Nicolle H. Packer

N-glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection in mass-spectrometric studies has remained elusive, but its low abundance has led to inconsistent detection in studies of human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N-glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N-glycan detection across multiple LC–MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC–MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N-glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N-glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC–MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, under the DMB-LC-FLR conditions and sample loading evaluated here, dynamic range was limited to two orders of magnitude, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that the DMB-LC-FLR assay’s specificity and sensitivity were insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC–MS to improve the measurement of Neu5Gc in clinical samples.

N -glycolylneuraminic acid (Neu5Gc) has been repeatedly associated with human cancer, but reliable detection has remained elusive, generating controversy regarding its presence in human samples. To address this, matrix-matched calibration curves, which have been pioneered in proteomics and metabolomics for assessing changes in complex mixtures, were measured of released N -glycans at four orders of magnitude dynamic range in defined mixtures, systematically benchmarking Neu5Gc-containing N -glycan detection across multiple LC-MS platforms and sites. Orthogonally, the gold-standard analytical method, consisting of fluorescence detection of labelled monosaccharides separated by LC, was applied to the same samples, yielding absolute concentrations of Neu5Gc. LC-MS demonstrated an extended detection range of three or more orders of magnitude while retaining intact N -glycan measurement, improving assay specificity and enabling detection of the variety of Neu5Gc-bearing N -glycans. By combining orthogonal dimensions of evidence, including chromatographic separation, isotopic distribution matching, and composition-confirming MS/MS, LC-MS confidently resolved Neu5Gc signals from noise, even at low abundance. In comparison, DMB-LC-FLR was limited to two orders of magnitude dynamic range, insufficient for detection of Neu5Gc in commercially available pooled human sera. These findings strongly support that DMB-LC-FLR assay specificity and sensitivity are insufficient for Neu5Gc detection in human samples due to noise overwhelming the Neu5Gc signal. By establishing a reusable benchmarking framework for future glycomic studies, we aim to use LC-MS to improve the measurement of Neu5Gc in clinical samples.

The emergence of mobile colistin resistance ( mcr ) genes threatens the efficacy of polymyxins as last-resort antibiotics for treating multidrug-resistant bacterial infections. Here, we identify a novel environmental mcr gene, mcr-12 , discovered in Pigmentiphaga litoralis from Australian sediment, and evaluate its potential role in the intersection between environmental resistance reservoirs and clinically relevant bacteria. Gene mcr-12 was located within a metal-resistance gene cluster on plasmid pPLE30.2, which also carried a predicted novel β-lactamase gene ( bla _OXA-1383 ). Removal of pPLE30.2 increased polymyxin susceptibility 32-fold, while reintroduction of mcr-12 restored resistance. Despite its low amino-acid identity to known MCR enzymes, MCR-12 confers polymyxin resistance by phosphoethanolamine transferase modification of lipid A. Expression of mcr-12 in Pseudomonas spp. and Acinetobacter baumannii conferred polymyxin resistance, suggesting that mcr-12 is compatible between environmental strains and clinical pathogens. The discovery of a new mcr gene from an environmental source and from outside Gammaproteobacteria highlights the need for further surveillance efforts within a One Health framework.

Adipogenesis is the process of adipose-derived stem cells (ADSCs) responding to extracellular signals from the stem cell niche to differentiate into adipocytes (fat cells) and may be studied in vitro using a cocktail of chemicals that promote adipogenic differentiation to produce differentiated ADSCs (dADSCs). The global membrane N - and O -glycosylation changes of this process have been previously analysed and compared to native adipocytes as a benchmark for a ‘true’ adipocyte profile, and revealed that bisecting GlcNAc type N -glycans are characteristic of adipogenesis. As stem cell differentiation has been widely reported to result in cellular protein changes, the same cells (ADSCs, dADSCs and mature adipocytes) were characterised for their membrane proteome here using label-free quantitative shotgun proteomics analysis. The membrane proteome displayed more differences in protein numbers between the cell types compared to the previously reported N -glycome which had shown high identical glycomes between stem cells and in vitro dADSCs, suggesting that the proteome is more dynamic during in vitro adipogenesis. Following the global shotgun proteomics analysis, a more targeted approach of carrying out proteomic analysis of de- N -glycosylated peptides of gel-separated proteins unearthed new glycoproteins not detected in the shotgun proteomic analysis. This approach identified the adipogenic marker, CD36, to be under-represented in the shotgun proteome analysis, but as the dominant (glyco)protein in the adipocyte membrane proteome that was also up-regulated at the mRNA transcript level in both the in vitro differentiated ADSCs (7.1-fold increase) and mature adipocytes (102.9-fold increase). A comparison of CD36 sequence coverage in the global shotgun analysis with the de- N -glycosylated CD36 revealed a 41% increase when N -glycans were removed prior to trypsin digestion, explaining its observed increased abundance and highlights the crucial need for de- N -glycosylation of proteins in proteomics experiments for increased identification of glycoproteins. The systems glycobiology approach by the integration of previously reported glycomics data and the proteomics and transcriptomics analyses in this work extended the investigation of membrane protein glycosylation changes in adipose-derived stem cell differentiation. The work provides a framework for future glycoproteomics-based investigations into the differentiation of stem cells into adipocytes, and will allow their related pathologies and potential therapeutic applications to be discovered. GRAPHICAL

The HUPO Human Proteome Project (HPP) aims to complete the human protein parts list by detecting evidence of expression and of function for all proteins in the human proteome, and make proteomics an integral part of multiomics studies of health and disease. Here we describe the state of the 2025 HPP reference proteome of 19,435 proteins, based on GENCODE v48, UniProtKB 2025_03, Human Protein Atlas 24, MassIVE-KB 2023, and PeptideAtlas 2025-01. We evaluate the progress in the past year, with 93.6% of the proteome detected, and examine the proteins that have not yet been detected to determine where further progress can be made. We also evaluate the progress in determining at least one function for every protein in the HPP target list, finding an increase of 288 proteins in the highest category (FE1) to 5562. Finally, we provide highlights from 12 Biology/Disease-based HPP initiatives, HPP resource pillars, and π-HuB.

The transport and Golgi organization 2 ( TANGO2 ) gene has been identified to cause a rare recessive genetic disorder known as TANGO2‐deficiency disorder (TDD). TDD is a multi‐systemic disorder that can affect different organs including the brain, heart, and muscles, presenting as recurrent metabolic crises, muscle weakness, and neurological symptoms including developmental delay and seizures. In a TANGO2‐KO Cal51 cell line model, we previously showed a significant change in the protein N‐glycomics profile, that was corroborated by RNAseq transcriptomics data that showed increased levels of the respective glycosyltransferases. To investigate the in vivo effects of TANGO2‐KO on the glycans in the brain of a mouse model, we profiled the polysialic acid (polySia), glycosaminoglycans (GAGs), glycolipid glycans (GSLs), and protein N‐ and O‐glycans (NGs, OGs) of the cortex of the TANGO2‐KO mice using the Same Sample Sequential Multi‐Glycomics workflow. Overall, we observed a significant decrease in chondroitin sulfate GAG disaccharides, and also a decrease in sialylation across the GSLs, NGs, and OGs, resulting in an overall large decrease in negatively charged glycoconjugates. When compared with a parallel data‐dependent label‐free proteomics of the tissue from the same mouse model, the glyco‐enzymes, sialic acid synthase (NANS), and chondroitin polymerizing factor (CHPF) were found to be significantly downregulated in the brain, correlating with the changes observed by the multi‐glycomics analysis. This overall decrease in negatively charged glycoconjugates due to the loss of TANGO2 function may thus relate to the neurological impairments of TDD.

A complex extracellular matrix (ECM) assembles around the mammalian oocyte during maturation and ovulation, comprising hyaluronan as well as proteoglycans. These proteoglycans are hypothesised to carry heparan and chondroitin sulfate side chains. This matrix is essential for ovulation, mediates signalling, and regulates solute diffusion to control the oocyte environment. In vivo, ECM formation is initiated by epidermal growth factor-like peptides released within the follicle, acting together with oocyte-derived growth factors. Although proteoglycans are known components, the specific glycosaminoglycan (GAG) composition remains poorly understood. Here, we characterised GAG abundance in murine cumulus-oocyte-complexes and assessed differences between in vivo and in vitro maturation. The latter is an assisted reproductive technology that requires less drugs than IVF, but oocytes have reduced developmental potential. We found that sulfated GAG abundance increased significantly in vivo but not during in vitro oocyte maturation. We also employed high performance liquid-chromatography to measure the abundance of specific GAGs—hyaluronan, chondroitin sulfate, and heparan sulfate—in this matrix at different stages of maturation. These were enriched within the ECM during in vivo maturation but reduced or undetectable in vitro. Reduced GAG abundance following in vitro maturation was associated with poorer oocyte developmental potential. GAG deficiency following in vitro maturation likely arises from the failure of in vitro conditions to replicate the signalling milieu that occurs in vivo. Altered GAG abundance during in vitro maturation may impair functions of the ECM, including growth factor binding and activity or the regulated diffusion of solutes, potentially contributing to decreased oocyte developmental potential.