Protein glycosylation is increasingly recognized as a common protein modification across bacterial species. Within the Neisseria genus O-linked protein glycosylation is conserved yet closely related Neisseria species express O-oligosaccharyltransferases (PglOs) with distinct targeting activities. Within this work, we explore the targeting capacity of different PglOs using Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) fractionation and Data-Independent Acquisition (DIA) to allow the characterization of the impact of changes in glycosylation on the proteome of N. gonorrhoeae. We demonstrate FAIMS expands the known glycoproteome of wild type N. gonorrhoeae MS11 and enables differences in glycosylation to be assessed across strains expressing different pglO allelic chimeras with unique substrate targeting activities. Combining glycoproteomic insights with DIA proteomics, we demonstrate that alterations within pglO alleles have widespread impacts on the proteome of N. gonorrhoeae. Examination of peptides known to be targeted by glycosylation using DIA analysis supports alterations in glycosylation occupancy occurs independently of changes in protein levels and that the occupancy of glycosylation is generally low on most glycoproteins. This work thus expands our understanding of the N. gonorrhoeae glycoproteome and the roles that pglO allelic variation may play in governing genus-level protein glycosylation.

The ability of trophectodermal cells (outer layer of the embryo) to attach to the endometrial cells and subsequently invade the underlying matrix are critical stages of embryo implantation during successful pregnancy establishment. Extracellular vesicles (EVs) have been implicated in embryo-maternal crosstalk, capable of reprogramming endometrial cells towards a pro-implantation signature and phenotype. However, challenges associated with EV yield and direct loading of biomolecules limit their therapeutic potential. We have previously established generation of cell-derived nanovesicles (NVs) from human trophectodermal cells (hTSCs) and their capacity to reprogram endometrial cells to enhance adhesion and blastocyst outgrowth. Here, we employed a rapid NV loading strategy to encapsulate potent implantation molecules such as HB-EGF (NVHBEGF). We show these loaded NVs elicit EGFR-mediated effects in recipient endometrial cells, activating kinase phosphorylation sites that modulate their activity (AKT S124/129, MAPK1 T185/Y187), and downstream signalling pathways and processes (AKT signal transduction, GTPase activity). Importantly, they enhanced target cell attachment and invasion. The phosphoproteomics and proteomics approach highlight NVHBEGF-mediated short-term signalling patterns and long-term reprogramming capabilities on endometrial cells which functionally enhance trophectodermal-endometrial interactions. This proof-of-concept study demonstrate feasibility in enhancing the potency of NVs in the context of embryo attachment and establishment.

Cancer remains one of the most complex and challenging diseases in mankind. To address the need for a personalized treatment approach for particularly complex tumor cases, molecular tumor boards (MTBs) have been initiated. MTBs are interdisciplinary teams that perform in-depth molecular diagnostics to cooperatively and interdisciplinarily advise on the best therapeutic strategy. Current routine molecular diagnostics are routinely performed on the transcriptomic and genomic levels, aiming for the identification of tumor-driving mutations. However, these approaches can only partially capture the actual phenotype as well as the molecular key players of tumor growth and progression. Thus, direct investigation of the expressed proteins and activated signaling pathways provide complementary information on the tumor-driving molecular characteristics of the tissue. Technological advancements in mass-spectrometry-based proteomics enable the robust, rapid, and sensitive detection of thousands of proteins in minimal sample amounts, paving the way for clinical proteomics and the probing of oncogenic signaling activity. Therefore, proteomics is currently being integrated into molecular diagnostics within MTBs and holds promising potential in aiding tumor classification and identifying personalized treatment strategies. This review gives an introduction to MTBs and describes current state-of-the-art clinical proteomics, its potential in precision oncology, and highlights the benefits of multi-omic data integration.

A document by Ayushi Verma. Click on the document to view its contents.

Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-actylation of residue K16, while also installing N-ε-acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of isomeric proteoforms and precise PTM localization.

MALDI mass spectrometry imaging (MALDI imaging) is uniquely suited to advance cancer research by measuring spatial distribution of endogenous and exogenous molecules directly from thin tissue sections. These molecular maps provide valuable insights into various aspects of basic and translational cancer research, including spatial tumor and tumor microenvironment biology, pharmacological interventions, and patient stratification. However, despite these advantages, the utilization of MALDI imaging in studying rare cancers, which comprise approximately 20% of all cancers, remains limited. Rare cancers pose unique challenges in medical research, resulting in understudied entities with suboptimal management and outcomes. In this review, we explore the value of MALDI imaging in sarcoma, as an example of a highly heterogeneous and challenging rare cancer. We summarize existing MALDI imaging studies in sarcoma and outline potential future applications. In addition, we address the specific challenges encountered when employing MALDI imaging to rare cancers, and propose solutions, including the utilization of formalin-fixed paraffin-embedded tissues, multi-site studies, implementation of multiplexed experiments, and considerations for data sharing practices. Through this review, we aim to inspire collaboration between MALDI imaging researchers and clinical colleagues, to deploy the unique capabilities of MALDI imaging in rare cancer research, particularly in the context of sarcoma.

Changes in the structure of biological macromolecules, such as RNA and protein, have an important impact on biological functions, and are even important determinants of disease pathogenesis and treatment. Some genetic variations, including copy number variation, single nucleotide variation, and so on, can lead to changes in biological function and increased susceptibility to certain diseases by changing the structure of biological macromolecules. Here, we reviewed the progress of research about the effects of genetic variation on the structure of macromolecules including RNAs and proteins, several typical methods and common tools, and the effect on several diseases. An online resource (http://www.onethird-lab.com/gems/) to support convenient retrieval of common tools is also built. Finally, the challenges and future development of effect prediction were discussed.