Specific N-Linked Glycosylation Patterns in Areas of Necrosis in Tumor Tissues.

Tissue necrosis is a form of cell death common in advanced and aggressive solid tumors, and is associated with areas of intratumoral chronic ischemia. The histopathology of necrotic regions appear as a scaffold of cellular membrane remnants, reflective of the hypoxia and cell degradation events associated with this cellular death pathway. Changes in the glycosylation of cell surface proteins is another common feature of cancer progression. Using a recently developed mass spectrometry imaging approach to evaluate N-linked glycan distributions in human formalin-fixed clinical cancer tissues, differences in the glycan structures of regions of tumor, stroma and necrosis were evaluated. While the structural glycan classes detected in the tumor and stromal regions are typically classified as high mannose or branched glycans, the glycans found in necrotic regions displayed limited branching, contained sialic acid modifications and lack fucose modifications. While this phenomenon was initially classified in breast cancer tissues, it has been also seen in cervical, thyroid and liver cancer samples. These changes in glycosylation within the necrotic regions could provide further mechanistic insight to necrotic changes in cancer tissue and provide new research directions for identifying prognostic markers of necrosis.

Control of pollen-mediated gene flow in transgenic trees.

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

Optimization of Multiple Glycosidase and Chemical Stabilization Strategies for N-Glycan Isomer Detection by Mass Spectrometry Imaging in Formalin-Fixed, Paraffin-Embedded Tissues.

The analysis of N-glycan distributions in formalin-fixed, paraffin-embedded (FFPE) tissues by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is an effective approach for characterization of many disease states. As the workflow has matured and new technology emerged, approaches are needed to more efficiently characterize the isomeric structures of these N-glycans to expand on the specificity of their localization within tissue. Sialic acid chemical derivatization can be used to determine the isomeric linkage (α2,3 or α2,6) of sialic acids attached to N-glycans, while endoglycosidase F3 (Endo F3) can be enzymatically applied to preferentially release α1,6-linked core fucosylated glycans, further describing the linkage of fucose on N-glycans. Here we describe workflows where N-glycans are chemically derivatized to reveal sialic acid isomeric linkages, combined with a dual-enzymatic approach of endoglycosidase F3 and PNGase F to further elucidate fucosylation isomers on the same tissue section.

Olfactory cleft mucus proteome in chronic rhinosinusitis: a case-control pilot study.

BACKGROUND: Mechanisms of smell loss in chronic rhinosinusitis (CRS) are still unclear and likely multifactorial. Little attention has been given to olfactory cleft (OC) mucus proteins involved in odorant binding and metabolizing enzymes and their potential role in smell loss. METHODS: Mucus from the OC was sampled from patients with CRS (n = 20) and controls (n = 10). Liquid chromatography and mass spectrometry were performed, followed by data processing so that protein groups could be identified, quantified, and compared. Hierarchical clustering and bioinformatic analysis were performed on significantly different proteins to explore for enrichment in known biologic pathways. RESULTS: A total of 2514 proteins were found in OC mucus from all 30 subjects. Significant differences in protein abundance were found between CRS and controls, including both CRSsNP (n = 351 proteins; log2 fold change range: -3.88 to 6.71) and CRSwNP (n = 298 proteins; log2 fold change range: -4.00 to -6.13). Significant differences were found between patients with normosmia and those with dysosmia (n = 183; log2 fold change range: -3.62 to -2.16) and across groups of interest for a number of odorant binding proteins and metabolizing enzymes. CONCLUSION: OC mucous in CRS displays a rich and abundant array of proteins, many of which have been implicated in odorant transport and metabolization in animal studies. Significant differences in the olfactory mucus proteome were seen between CRS subtypes and controls, as well as between those with normal and abnormal olfaction. Further study should confirm these findings and explore the role individual proteins play in odorant transport and metabolization. ©2020 ARSAAOA, LLC.

Collagen fiber regulation in human pediatric aortic valve development and disease.

Congenital aortic valve stenosis (CAVS) affects up to 10% of the world population without medical therapies to treat the disease. New molecular targets are continually being sought that can halt CAVS progression. Collagen deregulation is a hallmark of CAVS yet remains mostly undefined. Here, histological studies were paired with high resolution accurate mass (HRAM) collagen-targeting proteomics to investigate collagen fiber production with collagen regulation associated with human AV development and pediatric end-stage CAVS (pCAVS). Histological studies identified collagen fiber realignment and unique regions of high-density collagen in pCAVS. Proteomic analysis reported specific collagen peptides are modified by hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis reported significant regulation of collagen HYP sites across patient categories. Non-collagen type ECM proteins identified (26 of the 44 total proteins) have direct interactions in collagen synthesis, regulation, or modification. Network analysis identified BAMBI (BMP and Activin Membrane Bound Inhibitor) as a potential upstream regulator of the collagen interactome. This is the first study to detail the collagen types and HYP modifications associated with human AV development and pCAVS. We anticipate that this study will inform new therapeutic avenues that inhibit valvular degradation in pCAVS and engineered options for valve replacement.

In Situ Imaging of Tryptic Peptides by MALDI Imaging Mass Spectrometry Using Fresh-Frozen or Formalin-Fixed, Paraffin-Embedded Tissue.

Tryptic peptide imaging is a primary workflow for matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) and has led to new information reporting highly multiplexed protein localization. Technological advances within the last few years have produced robust tools for automated spraying of both matrix and enzymes. When combined with high-mass-resolution and high-mass-accuracy instrumentation, studies now generally result in two-dimensional mapping of well over 1,000 peptide peaks. This protocol describes sample preparation, spraying, and application of enzymes and matrices, and MALDI FT-ICR instrumental considerations for two-dimensional mapping of tryptic peptides from fresh-frozen or formalin-fixed, paraffin-embedded tissue sections. Procedures for extraction of tryptic peptides from tissue sections for LC-MS/MS identification are also described. © 2018 by John Wiley & Sons, Inc.

MALDI Imaging Mass Spectrometry of N-glycans and Tryptic Peptides from the Same Formalin-Fixed, Paraffin-Embedded Tissue Section.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique and well developed tool for probing the protein content of formalin-fixed, paraffin-embedded tissue (FFPE). Integral to this approach is the application of trypsin, and more recently peptide N-glycosidase F, to release tryptic peptides or N-glycans from tissue and report localization of distinct species. This is typically done on serial or adjacent tissue sections, and there is an emerging need to understand the colocalized protein population linked to the exact same regions of N-glycans. Here we describe an approach where N-glycans are first imaged from a tissue section followed by reprocessing of the same tissue section for tryptic peptide MALDI IMS. Strategies for colocalizing peptides to target N-glycans or N-glycan regions are described.

In Situ Imaging of N-Glycans by MALDI Imaging Mass Spectrometry of Fresh or Formalin-Fixed Paraffin-Embedded Tissue.

Glycosylation of cell surface, secreted, and circulating proteins is one of the most common types of post-translational modification. These modifications occur most commonly as one of three major classes: N-linked glycosylation on asparagine residues, O-linked glycosylation on serine or threonine residues, or as glycosaminoglycan oligosaccharide polymers on serine. Specifically, for N-linked glycans, an endoglycosidase enzyme, peptide N-glycosidase F (PNGase F), cleaves the attached oligosaccharides between the asparagine and first sugar. A method to analyze released N-glycans and map them to specific locations within a tissue is presented here. The PNGase F is applied by solvent sprayer as a molecular layer on frozen or formalin-fixed tissues and all released N-glycans in a given region of tissue are detected using matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (MALDI-IMS). Using the described MALDI-IMS protocol, at least 40 or more individual N-glycans can be mapped to tissue histopathology and extracted for further structural analysis approaches. © 2018 by John Wiley & Sons, Inc.