bims-mascan 2025-04-06 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2025–04–06
23 papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

A comparative study of data-independent acquisition and data-dependent acquisition in liquid chromatography-mass spectrometry-based untargeted metabolomics analysis of Panax genus sample.
Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.
High-Throughput Human Cerebrospinal Fluid Proteome Analysis with Direct Data-Independent Acquisition (dDIA).
HeuSMA: A Multigradient LC-MS Strategy for Improving Peak Identification in Untargeted Metabolomics.
Offset mass carrier proteome improves quantification of multiplexed single cell proteomics.
Optimization of Collagenase Proteomics for Improved Mass Spectrometry Imaging Peptide Identification.
Mass Spectrometry Applied to Human Cerebrospinal Fluid Lipidome (II).
Advances in Tandem Mass Spectrometry Imaging for Next-Generation Spatial Metabolomics.
OzMALDI: A Gas-Phase, In-Source Ozonolysis Reaction for Efficient Double-Bond Assignment in Mass Spectrometry Imaging with Matrix-Assisted Laser Desorption/Ionization.
How to Train a Postprocessor for Tandem Mass Spectrometry Proteomics Database Search While Maintaining Control of the False Discovery Rate.
Protocol for label-free quantitative lysine lactylproteome profiling.
A multi-omics reciprocal analysis for characterization of bacterial metabolism.
Rapid MALDI-MS/MS-Based Profiling of Lipid A Species from Gram-Negative Bacteria Utilizing Trapped Ion Mobility Spectrometry and mzmine.
Targeted Absolute Quantification of Protein Biomarkers in Cerebrospinal Fluid by FastCAT.
Application of Isotope Dilution Gas Chromatography-Mass Spectrometry Using Selected Ion-Monitoring to Evaluate Nonenzymatic Protein Damage in Cerebrospinal Fluid.
Global analysis of protein turnover dynamics in single cells.
Examining Instrumental Factors Influencing the Performance of Data-Independent Acquisition Methods in Hydrogen/Deuterium Exchange Mass Spectrometry.
Pentafluorobenzyl bromide - A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents.
An LC-MS/MS Multiplexed Method to Quantify 14 Sphingolipids in Human Cerebrospinal Fluid (CSF).
Norm ISWSVR Enhanced Data Repeatability and Accuracy in Large-Scale Targeted Quantification Metabolomics.
Establishment of a pseudotargeted LC‒MS/MS workflow for analyzing triglycerides in biological samples.
Data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) analysis of the separated zebrafish lens improves identifications.
Evaluating Ion Mobility Data Acquisition, Calibration, and Processing for Small Molecules: A Cross-Platform Assessment of Drift Tube and Traveling Wave Methodologies.

Anal Bioanal Chem. 2025 Apr 02.

A comparative study of data-independent acquisition and data-dependent acquisition in liquid chromatography-mass spectrometry-based untargeted metabolomics analysis of Panax genus sample.

Yi Wu, Yang Wang.

  Data-independent acquisition (DIA) and data-dependent acquisition (DDA) are frequently employed in the execution of tandem mass spectrometry (MS2) analyses. This study explored the application of DIA (MSe) and DDA (fast-DDA) in liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomics using Panax genus samples. MSe provided comprehensive sample information, extracting more ion peaks with better peak shape and increased scan points compared to fast-DDA. Features from MSe data are four times more than those from fast-DDA data. Fast-DDA, however, delivered high-quality MS2 data, enhancing compound annotation via the GNPS web tool. Database matches with fast-DDA data were nearly 35 times greater than those using MSe data. Therefore, combining MSe and fast-DDA can improve data analysis and metabolite annotation. An improved workflow integrating DIA and DDA was proposed, requiring additional QC sample injections for DDA analysis but eliminating the need for sample reprocessing and re-analysis, thus saving time and resources. The established workflow was applied to the Panax genus samples analysis to confirm its applicability. This study offers a deeper understanding of DIA and DDA, guiding the selection of data acquisition strategies for LC-MS-based untargeted metabolomics.

Keywords:   Panax genus; GNPS; Liquid chromatography-mass spectrometry; Molecular networking; Untargeted metabolomics

DOI:  https://doi.org/10.1007/s00216-025-05861-9
bioRxiv. 2025 Mar 13. pii: 2025.03.10.642426. [Epub ahead of print]

Microenvironmental arginine restriction sensitizes pancreatic cancers to polyunsaturated fatty acids by suppression of lipid synthesis.

Patrick B Jonker, Mumina Sadullozoda, Guillaume Cognet, Juan J Apiz Saab, Kelly H Sokol, Violet X Wu, Deepa Kumari, Colin Sheehan, Mete E Ozgurses, Darby Agovino, Grace Croley, Smit A Patel, Althea Bock-Hughes, Kay F Macleod, Hardik Shah, Jonathan L Coloff, Evan C Lien, Alexander Muir.

Nutrient limitation is a characteristic feature of poorly perfused tumors. In contrast to well-perfused tissues, nutrient deficits in tumors perturb cellular metabolic activity, which imposes metabolic constraints on cancer cells. The metabolic constraints created by the tumor microenvironment can lead to vulnerabilities in cancers. Identifying the metabolic constraints of the tumor microenvironment and the vulnerabilities that arise in cancers can provide new insight into tumor biology and identify promising antineoplastic targets. To identify how the microenvironment constrains the metabolism of pancreatic tumors, we challenged pancreatic cancer cells with microenvironmental nutrient levels and analyzed changes in cell metabolism. We found that arginine limitation in pancreatic tumors perturbs saturated and monounsaturated fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1. Synthesis of these fatty acids is critical for maintaining a balance of saturated, monounsaturated, and polyunsaturated fatty acids in cellular membranes. As a consequence of microenvironmental constraints on fatty acid synthesis, pancreatic cancer cells and tumors are unable to maintain lipid homeostasis when exposed to polyunsaturated fatty acids, leading to cell death by ferroptosis. In sum, arginine restriction in the tumor microenvironment constrains lipid metabolism in pancreatic cancers, which renders these tumors vulnerable to polyunsaturated-enriched fat sources.

DOI: https://doi.org/10.1101/2025.03.10.642426
Methods Mol Biol. 2025 ;2914 129-139

High-Throughput Human Cerebrospinal Fluid Proteome Analysis with Direct Data-Independent Acquisition (dDIA).

Ramón Díaz-Peña, Pol Andrés-Benito, Erica Peral, Raúl Domínguez, Mónica Povedano, Enrique Santamaría, Joaquín Fernández-Irigoyen.

  Cerebrospinal fluid (CSF) is a low-risk, rapid, and mid-invasive sampling for diagnosis, prognosis, and treatment of neurological pathologies. The CSF liquid biopsies disponibility and sampling homogeneity foster the research for biomarker discovery for neurological disorders and pathologies and, importantly, enable extensive population studies. Liquid chromatography-mass spectrometry (LC-MS) proteomics is a powerful tool for biomarker discovery. Proteomics large studies provide more robust and reliable results, offering exceptional FDR control and outlier identification, allowing high-precision results. Consequently, a robust and reliable pipeline proteomics methodology must be required to analyze hundreds of samples, from sample preparation to data analysis. Here, we describe a detailed workflow for analyzing human CSF samples for large studies by direct data-independent (dDIA).

Keywords:  Cerebrospinal fluid; Mass-spectrometry; Proteomics; dDIA

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_11
Anal Chem. 2025 Apr 03.

HeuSMA: A Multigradient LC-MS Strategy for Improving Peak Identification in Untargeted Metabolomics.

Yao-Yu Chen, Na An, Yan-Zhen Wang, Peng-Cheng Mei, Jun-Di Hao, Song-Mei Liu, Quan-Fei Zhu, Yu-Qi Feng.

Metabolomics, which involves the comprehensive analysis of small molecules within biological systems, plays a crucial role in elucidating the biochemical underpinnings of physiological processes and disease conditions. However, current coverage of the metabolome remains limited. In this study, we present a heuristic strategy for untargeted metabolomics analysis (HeuSMA) based on multiple chromatographic gradients to enhance the metabolome coverage in untargeted metabolomics. This strategy involves performing LC-MS analysis under multiple gradient conditions on a given sample (e.g., a pooled sample or a quality control sample) to obtain a comprehensive metabolomics data set, followed by constructing a heuristic peak list using a retention index system. Guided by this list, heuristic peak picking in quantitative metabolomics data is achieved. The benchmarking and validation results demonstrate that HeuSMA outperforms existing tools (such as MS-DIAL and MZmine) in terms of metabolite coverage and peak identification accuracy. Additionally, HeuSMA improves the accessibility of MS/MS data, thereby facilitating the metabolite annotation. The effectiveness of the HeuSMA strategy was further demonstrated through its application in serum metabolomics analysis of human hepatocellular carcinoma (HCC). To facilitate the adoption of the HeuSMA strategy, we also developed two user-friendly graphical interface software solutions (HPLG and HP), which automate the analysis process, enabling researchers to efficiently manage data and derive meaningful conclusions (https://github.com/Lacterd/HeuSMA).

DOI: https://doi.org/10.1021/acs.analchem.4c05315
Mol Cell Proteomics. 2025 Apr 01. pii: S1535-9476(25)00057-X. [Epub ahead of print] 100959

Offset mass carrier proteome improves quantification of multiplexed single cell proteomics.

Tommy K Cheung, Ying Zhu, Christopher M Rose.

Multiplexed single cell proteomics by mass spectrometry (scpMS) approaches currently offer the highest throughput as measured by cells analyzed per day. These methods employ isobaric labels and typically a carrier proteome - a sample added at 20-500x the single cell level that improves peptide sampling and identification. Peptides from the carrier and single cell proteomes exist within the same precursor isotopic cluster and are co-isolated for identification and quantification. This represents a challenge as high levels of carrier proteome limit the sampling of peptide ions from single cell samples and can potentially lead to decreased accuracy of quantitative measurements. Here, we address this limitation by introducing a triggered by offset mass acquisition method for scpMS (toma-scpMS) that utilizes a carrier proteome labeled with non-isobaric tags that have the same chemical composition but different mass as the labels used for quantitative multiplexing. Within toma-scpMS the carrier proteome and single cell proteome are separated at the precursor level, enabling separate isolation, fragmentation, and quantitation of the single cell samples. To enable this workflow we implemented a custom data acquisition scheme within inSeqAPI, an instrument application programming interface program that performed real-time identification of carrier proteome peptides and subsequent triggering of offset single cell quantification scans. We demonstrate that toma-scpMS is more robust to high-levels of carrier proteome and offers superior quantitative accuracy as compared to traditional multiplexed scpMS approaches when similar carrier proteome levels are employed.

DOI: https://doi.org/10.1016/j.mcpro.2025.100959
Anal Chem. 2025 Mar 28.

Optimization of Collagenase Proteomics for Improved Mass Spectrometry Imaging Peptide Identification.

Jade K Macdonald, Stephen C Zambrzycki, Harrison B Taylor, Jaclyn B Dunne, Montana Quick, Anand S Mehta, Richard R Drake, Peggi M Angel.

The extracellular matrix (ECM) is composed of a dynamically regulated collagenous scaffold that provides structure, conveys cellular and environmental communication, and contributes to disease progression. Collagen proteins derived from clinically archived formalin-fixed, paraffin-embedded (FFPE) tissues are analytically challenging due to dense post-translational modifications, high proline content, and insolubility. A recent advancement in ECM proteomics is the use of collagenase type III, an ECM-specific bacterial protease, to target native collagenous structures on-tissue for peptide imaging. The resulting collagenase-generated peptides have biochemical differences compared to tryptic peptides, creating analytical challenges in elucidating peptide sequence information. In this study, we characterize collagenase as a proteomic enzyme for ECM-targeted liquid chromatography trapped ion mobility spectrometry tandem mass spectrometry (LC-TIMS-MS/MS) and matrix-assisted laser/desorption ionization mass spectrometry imaging (MALDI-MSI) proteomic workflows. We then optimized collagenase-generated peptide sequencing for MALDI-MSI peptide identification from clinically archived FFPE tissue sections. Soluble rat tail collagen solution is used as a collagen standard to elucidate tryptic and collagenase cleavage sites within collagen. Proteomic readouts of FFPE tissue are compared across trypsin and collagenase digests to assess for ECM enrichment by collagenase in biologically complex samples. Optimized methods for MALDI-MSI peptide identification are comprehensively detailed from sample preparation to MS data acquisition and MS data analysis for reproducible implementation. On-tissue digestion followed by liquid surface extraction (LSE), inclusion of singly charged peptides during data acquisition, and implementation of nonspecific cleavage during database searching resulted in the most collagenase-generated peptide spectrum matches as well as MALDI-MSI peptide identifications. This research establishes parameters for the optimal identification of peptides from collagenase-directed ECM proteomic workflows for targeted spatial analysis of the ECM.

DOI: https://doi.org/10.1021/acs.analchem.4c04818
Methods Mol Biol. 2025 ;2914 203-212

Mass Spectrometry Applied to Human Cerebrospinal Fluid Lipidome (II).

Laura Millán, Joaquín Fernández-Irigoyen, Enrique Santamaría, Rebeca Mayo.

  Lipidomics aims at characterizing lipid profiles and their biological role with respect to protein expression involved in lipid metabolism. Specifically, cerebrospinal fluid (CSF) lipidomics is offering a new perspective in the search for surrogate biomarkers to facilitate early diagnosis of psychiatric and neurodegenerative diseases. In this chapter, we describe a nontargeted approach to profile lipid molecular species present in human CSF using ultrahigh-performance liquid chromatography-electrospray ionization-time-of-flight mass spectrometry (UPLC-ESI-ToF-MS). This workflow complements the toolbox useful for the exploration and monitoring of neurodegenerative mechanisms associated with dysregulation in lipid metabolism.

Keywords:  CSF; Lipidomics; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_15
Anal Chem. 2025 Apr 02.

Advances in Tandem Mass Spectrometry Imaging for Next-Generation Spatial Metabolomics.

Yao Qian, Xiaoxiao Ma.

Spatial metabolomics based on mass spectrometry imaging (MSI) is a promising approach for fundamental biological research and disease biomarker discovery. It simultaneously reveals the spatial distributions of hundreds of metabolites across tissue sections. While previous MSI experiments predominantly rely on high-resolution mass analysis for metabolite annotation, the high specificity in resolving molecular structures is essential to distinguish isomers or isobars to obtain ultimate identities of the metabolites. This is also critical for correlating their biological functions with spatial distribution patterns. Tandem mass spectrometry (MS/MS) is effectively used to obtain molecular structural information and has been integrated into MSI for spatial mapping of structurally distinct biomolecules, though typically with low coverage. The main technical challenge in achieving high-coverage, high-structure-resolving spatial mapping of biomolecules lies in the limited amount of sample available from each tissue pixel in conventional MS/MS analysis, which restricts the number of MS/MS scans that can be conducted on the metabolite precursors of interest. In this Perspective, we highlight recent developments in advanced MS/MS imaging strategies aimed at achieving high-coverage spatial metabolomics.

DOI: https://doi.org/10.1021/acs.analchem.5c00157
Anal Chem. 2025 Mar 31.

OzMALDI: A Gas-Phase, In-Source Ozonolysis Reaction for Efficient Double-Bond Assignment in Mass Spectrometry Imaging with Matrix-Assisted Laser Desorption/Ionization.

Josiah J Rensner, Hyojin Kim, Kiyoul Park, Edgar B Cahoon, Young Jin Lee.

Lipids make up an important class of biomolecules with diverse structures and varied chemical functions. This diversity is a major challenge in chemical analysis and limits our understanding of biological functions and regulation. A major way lipid isomers differ is by double-bond (db) position, and analyzing db-isomers is especially challenging for mass spectrometry imaging (MSI). Ozonolysis can be used to determine the db-position and has been paired with MSI before. However, previous techniques require increased analysis time to allow for gas-phase reactions within an ion trap or ion mobility cell or additional sample preparation time to allow for offline ozonation. Here, we introduce a new ozonolysis method inside the matrix-assisted laser desorption-ionization (MALDI) source, termed OzMALDI, that simultaneously produces ozonides from all unsaturated lipids. This allows us to determine db-positions without adding additional reaction time while maintaining the high mass resolution provided by Orbitrap MS. This new technique is especially effective at determining multiple db-positions in lipids containing polyunsaturated fatty acids, which is a limitation of many previous techniques. OzMALDI-MSI was applied to the analysis of rat brain and genetically engineered Camelina and soybean seed samples, demonstrating the utility of this method and uncovering novel biological information.

DOI: https://doi.org/10.1021/acs.analchem.5c00284
J Proteome Res. 2025 Mar 31.

How to Train a Postprocessor for Tandem Mass Spectrometry Proteomics Database Search While Maintaining Control of the False Discovery Rate.

Jack Freestone, Lukas Käll, William Stafford Noble, Uri Keich.

  Decoy-based methods are a popular choice for the statistical validation of peptide detection in tandem mass spectrometry and proteomics data. Such methods can achieve a substantial boost in statistical power when coupled with postprocessors such as Percolator that use auxiliary features to learn a better-discriminating scoring function. However, we recently showed that Percolator can struggle to control the false discovery rate (FDR) when reporting the list of discovered peptides. To address this problem, we introduce Percolator-RESET, which is an adaptation of our recently developed RESET meta-procedure to the peptide detection problem. Specifically, Percolator-RESET fuses Percolator's iterative SVM training procedure with RESET's general framework to provide valid false discovery rate control. Percolator-RESET operates in both a standard single-decoy mode and a two-decoy mode, with the latter requiring the generation of two decoys per target. We demonstrate that Percolator-RESET controls the FDR in both modes, both theoretically and empirically, while typically reporting only a marginally smaller number of discoveries than Percolator in the single-decoy mode. The two-decoy mode is marginally more powerful than both Percolator and the single-decoy mode and exhibits less variability than the latter.

Keywords:  false discovery rate control; proteomics; tandem mass spectrometry

DOI:  https://doi.org/10.1021/acs.jproteome.4c00742
STAR Protoc. 2025 Apr 03. pii: S2666-1667(25)00132-7. [Epub ahead of print]6(2): 103726

Protocol for label-free quantitative lysine lactylproteome profiling.

Yu Ran, Qiqing Yang, Lianqi Zhou, Heyu Li, Bing Yang, Long Zhang.

  L-lactate has been recognized as an essential molecule for signaling and metabolic balance. Lactylation, a post-translational modification (PTM) derived from L-lactate, is commonly observed on various proteins and plays essential roles in cellular processes. Here, we present a protocol to globally profile the lactylation proteome and perform label-free quantification. We provide steps for cell preparation, protein extraction, digestion, peptide desalting, and lactylpeptide enrichment. Additionally, we outline the parameters for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Keywords:  Mass spectrometry; Protein expression and purification; Proteomics

DOI:  https://doi.org/10.1016/j.xpro.2025.103726
Front Mol Biosci. 2025 ;12 1515276

A multi-omics reciprocal analysis for characterization of bacterial metabolism.

Gabriel Santos Arini, Tiago Cabral Borelli, Elthon Góis Ferreira, Rafael de Felício, Paula Rezende-Teixeira, Matheus Pedrino, Franciene Rabiço, Guilherme Marcelino Viana de Siqueira, Luiz Gabriel Mencucini, Henrique Tsuji, Lucas Sousa Neves Andrade, Leandro Maza Garrido, Gabriel Padilla, Alberto Gil-de-la-Fuente, Mingxun Wang, Norberto Peporine Lopes, Daniela Barretto Barbosa Trivella, Letícia Veras Costa-Lotufo, María-Eugenia Guazzaroni, Ricardo Roberto da Silva.

   Introduction: Exploiting microbial natural products is a key pursuit of the bioactive compound discovery field. Recent advances in modern analytical techniques have increased the volume of microbial genomes and their encoded biosynthetic products measured by mass spectrometry-based metabolomics. However, connecting multi-omics data to uncover metabolic processes of interest is still challenging. This results in a large portion of genes and metabolites remaining unannotated. Further exacerbating the annotation challenge, databases and tools for annotation and omics integration are scattered, requiring complex computations to annotate and integrate omics datasets.
Methods: Here we performed a two-way integrative analysis combining genomics and metabolomics data to describe a new approach to characterize the marine bacterial isolate BRA006 and to explore its biosynthetic gene cluster (BGC) content as well as the bioactive compounds detected by metabolomics.
Results and Discussion: We described BRA006 genomic content and structure by comparing Illumina and Oxford Nanopore MinION sequencing approaches. Digital DNA:DNA hybridization (dDDH) taxonomically assigned BRA006 as a potential new species of the Micromonospora genus. Starting from LC-ESI(+)-HRMS/MS data, and mapping the annotated enzymes and metabolites belonging to the same pathways, our integrative analysis allowed us to correlate the compound Brevianamide F to a new BGC, previously assigned to other function.

Keywords:  bioinformactics; genomics; microbiology; multi-omics analysis; untargeted metabolomics

DOI:  https://doi.org/10.3389/fmolb.2025.1515276
Anal Chem. 2025 Apr 01.

Rapid MALDI-MS/MS-Based Profiling of Lipid A Species from Gram-Negative Bacteria Utilizing Trapped Ion Mobility Spectrometry and mzmine.

Edward Rudt, Matti Froning, Steffen Heuckeroth, Lucas Ortmann, Julia Diemand, Linus Hörnschemeyer, Alexander Pleger, Max Vinzelberg, Robin Schmid, Tomáš Pluskal, Ulrich Dobrindt, Heiko Hayen, Ansgar Korf.

Lipid A, a crucial component of lipopolysaccharides (LPS), plays a pivotal role in the pathogenesis of Gram-negative bacteria. Lipid A patterns are recognized by mammals and can induce immunostimulatory effects. However, the outcome of the interaction is highly dependent on the chemical composition of individual lipid A species. The diversity of potential fatty acyl and polar headgroup combinations in this complex saccharolipid presents a significant analytical challenge. Current mass spectrometry (MS)-based lipid A methods are focused on either direct matrix-assisted laser desorption/ionization (MALDI)-MS screening or comprehensive structural elucidation by tandem mass spectrometry (MS/MS) hyphenated with separation techniques. In this study, we developed an alternative workflow for rapid lipid A profiling covering the entire analysis pipeline from sample preparation to data analysis. This workflow is based on microextraction and subsequent MALDI-MS/MS analysis of uropathogenic Escherichia coli utilizing trapped ion mobility spectrometry (TIMS), followed by mzmine data processing. The additional TIMS dimension served for enhanced sensitivity, selectivity, and structural elucidation through mobility-resolved fragmentation via parallel accumulation-serial fragmentation (PASEF) in parallel reaction monitoring (prm)-mode. Furthermore, mzmine enabled automated MS/MS acquisition by adapting the spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF) strategy for MALDI spot analysis. It also facilitated robust lipid A annotation through a newly developed extension of the rule-based lipid annotation module, allowing for the custom generation of lipid classes, including specific fragmentation rules. In this study, the first publication of lipid A species' collision cross section (CCS) values is reported, which will enhance high-confidence lipid A annotation in future studies.

DOI: https://doi.org/10.1021/acs.analchem.4c05989
Methods Mol Biol. 2025 ;2914 65-74

Targeted Absolute Quantification of Protein Biomarkers in Cerebrospinal Fluid by FastCAT.

Ignacy Rzagalinski, Viditha Rao, Aliona Bogdanova, Lena Hersemann, Andrej Shevchenko.

  Absolute (molar) quantification of proteins in cerebrospinal fluid (CSF) provides reference values and ranges of their physiological variation for perspective biomarkers across a broad spectrum of neurological disorders. FastCAT (for Fast-track QconCAT) method employs targeted mass spectrometry and multiple recombinant stable-isotope labeled chimeric proteins (CPs) as internal calibrants for the accurate and consistent determination of molar concentrations of dozens of CSF proteins. No purification or external quantification of CPs is required. We demonstrate that FastCAT, powered by parallel reaction monitoring (PRM) detection of multiple quantotypic peptides per target proteins, can quantify the molar concentration of neurological biomarkers from neat CSF over a dynamic range of more than 1000-fold, with high precision (coefficient of variation <20%) and low detection limits (<1 nmol/L) in a single LC-MS/MS analysis.

Keywords:  Absolute quantification; Cerebrospinal fluid; FastCAT; Protein biomarkers; Targeted proteomics

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_6
Methods Mol Biol. 2025 ;2914 99-113

Application of Isotope Dilution Gas Chromatography-Mass Spectrometry Using Selected Ion-Monitoring to Evaluate Nonenzymatic Protein Damage in Cerebrospinal Fluid.

Natalia Mota-Martorell, Mariona Jové Font, Anna Fernàndez-Bernal, Idoia Company-Marín, Alba Juanes-Casado, Reinald Pamplona.

  Mass spectrometry (MS)-based approach for the detection and quantification of nonenzymatic posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome damage with high throughput and deep coverage. Here, we describe a gas chromatography-mass spectrometry with a selected ion-monitoring mode (SIM-GC/MS) method for protein damage profiling of biological and clinical samples. This approach can be carried out on materials ranging from isolated proteins to tissue/biofluid samples. In particular, the SIM-GC/MS method is applied to detect and quantify diverse protein damage markers in the human cerebrospinal fluid (CSF) proteome. The protocol includes protein extraction, digestion, derivatization, PTMs detection and quantification by GC/MS, followed by computational data processing. Different protein damage markers of oxidation, glycoxidation, lipoxidation and succination can be confidently detected and quantified simultaneously. This protocol provides a general protein nonenzymatic modification measurement tool, enabling the dissection of protein and proteome modifications in a particular biofluid, the CSF.

Keywords:  Cerebrospinal fluid; Gas chromatography-mass spectrometry; Glycoxidation; Lipoxidation; Oxidation; Proteome; Selected ion-monitoring; Succination; Trifluoroacetic acid methyl esters

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_9
Cell. 2025 Mar 26. pii: S0092-8674(25)00275-2. [Epub ahead of print]

Global analysis of protein turnover dynamics in single cells.

Pierre Sabatier, Maico Lechner, Ulises H Guzmán, Christian M Beusch, Xinlei Zeng, Longteng Wang, Fabiana Izaguirre, Anjali Seth, Olga Gritsenko, Sergey Rodin, Karl-Henrik Grinnemo, Zilu Ye, Jesper V Olsen.

  Single-cell proteomics (SCPs) has advanced significantly, yet it remains largely unidimensional, focusing primarily on protein abundances. In this study, we employed a pulsed stable isotope labeling by amino acids in cell culture (pSILAC) approach to simultaneously analyze protein abundance and turnover in single cells (SC-pSILAC). Using a state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from ∼4,000 proteins in single HeLa cells recapitulating known biology. We performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSCs) encompassing 6 sampling times over 2 months and analyzed >1,000 cells. Protein turnover dynamics highlighted differentiation-specific co-regulation of protein complexes with core histone turnover, discriminating dividing and non-dividing cells. Lastly, correlating cell diameter with the abundance of individual proteins showed that histones and some cell-cycle proteins do not scale with cell size. The SC-pSILAC method provides a multidimensional view of protein dynamics in single-cell biology.

Keywords:  Chip-Tip; Evosep; Orbitrap Astral; cellenONE; histone; iPSC differentiation; mass spectrometry; protein turnover; pulsed SILAC; single-cell proteomics

DOI:  https://doi.org/10.1016/j.cell.2025.03.002
Anal Chem. 2025 Apr 01.

Examining Instrumental Factors Influencing the Performance of Data-Independent Acquisition Methods in Hydrogen/Deuterium Exchange Mass Spectrometry.

Frantisek Filandr, Morgan Hepburn, Vladimir Sarpe, D Alex Crowder, Maryam Hassannia, Stephen Coales, Yuqi Shi, Rosa Viner, Martin A Rossotti, Joey G Sheff, Jamshid Tanha, David C Schriemer.

Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS2 technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy settings typical of a proteomics DIA experiment generate maximal peptide retrieval from the DIA library. As few as three MS2 sequence ions are sufficient to generate a deuteration measurement with a precision that exceeds what is possible in traditional HX-MS. DIA window sizes are based on the chromatographic resolution of the method. An inter-scan window offset method is the recommended default configuration for most HX-DIA applications but an intra-scan overlap method can be tuned for highest performance and is recommended when maximum peptide retrieval is desired. We demonstrate the robustness of one HX-MS2 configuration (consisting of Trajan HDX automation technology, an Orbitrap Eclipse mass spectrometer and AutoHX software) on an extensive time-course analysis of phosphorylase B and an epitope analysis of single-domain antibodies (VHHs, nanobodies) specific to the receptor binding domain of SARS-CoV-2 spike protein.

DOI: https://doi.org/10.1021/acs.analchem.5c00429
J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Mar 28. pii: S1570-0232(25)00130-8. [Epub ahead of print]1257 124578

Pentafluorobenzyl bromide - A versatile derivatization agent in chromatography and mass spectrometry: II. Analysis of organic acids and bases, and comparison with other perfluorinated reagents.

Dimitrios Tsikas.

  Analytical derivatization is an important for the vast majority of substances an indispensable sample preparation step for their quantitative GC-MS and GC-MS/MS analysis in biological samples. Pentafluorobenzyl bromide (PFB-Br), pentafluorobenzoyl chloride (PFB-COCl), pentafluorobenzyl hydroxylamine (PFB-NHNH2), pentafluorophenyl hydrazine (PFPh-ONH2), pentafluoropropionic anhydride (PFPA), and heptafluorobutyric anhydride (HFBA) are versatile derivatization reagents in analytical chemistry. In the present work, the utility of the above mentioned derivatization reagents for the GC-MS analysis of carboxylic, aldehydic, hydroxylic and amine groups containing analytes including amino acids is reviewed and discussed. Derivatization requires different conditions for solvents, reaction temperature and time, and possibly for catalysts. The perfluorinated derivatives are electrically neutral and best soluble in water-immiscible organic solvents such as toluene. Under negative-ion chemical ionization (NICI) conditions, the perfluorinated derivatives readily and abundantly ionize that allows for sensitive analysis. In addition, the perfluorinated analyte derivatives emerge earlier from GC columns than protiated, thus enabling shorter analysis times. Externally added 2H-, 13C-, 15N and 18O-isotopologs for use as internal standards undergo similar changes during derivatization, extraction by organic solvents, ionization in the ion-source of GC-MS apparatus and have almost identical retention times with the analytes. Due to selective analytical derivatization, almost all classes of endogenous and exogenous low-molecular-mass analytes, including drugs and inorganic anions such as nitrite, nitrate, carbonate, and (pseudo)halogenides, become accessible to quantitative GC-MS and GC-MS/MS analysis. Thanks the high sensitivity of quantitative analytical methods based on GC-MS and GC-MS/MS, very low amounts of perfluorinated derivatization reagents are consumed. In consideration of the enormously high global warming potential (GWP) of F-containing derivatization reagents, this article discussed a potential abandonment of the use of perfluorinated reagents and their replacement by F-free reagents in GC-MS and GC-MS/MS.

Keywords:  Derivatization; Fluorine; GC–MS; GC–MS/MS; Global warming potential; Perfluorinated reagents

DOI:  https://doi.org/10.1016/j.jchromb.2025.124578
Methods Mol Biol. 2025 ;2914 251-258

An LC-MS/MS Multiplexed Method to Quantify 14 Sphingolipids in Human Cerebrospinal Fluid (CSF).

Yadira Perez Paramo, Rathna Veeramachaneni, Emily Parkhurst, Dawn Dufield, W Rodney Mathews, Veronica G Anania.

  Sphingolipids are bioactive lipids comprised of a sphingoid base linked to various long-chain fatty acids and phosphocholine and, in some cases, may also contain one or more sugars. A few species of sphingolipids share similar structures and may even have identical chemical composition but differ in their biological function. For this reason, they can only be accurately quantified using liquid chromatography coupled with mass spectrometry (LC-MS). This technique can separate sphingolipids based on their hydrophobicity and quantify them based on their mass-to-charge ratio (m/z). This is especially important when measuring isomeric species that have identical m/z but differ in their retention time. Here, we describe the development of a new LC-MS method to measure 14 sphingolipid species, including 7 ceramides (C14, C16, C18, C20, C24, C18:1, C24:1), 4 glycosphingolipids (C16 Glucosyl(ß) Ceramide (d18:1/16:0, C16 Galactosyl(ß) Ceramide (d18:1/16:0), C16 Lac Cer, C18 Lac Cer) and 3 sphingosines (Sphingosine (Sph), Glucosyl(ß) Sphingosine (d18:1), Galactosyl(ß) Sphingosine (d18:1)) in human cerebrospinal fluid (CSF). Using this 7-min method, chromatographic resolution of isomeric species glucosylceramide (Glc Cer) and galactosylceramide (Gal Cer) was achieved. This resolution is important as these two sphingolipid classes have distinct biological functions with important implications in neurological diseases like multiple sclerosis.

Keywords:  CSF; Gal Cer; Glc Cer; Isomers; LC-MS; Neurodegenerative diseases; Sphingolipids

DOI:  https://doi.org/10.1007/978-1-0716-4462-1_18
J Am Soc Mass Spectrom. 2025 Apr 03.

Norm ISWSVR Enhanced Data Repeatability and Accuracy in Large-Scale Targeted Quantification Metabolomics.

Jinpeng Bai, Chenxi Li, Mingmin Qian, Xian Ding, Qian Li, Yanhua Chen, Zhaoying Wang, Zeper Abliz.

  Targeted quantification metabolomics provides dynamic insights across various domains within the life sciences. Nevertheless, maintaining high-quality data obtained through liquid chromatography-mass spectrometry presents ongoing challenges. It is essential to develop normalization methods to correct for unwanted variations in metabolomic profiling such as batch effects and analytical drift. In this study, we assessed the normalization efficacy of Norm ISWSVR in targeted quantification metabolomics by comparing it with IS normalization and SERRF normalization. Consequently, Norm ISWSVR demonstrated exceptional efficacy in mitigating batch effects and reducing the relative standard deviation of quality control samples, in addition to correcting signal drift. Following normalization with Norm ISWSVR, the number of metabolites suitable for quantification increased with high precision. Collectively, Norm ISWSVR proves to be a robust and reliable method for enhancing data quality in targeted metabolomics, establishing itself as a promising approach for metabolomics research.

Keywords:  Norm ISWSVR; batch effects; signal drift; targeted metabolomics

DOI:  https://doi.org/10.1021/jasms.4c00467
Anal Chim Acta. 2025 May 08. pii: S0003-2670(25)00268-5. [Epub ahead of print]1350 343874

Establishment of a pseudotargeted LC‒MS/MS workflow for analyzing triglycerides in biological samples.

Wei-Chieh Wang, Chin-Yi Wang, Ta-Chen Su, Po-Chih Lin, Wen-Chi Chang, Kuei-Pin Chung, Ching-Hua Kuo.

   BACKGROUND: Triglycerides (TGs) play a crucial role in various physiological processes through the breakdown of their fatty acyl (FA) side chains. It has been demonstrated that not only the total levels of TGs but also the specific composition of FA side chains are vital for biological functions. However, biomedical studies that comprehensively identify FA compositions remain very limited. One of the reasons is the structural heterogeneity of TGs, with variability in their three fatty acyl chains posing significant challenges for TG analysis.
RESULTS: This study proposed a pseudotargeted TG analytical workflow that generated a unique dynamic multiple reaction monitoring (dMRM) acquisition list tailored to different biological sample types.TG profiles were acquired in full scan mode using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-qToF), while LC-triple quadrupole mass spectrometry (LC-QqQ) with PIS was applied to identify fatty acyl chains. Finally, dMRM transitions were derived from confirmed ion pairs of TGs with specific FAs. Two demonstration samples, murine type 2 alveolar epithelial cell line, MLE12, with fatty acid synthase deletion, and hypertriglyceridemia plasma, were used to display the capability of the platform. While more TG species were identified in the MLE12 cell samples compared to human plasma samples (53 vs. 47), a more complex and diverse range of FA compositions in TGs was observed in human plasma compared to MLE12 cell samples (379 vs. 167).
SIGNIFICANCE: Our results emphasize the need for customized MRM acquisition tailored to different biological samples, and the pseudotargeted TG analytical workflow proves effective in improving the understanding of TG regulation in biological systems. This study offers a novel and effective solution to address the complex challenges of TG analysis, enhancing accuracy, specificity, and interpretative strength.

Keywords:  Hypertriglyceridemia (HTG); Liquid chromatography-mass spectrometry (LC-MS); Pseudotargeted analysis; Triglyceride (TG)

DOI:  https://doi.org/10.1016/j.aca.2025.343874
bioRxiv. 2025 Mar 15. pii: 2025.03.13.642445. [Epub ahead of print]

Data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) analysis of the separated zebrafish lens improves identifications.

Sarah R Zelle, W Hayes McDonald, Kristie L Rose, Hassane S Mchaourab, Kevin L Schey.

Ocular lens fiber cells degrade their organelles during differentiation to prevent light scattering. Organelle degradation occurs continuously throughout an individual's lifespan, creating a spatial gradient of young cortical fiber cells in the lens periphery to older nuclear fiber cells in the center of the lens. Therefore, separation of cortical and nuclear regions enables examination of protein aging. Previously, the human lens cortex and nucleus have been studied using data-independent acquisition (DIA) proteomics, allowing for the identification of low-abundance protein groups. In this study, we employed data-independent acquisition parallel accumulation-serial fragmentation (diaPASEF) proteomics on a timsTOF HT instrument to study the zebrafish lens proteome and compared results to a standard DIA method employed on an Orbitrap Exploris 480. Using the additional ion mobility gas phase separation of diaPASEF, peptide and protein group identifications increased by over 200% relative to an orbitrap DIA method in the zebrafish lens. With diaPASEF, we identified 13,721 and 11,996 unique peptides in the zebrafish lens cortex and nucleus, respectively, which correspond to 1,537 and 1,389 protein groups. Thus, separation of the zebrafish lens into cortical and nuclear regions followed by diaPASEF analysis produced the most comprehensive zebrafish lens proteomic dataset to date.

DOI: https://doi.org/10.1101/2025.03.13.642445
J Am Soc Mass Spectrom. 2025 Apr 03.

Evaluating Ion Mobility Data Acquisition, Calibration, and Processing for Small Molecules: A Cross-Platform Assessment of Drift Tube and Traveling Wave Methodologies.

James N Dodds, Lucie C Ford, Jack P Ryan, Amie M Solosky, Ivan Rusyn, Erin S Baker.

As ion mobility spectrometry (IMS) separations continue to be added to analytical workflows due to their power in environmental and biological sample analyses, harmonization and capability understanding between existing and newly released instruments are desperately needed. Developments in IMS platforms often exhibit focus on increasing resolving power (Rp) to better separate molecules of similar structure. While the additional separation capacity is advantageous, ensuring these developments coincide with appropriate data extraction and analysis methods is imperative to ensure routine adoption. Herein, we assess the performance of the MOBILion MOBIE in relation to a commercially available drift tube IMS-MS, the Agilent 6560, and evaluate feature extraction and analysis pipelines. Both instruments were operated using matched conditions when possible, and performance metrics of scan speed, Rp, limits of detection (LOD), and propensity for isomer separation via LC-IMS-MS were evaluated. Similar scan speeds pertaining to IMS-MS frame generation were noted for both platforms, and collision cross section (CCS) values for the MOBIE were generally within ≤ 1% difference from previously reported drift tube values. Both platforms were also able to generate quantitative data (comparable limits of detection) in experiments with perfluoroalkyl substances (PFAS) mixtures in a cell-based model (both medium and cell lysates), as demonstrated in Skyline with adjusted mobility filtering parameters. Higher Rp was, however, noted on the MOBIE in comparison to the 6560 (200-300 vs 45-60 CCS/ΔCCS without data processing), allowing the detection of more PFAS isomers and indicating promise toward future applications in chemical exposomics studies and biomarker discovery when molecules exhibit similar structures.

DOI: https://doi.org/10.1021/jasms.5c00056