Uritboonthai W, Aisporna AE, Hoang L, Hoang C, Billings EM, Siuzdak G.

Anal Chem

PMID:42012876

Free PMC article

METLIN 960 K represents the largest collection of experimentally acquired small-molecule MS/MS spectra currently available. We introduce a reengineered publicaly accessible METLIN platform integrating high-resolution tandem mass spectrometry (MS/MS) data for over 960,000 empirically validated molecular standards. This scale was enabled by a high-throughput experimental framework integrating acoustic droplet ejection with high-throughput LC-MS/MS acquisition, allowing systematic empirical generation of MS/MS spectra from authentic standards. In addition to scale, METLIN 960 K provides a uniquely standardized MS/MS data set, with spectra acquired under controlled and consistent conditions across ionization modes and collision energies, enabling reproducible spectral comparison and machine-learning applications. Each compound is characterized by MS/MS spectra acquired in both positive and negative ionization modes across four collision energies (0, 10, 20, and 40 eV), enabling comprehensive fragmentation coverage and improved structural annotation. Designed as a reference library for XCMS-METLIN and compatible with machine-learning workflows, METLIN 960 K supports high-fidelity spectral matching, neutral loss analysis, and filtering of misannotations, including annotation of in-source fragments and biologically synchronized ranking of candidate metabolites. The platform also provides empirically derived MRM transitions on all standards (via METLIN-MRM), supporting quantitative method development across a chemically diverse range of metabolites, natural products, lipids, peptides, pharmaceuticals, and toxicants. A redesigned interface enables efficient querying by exact mass, formula, or structure with direct access to curated spectra and metadata. Two additional resources enhance identification: (1) METLIN Core, a high-frequency-use subset for rapid searching, and (2) > 1.02 million additional structures without MS/MS data for hypothesis generation. Derived exclusively from authentic standards, METLIN 960 K (https://metlin.scripps.edu) provides the largest publicly available empirical MS/MS database, delivering high-confidence annotation for both untargeted and targeted mass spectrometry workflows.

Rellin KF, Witting M.

Anal Bioanal Chem

PMID:42010161

Free PMC article

Lipidomics provides detailed insight into lipid metabolism and cellular function, but conventional workflows typically rely on bulk samples that mask cellular heterogeneity. Advances in analytical chemistry are enabling lipid analysis from extremely limited material, driving the development of miniaturized chromatography-mass spectrometry (LC-MS) workflows for low-input and single-cell studies. This review summarizes recent progress in miniaturized chromatography-based lipidomics. Reducing chromatographic scale improves ionization efficiency, sensitivity, and separation performance while minimizing sample consumption. We discuss key enabling technologies, such as low-flow electrospray interfaces and the integration of ion mobility (IM) spectrometry as an orthogonal separation dimension for lipid identification. Methodological considerations for low-input lipidomics are also addressed, particularly sample preparation and quantitative challenges at picogram-scale analyte levels. Finally, we highlight future directions in automation, microfluidics, and multidimensional separations. Together, these developments position miniaturized chromatography as a critical platform for advancing single-cell lipidomics and high-resolution studies of lipid metabolism.

Huang T, Zou C, Qi G, Zhao Z, Zhang Y, He SX, Huang K.

Prep Biochem Biotechnol

PMID:42013000

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Host cell proteins (HCPs) are important process-related safety concern factors during drug development, clinical trials and manufacturing, and their removal becomes particularly challenging when associated with antibody aggregates. Residual HCPs in drug products could trigger adverse effects or compromise drug efficacy and stability. This study aims to enhance the HCP removal specifically in monoclonal antibodies (mAbs) with high aggregate content before protein A chromatography by introducing reduction and denaturation steps. A model mAb was purified by different processes, and the impact on HCP removal was evaluated by enzyme-linked immunosorbent assay (ELISA) and Liquid chromatography-mass spectrometry (LC-MS). The HCP profiles in high aggregate, low aggregate and monomeric mAbs were analyzed by LC-MS. The additional reduction and denaturation steps improved HCP removal rate by 1.08 log10 reduction value (LRV) compared to the control process. For the relatively abundant high-risk HCPs, 45% were completely removed. HCP removal in the model mAb was significantly enhanced, especially in high aggregate forms. This method can reduce HCP carry-over from mAbs aggregates in subsequent downstream processing and can provide an efficient HCP removal strategy for manufacturing to improve patient safety and drug stability.

Tengsuttiwat T, Burke A, Kaderbhai NN, Gallagher J, Muhamadali H, Goodacre R.

PMID:PPR1179962

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Abstract Introduction: Recombinant protein production is pivotal across diverse industries, necessitating efforts to enhance both quantity and quality. Objectives: We employed gas chromatography coupled with mass spectrometry (GC-MS) to investigate the metabolic effects of producing mammalian cytochrome b 5 in the bacterial host E. coli N4830-1. The model system studied involved the cyt b 5 gene being introduced on plasmids into the host with varying copy numbers (0-6) under a λP L heat-sensitive promoter. Methods: Metabolic profiling involved GC-MS analysis with quality assessments using pooled QCs and multivariate chemometric analysis. This approach revealed specific metabolic features (identified to Level 2 of the Metabolomics Standards Initaive) correlating significantly with different cultivation conditions and variable copy numbers of the cyt b 5 gene among the examined bacterial strains. Results: Our study shows that CYT b 5 production imposed a substantial energetic burden, reflected by depletion of metabolites associated with the tricarboxylic acid (TCA) cycle, glycolysis/gluconeogenesis, pentose phosphate pathway, and glyoxylate metabolism, indicating increased ATP demand. Concurrent reductions in unsaturated fatty acids and changes in lipid-related metabolites suggest membrane remodeling in response to temperature induction (needed to initiate production of CYT b 5 ), as well as metabolic stress. Alterations in glyoxylate shunt and pentose phosphate pathway intermediates further indicate metabolic reprogramming to maintain carbon homeostasis, while significant changes in nucleotide- and amino acid-related metabolites suggest impacts on biosynthetic capacity associated with recombinant expression and gene copy number variation. Conclusion: Overall, these findings demonstrate that CYT b 5 production induces coordinated metabolic adjustments affecting energy-related metabolic pathways, bacterial cell wall and membrane biosynthesis, nucleotide metabolisms and bacterial stress responses, providing valuable insights that may be useful for the optimisation of recombinant protein production processes.

Boddu V, Mandapaka MK.

Anal Methods

PMID:42011713

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Riociguat, a vasodilatory drug for treating pulmonary hypertension (PH), might generate a nitrosamine drug substance-related impurity (NDSRI), reported as N -nitroso-desformyl riociguat (NNDFR). NDSRIs are a class of nitrosamine impurities that are specific to each drug and are structurally related to the active pharmaceutical ingredient (API) either or both sharing a common molecular backbone but differing by specific functional groups. Such impurities have drawn growing regulatory attention due to their recognized genotoxic and carcinogenic potential, in which the identification and quantification in the product is mandatory. Thus, a sensitive and reliable Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) method is developed employing a Shimpack-GIS CN column (150 mm × 4.6 mm × 3 µm) with a gradient of 0.1% formic acid in water, and 0.1% formic acid in methanol for chromatography separation, and in multiple reaction monitoring (MRM) mode in an electrospray ionization (ESI) environment. Optimized conditions focused on chromatographic selectivity, ionization efficiency, and matrix interference reduction. Method validation for the identification of NNDFR in the riociguat drug product confirmed that this method achieved baseline separation of NNDFR isomers with retention times of 10.037 (Isomer-1) and 10.579 (Isomer-2) minutes, prohibiting interferences from other compounds. Validation also confirmed excellent linearity ( R 2 > 0.99), precision (RSD < 1.3%), and accuracy (recoveries from 100.5% to 110.4%). The LOD and LOQ were determined to be 0.4 ppm and 1.22 ppm, respectively. A 24-hour stability study confirmed the analyte integrity and method robustness at various flow rates and column temperatures, and under various filtration conditions, ensuring consistent performance. This validated UHPLC-MS/MS method is robust, sensitive, and reliable for routinely quantifying NNDFR in riociguat drug products. Subsequently, the analytical method developed was assessed for environmental sustainability using established greenness assessment tools. The AGREE score was calculated to be 0.73, AGREEprep to be 0.69, and the BAGI value to be 70.0. These values reflected a favorable environmental profile, efficient sample preparation, and practical sustainability. Together, these metrics confirmed the method's suitability for both regulatory compliance and routine application in industrial quality control laboratories.