NCL (CAN), in which peptides are incorporated with traceless "helping hand" lysine linkers that enable addition of dibenzocyclooctyne (DBCO) and azide handles. The resulting strain-promoted alkyne-azide cycloaddition (SPAAC) increases their effective concentration to greatly accelerate ligations. We demonstrate that copper(I) protects DBCO from acid-mediated rearrangement during acidic peptide cleavage, enabling direct production of DBCO synthetic peptides. Excitingly, triazole-linked model peptides ligated rapidly and accumulated little side product due to the fast reaction time. Using the E. coli ribosomal subunit L32 as a model protein, we further demonstrate that SPAAC, ligation, desulfurization, and linker cleavage steps can be performed in one pot. CAN is a useful method for overcoming challenging ligations involving sterically hindered junctions. Additionally, CAN is anticipated to be an important stepping stone toward a multisegment, one-pot, templated ligation system.

Fatal Coronary Artery Vasculitis With Mixed Features.

ABSTRACT: Atherosclerotic cardiovascular disease (ASCVD) is often investigated by medical examiners as a cause of sudden death. Because of the variation in presentation of atherosclerotic cardiovascular disease, the examiner must be cautious when assigning a final diagnosis. The presented case depended upon histologic examination of coronary artery lesions to reach an appropriate final diagnosis of vasculitis with mixed features. Autopsy findings showed hepatosplenic vasculitis with noncaseating granulomas, and multifocal diffuse coronary fibrosis with histologic findings consistent with late-stage polyarteritis nodosa (PAN). However, the patient lacked the hallmark renal involvement observed in PAN. Furthermore, the vasculitis within the liver showed a highly granulomatous appearance, more consistent with IgG4 disease. In these mixed-appearance cases with limited history, exact categorization of the disease may prove difficult to impossible. Herein, we review a differential diagnosis of classic vasculitides with a focus on those that commonly affect the coronary arteries in adults, namely, PAN.

Glyphosate and Polyoxyethyleneamine Ingestion Leading to Renal, Hepatic, and Pulmonary Failure.

ABSTRACT: Glyphosate is an organophosphorus compound and the active ingredient in commonly used herbicides, whereas polyoxyethyleneamine (POEA) is a nonionic surfactant often coupled with glyphosate in these herbicides to increase their efficacy. Cases of glyphosate-POEA ingestion have shown a variety of outcomes, ranging from skin and mucosal surface irritation to death. Here, we report mortality after ingestion of at least 237 mL of an herbicide confirmed to contain both glyphosate and POEA. The decedent's electronic medical record indicates presentation to the emergency department shortly after ingestion and rapid decompensation, with death occurring on the fourth day of admission. The autopsy report showed extensive pulmonary edema and congestion with no alimentary tract abnormalities. Microscopically, airway inflammation, edema, and hemorrhage were shown as well as pericentral necrosis and macrovascular hepatic steatosis. This case is unusual for several reasons including the fatal outcome in a young 30-year-old patient, the large volume of the herbicide consumed, the associated large volume aspirated, and the lung pathology associated with exposure to glyphosate-POEA since inhalation, and in this case, aspiration is an uncommon route of glyphosate-POEA exposure. This report therefore offers rare respiratory tract pathological findings and the clinical course after aspiration of a large volume of glyphosate-POEA.

Acanthamoeba spp. and Balamuthia mandrillaris leading to fatal granulomatous amebic encephalitis.

Acanthamoeba spp. and Balamuthia mandrillaris are free-living amebae known to cause disseminated and fatal central nervous system dysfunction which manifests as granulomatous amebic encephalitis (GAE) with exceedingly rare frequency. We report two lethal cases of infection with free-living amebae: an acute case of Acanthamoeba spp. infection in an immunocompromised female and a subacute case of B. mandrillaris in a Hispanic male. The Acanthamoeba spp. infection presented with an atypical lesion in the thalamus that caused rapid deterioration of the patient while the case of B. mandrillaris had a prolonged clinical course with multifocal lesions beginning in the frontal lobe. Cerebrospinal fluid results were non-specific in both cases, however, post-mortem histology demonstrated the presence of trophozoites along a perivascular distribution of necrosis and infiltrate composed primarily of neutrophils. In addition to detailing the clinical presentations of these infrequent amebic infections, we offer insight into the difficulties surrounding their diagnoses in order to aid the clinician in accurate and timely identification.

Chemical synthesis of Shiga toxin subunit B using a next-generation traceless "helping hand" solubilizing tag.

The application of solid-phase peptide synthesis and native chemical ligation in chemical protein synthesis (CPS) has enabled access to synthetic proteins that cannot be produced recombinantly, such as site-specific post-translationally modified or mirror-image proteins (D-proteins). However, CPS is commonly hampered by aggregation and insolubility of peptide segments and assembly intermediates. Installation of a solubilizing tag consisting of basic Lys or Arg amino acids can overcome these issues. Through the introduction of a traceless cleavable linker, the solubilizing tag can be selectively removed to generate native peptide. Here we describe the synthesis of a next-generation amine-reactive linker N-Fmoc-2-(7-amino-1-hydroxyheptylidene)-5,5-dimethylcyclohexane-1,3-dione (Fmoc-Ddap-OH) that can be used to selectively introduce semi-permanent solubilizing tags ("helping hands") onto Lys side chains of difficult peptides. This linker has improved stability compared to its predecessor, a property that can increase yields for multi-step syntheses with longer handling times. We also introduce a new linker cleavage protocol using hydroxylamine that greatly accelerates removal of the linker. The utility of this linker in CPS was demonstrated by the preparation of the synthetically challenging Shiga toxin subunit B (StxB) protein. This robust and easy-to-use linker is a valuable addition to the CPS toolbox for the production of challenging synthetic proteins.

Acute Methamphetamine-Induced Hepatic and Pancreatic Ischemia.

Methamphetamine is a central nervous system stimulant that induces arousal, a positive mood, cardiac stimulation, and an acute improvement in cognitive domains. Its illicit exploitation is rapidly growing in North America. Typically, extended use of the drug induces organ damage via vasoconstriction and subsequent ischemia. This case specifically discusses hepatic and pancreatic pathology resulting from methamphetamine overdose alongside an unusual discovery of globally necrotic von Meyenburg complexes.

Embolization of Endocardial Vegetation With Stroke Presentation.

This case report details the unique cause of death of a 37-year-old Caucasian woman with a history significant for intravenous drug abuse. Before her death, she complained of extremity weakness and pain. Although her death was discovered to be the result of endocarditis, her symptoms were similar to that of a stroke. Autopsy revealed a large endocardial vegetation infecting both the tricuspid and mitral valves and a patent foramen ovale. The subsequent embolization of this vegetation caused blockages in the lungs, liver, and brain. An acute embolization of these vegetations to the bilateral middle cerebral arteries is the cause of the stroke presentation. Other comorbidities, such as cardiomegaly, microscopic evidence of myocardial infarction, and atherosclerotic disease, also contributed to the cause of death. As the opioid crisis continues in the United States, it is important to review cases involving the effects of drug use. The multiple interactions between endocarditis and the aforementioned conditions are documented to not only serve as references for future autopsies but also for the treatment of patients who have similar symptoms and comorbidities.

Miliary pattern MRSA sepsis following clandestine intravenous infusion.

Intravascular devices aid in drug administration and fluid replacement for hospitalized patients and are thus an integral part of modern medical care; however, poor aseptic technique and improper manipulation of infusion devices increase the risk of infections secondary to catheterization that can progress to sepsis and septic shock. We report the case of a woman who presented with altered mental status after receiving normal saline through an intravenous catheter placed by a medically untrained individual. Less than 24 h following her initial presentation to emergency medical services the patient became unresponsive to multiple vasopressors and broad-spectrum antibiotics and succumbed to septic shock. At autopsy, the decedent had enumerable hemorrhagic lesions consistent with septic emboli, and microscopic examination revealed clusters of coccoid-appearing bacteria. Cultures of the intravenous fluid and IV tubing collected at the decedent's home grew methicillin-resistant Staphylococcus aureus (MRSA), which was consistent with ante-mortem cultures. This case highlights the rapid clinical deterioration and autopsy presentation of MRSA sepsis due to contamination of the intravenous delivery system.

Mortality from neonatal herpes simplex viremia causing severe hepatitis.

Neonatal herpes simplex viral infections are rare in the setting of appropriate prenatal care; however, under circumstances where prenatal care is not delivered, these infections can lead to significant disease. We report a fatal case of herpes simplex virus with severe herpes hepatitis in a 14-day old male neonate. The clinical history was limited and nonspecific, however there was no prenatal care and a known history of drug abuse in the family. Autopsy revealed extensive necrosis and hemorrhage of the liver and cerebellum. Histologically, the liver revealed viral intranuclear ground glass inclusions, characteristic of herpes virus. Immunohistochemistry for herpes simplex virus performed on the both the liver and cerebellum showed strong diffuse staining in the liver and negative staining in the cerebellum. Neonatal herpes simplex virus infection is a disease of low prevalence with significant morbidity and mortality, and an exceptionally high rate of fatality in those with disseminated disease with associated fulminant hepatic failure.

An Unusual Presentation of Pituitary Gland Apoplexy With Noninfectious Meningitis.

Pituitary adenomas make up 10% of intracranial tumors, but because of their location, they may go undetected for long periods. In this article, we report the case of a 68-year-old white man found deceased in his residence, who died of acute pituitary tumor apoplexy. He was known to have severe symptoms including acute headache, vision loss, and altered behavior. When found, his home was in extreme disarray, mimicking a possible assault. At autopsy, the decedent had multiple superficial abrasions about the upper and lower extremities, as well as a 2.5 × 3-cm pituitary adenoma compressing the carotid arteries and optic nerves. Initial coroner and police investigators were strongly considering homicide with robbery as a motive, given the disarray present at the scene. This case highlights the importance of postmortem examination of the pituitary gland in all cases where neurological symptoms are reported prior to death.

Pulmonary Tumor Thrombotic Microangiopathy: Case Report and Review of Literature.

Pulmonary tumor thrombotic microangiopathy (PTTM) is a rare rapidly progressive fatal disease that is difficult to diagnosis antemortem. Activation of the coagulation cascade and fibrocellular intimal thickening caused by embolism of tumor cells into pulmonary vasculature leads to extensive pulmonary hypertension and eventually death. We describe a case of PTTM with association of a presumed lung adenocarcinoma primary. Although rare in nature, PTTM should be a considered diagnosis with chronic dyspnea of unknown origin, severe pulmonary hypertension coupled with right-side heart failure, in the absence of pulmonary embolism.

Methicillin-Resistant Staphylococcus aureus Parotitis Leading to Mortality in an Adolescent Male.

A case of toxic shock syndrome associated with methicillin-resistant Staphylococcus aureus parotitis in a 13-year-old male is presented. He was initially diagnosed with left-sided parotitis by his primary care physician, was started on sulfamethoxazole/trimethoprim, and became severely ill the following day. He was transported to the hospital after a syncopal episode at home and was found to have altered mental status, hypotension, and hypoxia. He was transferred to a larger care facility and died en route despite aggressive resuscitation. At autopsy, he was found to have a severe left-sided parotitis, severe pulmonary congestion, edema, and pneumonia, as well as bilateral lower limb hemorrhagic lesions. Blood cultures from the time of admission and at autopsy grew methicillin-resistant Staphylococcus aureus, which is rarely reported as the sole cause of parotitis. In addition, although S. aureus bacteremia is not necessarily a rare complication of a parotid gland infection, it is exceedingly rare in an immunocompetent adolescent.

GFI1 functions in transcriptional control and cell fate determination require SNAG domain methylation to recruit LSD1.

Proper hematopoietic cell fate decisions require co-ordinated functions of transcription factors, their associated co-regulators, and histone-modifying enzymes. Growth factor independence 1 (GFI1) is a zinc finger transcriptional repressor and master regulator of normal and malignant hematopoiesis. While several GFI1-interacting proteins have been described, how GFI1 leverages these relationships to carry out transcriptional repression remains unclear. Here, we describe a functional axis involving GFI1, SMYD2, and LSD1 that is a critical contributor to GFI1-mediated transcriptional repression. SMYD2 methylates lysine-8 (K8) within a -(8)KSKK(11)- motif embedded in the GFI1 SNAG domain. Methylation-defective GFI1 SNAG domain lacks repressor function due to failure of LSD1 recruitment and persistence of promoter H3K4 di-methyl marks. Methylation-defective GFI1 also fails to complement GFI1 depletion phenotypes in developing zebrafish and lacks pro-growth and survival functions in lymphoid leukemia cells. Our data show a discrete methylation event in the GFI1 SNAG domain that facilitates recruitment of LSD1 to enable transcriptional repression and co-ordinate control of hematopoietic cell fate in both normal and malignant settings.

Coprophagic Asphyxiation in an Intellectually Disabled Woman.

The ingestion of one's own feces is referred to as coprophagia. In this report, the author discusses a case of coprophagia-associated death in a 36-year-old woman. The woman had a history of intellectual disability, bipolar disorder, and schizoaffective disorder and was living in a group home. At autopsy, examination of the upper airway revealed 4 distinct spherical boli of impacted fecal matter in the oral cavity and pharynx. Death was attributed to asphyxia due to aspiration of stool bolus during an episode of coprophagia. This is now the second reported case of coprophagic-related death, illustrating the need for careful monitoring of eating activities in individuals with mental disorders.