bims-mascan 2025-06-22 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2025–06–22
sixteen papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Assessment of false discovery rate control in tandem mass spectrometry analysis using entrapment.
Protocol for spatial proteomic profiling of tonsil cancer microenvironments using multiplexed imaging-powered deep visual proteomics.
High-resolution accurate mass- mass spectrometry based- untargeted metabolomics: Reproducibility and detection power across data-dependent acquisition, data-independent acquisition, and AcquireX.
LC-MS Orbitrap-based metabolomics using a novel hybrid zwitterionic hydrophilic interaction liquid chromatography and rigorous metabolite identification reveals doxorubicin-induced metabolic perturbations in breast cancer cells.
Comprehensive lipid structure annotation via photochemical epoxidation and mass spectrometry.
A unified pan-cancer proteome atlas.
Single-Cell Lipidomics by LC-MS Interlaboratory Study Reveals the Impact of X-ray Irradiation on a Pancreatic Cancer Cell Line and Its Bystanders.
Activity-Based Protein Profiling for Functional Cysteines and Protein Target Identification.
Targeting amino acid in tumor therapy.
Untargeted analysis of hydrophilic metabolites using enhanced LC-MS separation with a pentafluoro phenylpropyl-functionalized column and prediction-based MS/MS spectrum annotation.
Untargeted and semi-targeted metabolomics approach for profiling small intestinal and fecal metabolome using high-resolution mass spectrometry.
Quantification of Endogenous Steroids and Hormonal Contraceptives in Human Plasma via Surrogate Calibration and UHPLC-MS/MS.
Faecal amino acids stability: investigating optimal sampling conditions for analysis.
Quantitative neuropeptide analysis by mass spectrometry: advancing methodologies for biological discovery.
Activity-Based Profiling of Reactive Nucleophilic Cysteine Sites from Tissue Proteomes for Drug Discovery Applications.
Screening of Protein Carbonylation Sites in Human Serum by Ion Mobility Mass Spectrometry.

Nat Methods. 2025 Jun 16.

Assessment of false discovery rate control in tandem mass spectrometry analysis using entrapment.

Bo Wen, Jack Freestone, Michael Riffle, Michael J MacCoss, William S Noble, Uri Keich.

A critical challenge in mass spectrometry proteomics is accurately assessing error control, especially given that software tools employ distinct methods for reporting errors. Many tools are closed-source and poorly documented, leading to inconsistent validation strategies. Here we identify three prevalent methods for validating false discovery rate (FDR) control: one invalid, one providing only a lower bound, and one valid but under-powered. The result is that the proteomics community has limited insight into actual FDR control effectiveness, especially for data-independent acquisition (DIA) analyses. We propose a theoretical framework for entrapment experiments, allowing us to rigorously characterize different approaches. Moreover, we introduce a more powerful evaluation method and apply it alongside existing techniques to assess existing tools. We first validate our analysis in the better-understood data-dependent acquisition setup, and then, we analyze DIA data, where we find that no DIA search tool consistently controls the FDR, with particularly poor performance on single-cell datasets.

DOI: https://doi.org/10.1038/s41592-025-02719-x
STAR Protoc. 2025 Jun 18. pii: S2666-1667(25)00307-7. [Epub ahead of print]6(3): 103901

Protocol for spatial proteomic profiling of tonsil cancer microenvironments using multiplexed imaging-powered deep visual proteomics.

Xiang Zheng, Andreas Mund, Matthias Mann.

  Here, we present a protocol for spatial proteomic profiling of the tumor microenvironment in tonsil cancer using multiplexed imaging-powered deep visual proteomics (mipDVP). We describe steps for automated 22-plex immunofluorescence staining and imaging on formalin-fixed paraffin-embedded (FFPE) tissue sections, automated single-cell laser microdissection, and single-cell-type mass spectrometry. This workflow enables the spatially resolved isolation of distinct cell populations for proteomic analysis. We optimized this protocol for studying tumor-immune interactions, where it facilitates the systematic identification of biomarkers and functional cellular networks. For complete details on the use and execution of this protocol, please refer to Zheng et al.1.

Keywords:  Biotechnology and bioengineering; Cancer; Microscopy

DOI:  https://doi.org/10.1016/j.xpro.2025.103901
Comput Struct Biotechnol J. 2025 ;27 2412-2423

High-resolution accurate mass- mass spectrometry based- untargeted metabolomics: Reproducibility and detection power across data-dependent acquisition, data-independent acquisition, and AcquireX.

Hanane El Boudlali, Laura Lehmicke, Uta Ceglarek.

  Untargeted metabolomics aims at the unbiased metabolic profiling and biomarker discovery but requires methods with high sensitivity and reproducibility. Here, we compare three acquisition modes-Data-Dependent Acquisition (DDA), Data-Independent Acquisition (DIA), and AcquireX -to evaluate performance and reproducibility in detecting low-abundance metabolites in a complex matrix. A system suitability test (SST) based on 14 eicosanoid standards was implemented to evaluate the suitability of our instrumental setup prior to conducting untargeted metabolomics analyses and monitor long-term system performance. Bovine liver total Lipid Extract (TLE) was spiked with decreasing levels (10-0.01 ng/mL) of the eicosanoid standard mix (StdMix) to compare the detection power of each mode. Reproducibility was evaluated over three independent measurements, spaced one week apart. Chromatographic separation was performed on a C18-Kinetex Core-Shell column and HRAM-MS/MS data were acquired using an Orbitrap Exploris 480. DIA detected and identified the highest number of metabolic features, (averaging 1036 metabolic features over three measurements), followed by DDA (18 % fewer) and AcquireX (37 % fewer). Moreover, DIA demonstrated superior reproducibility, with a coefficient of variance of 10 % across detected compounds over three measurements, compared to 17 % for DDA and 15 % for AcquireX. DIA further exhibited better compound identification consistency, with 61 % overlap between two days, compared to DDA (43 %) and AcquireX (50 %). DIA reproduced fragmentation spectra patterns with high consistency, contributing to higher reproducibility in compound identification. DIA showed the best detection power for all spiking eicosanoids at 10 and 1 ng/mL in TLE matrix. At low spiking levels, 0.1 and 0.01 ng/mL, a general cut-off was observed for the three acquisition modes. None of this assessed acquisition modes was able to detect and/or identify eicosanoids at physiologically relevant concentrations, explaining their frequent omission in routine untargeted analyses.

Keywords:  AcquireX; DDA; DIA; detection power; reproducibility

DOI:  https://doi.org/10.1016/j.csbj.2025.05.046
RSC Adv. 2025 Jun 16. 15(26): 20745-20759

LC-MS Orbitrap-based metabolomics using a novel hybrid zwitterionic hydrophilic interaction liquid chromatography and rigorous metabolite identification reveals doxorubicin-induced metabolic perturbations in breast cancer cells.

Salah Abdelrazig, Áine McCabe, Alia Yasin, Rajneil Chaudhary, Michael A Ochsenkühn, David Scicchitano, Shady A Amin.

The identification of metabolites in biological samples presents a challenge in untargeted metabolomics, mainly due to limited databases and inadequate chromatography. Current LC columns suffer from high pH instability (silica-based), low efficiencies and pressure limitations (polymer-based), or inadequate retention of polar/semi-polar metabolites (reverse-phase). In this study, a comprehensive LC-MS workflow was developed to address these limitations using a novel zwitterionic HILIC (Z-HILIC), high-resolution MS, deep-scan data-dependent acquisition (DDA), and a large chemical library comprising 990 standards. The method performance was evaluated and compared with a widely-used ZIC-pHILIC method. Z-HILIC detected 707 (71%) of the standards compared to 543 (55%) standards with the ZIC-pHILIC showing enhanced resolution, sensitivity, selectivity and retention time (RT) distribution. In triple-negative Hs578T breast cancer cell extracts spiked with the standards, Z-HILIC annotated 79.1% of the detected standards versus 66.6% with ZIC-pHILIC, demonstrating improved sensitivity, stability, and reduced matrix effects for metabolite profiling. Deep-scan DDA of the spiked cell extracts increased the number of the identified metabolites using RT, m/z and MS/MS by more than 80% compared to standard DDA. The workflow was used to investigate the metabolic signature of doxorubicin-treated Hs578T cells (n = 15). The analysis resulted in identifying 173 metabolites, of which 26 metabolites and 20 metabolic pathways were significantly altered in doxorubicin treated cells compared to controls. These pathways were associated with oxidative stress, mitochondrial dysfunction, and impaired biosynthesis, consistent with prior knowledge about the action of doxorubicin. This comprehensive workflow promises to enhance metabolite profiling across diverse metabolomics studies.

DOI: https://doi.org/10.1039/d5ra01044f
Anal Bioanal Chem. 2025 Jun 20.

Comprehensive lipid structure annotation via photochemical epoxidation and mass spectrometry.

Jing Yu, Belal Alshaar, Sven Heiles.

  Cells tightly regulate lipid structures to fulfill cellular functions and to respond to external stimuli. The biochemical details of the processes that determine lipid structure alterations are often not fully understood. In this manuscript, we present a new epoxidation strategy of unsaturated lipids, which allows the annotation of lipid head group, fatty acid composition, C = C bond position, C = C bond geometry, and sn-isomerism when derivatized lipids are analyzed with corresponding separation and mass spectrometry methods. Tandem mass spectra of epoxidized deprotonated or protonated lipids provide information on head groups, fatty acids, and C = C positions, while MSⁿ of alkali metal adducts reveals sn-isomer compositions. Separation of epoxidation products via reversed-phase liquid chromatography (RPLC) not only distinguishes lipid C = C position and sn-isomers, remaining non-reacted unsaturated lipid C = C bonds photo-isomerize to reveal C = C E/Z configurations. To demonstrate the capabilities of the methodology, C = C positions of LPEs, LPCs, TGs, DGs, PCs, PSs, and PEs are annotated for bovine heart and liver extracts in RPLC-MS2 experiments, and shotgun MSn is employed to characterize 56 PC sn-isomers in HeLa and H9c2 cell lines.

Keywords:  Epoxidation; Isomers; Lipid derivatization; Mass spectrometry; Tandem mass spectrometry

DOI:  https://doi.org/10.1007/s00216-025-05953-6
Cancer Cell. 2025 Jun 04. pii: S1535-6108(25)00221-1. [Epub ahead of print]

A unified pan-cancer proteome atlas.

Yang Du, Jianmin Wu.

In this issue of Cancer Cell, Knol et al. present the Pan-Cancer Proteome Atlas (TPCPA), a proteomic resource developed using single-shot data-independent acquisition mass spectrometry (DIA-MS). TPCPA provides proteome-scale quantifications of 999 tumors across 22 cancer types in a unified manner, for discovering tumor biology, biomarkers, and therapeutic targets.

DOI: https://doi.org/10.1016/j.ccell.2025.05.012
Anal Chem. 2025 Jun 17.

Single-Cell Lipidomics by LC-MS Interlaboratory Study Reveals the Impact of X-ray Irradiation on a Pancreatic Cancer Cell Line and Its Bystanders.

Kyle D G Saunders, Johanna von Gerichten, Rahul Deshpande, Matt Spick, Susan Bird, Giuseppe Schettino, Hannah Bolland, Anthony Whetton, Eirini Velliou, Melanie J Bailey.

Live single-cell lipidomics by liquid chromatography mass spectrometry (LC-MS) is a nascent and rapidly growing field which can shed new light on infectious diseases, cancer, immunology, and drug delivery. There are now a growing number of laboratories that can isolate single cells and laboratories that can perform lipidomics analysis at correspondingly low sample volumes, but there is a lack of validation data. We have carried out the first interlaboratory LC-MS lipidomics experiment for single cells, aimed at filling this gap. We present a novel workflow to enable interlaboratory studies, comprising live-cell imaging and single-cell isolation, followed by freeze-drying, international shipping, reconstitution, and untargeted lipidomics analysis. We applied this methodology to reveal radiation-induced bystander effects in pancreatic cancer cells. X-ray irradiated cells and their bystanders sampled live 48 h postirradiation demonstrated reduced lipid abundance compared to controls, with distinct changes in molar ratios of several polyunsaturated lipids. This demonstrates for the first time that radiation can cause considerable cellular lipid remodelling, not only at the site of delivery. A striking similarity in lipid changes was observed between the two participating laboratories despite differences in sample preparation and analysis methods. Our results are further corroborated by live-cell imaging analysis of lipid droplets. This work serves as an important validation and demonstration of the nascent and rapidly growing field of live single-cell lipidomics.

DOI: https://doi.org/10.1021/acs.analchem.5c02010
Methods Mol Biol. 2025 ;2921 331-344

Activity-Based Protein Profiling for Functional Cysteines and Protein Target Identification.

Tin-Yan Koo, Clive Yik-Sham Chung.

  Activity-based protein profiling (ABPP) has been widely used for proteome-wide cysteine profiling to study functional cysteines in biological systems. ABPP can also be performed in a competitive manner for lead compound discovery and target identification of the lead compound. Recently, we reported a new class of acrylamide-based cysteine probe, NAIA, which has superior cysteine reaction kinetics to capture more functional cysteines. We further established the workflow of using NAIA for cysteine profiling by mass spectrometry (MS)-based ABPP. Here, we describe a step-by-step protocol using NAIA in the MS-based ABPP experiments for profiling functional cysteines in cancer cells/cell lysates and protein target identification of covalent ligands.

Keywords:  Acrylamide; Activity-based probes; Activity-based protein profiling; Chemoproteomics; Cysteine profiling; Cysteinome; Mass spectrometry; Target identification

DOI:  https://doi.org/10.1007/978-1-0716-4502-4_18
Front Oncol. 2025 ;15 1582116

Targeting amino acid in tumor therapy.

Mingpai Ge, Yuan Xu, Lu Cui, Enyuan Huang, Zhaorui Liu, Kai Yin.

  Tumor cells undergo profound metabolic reprogramming to sustain their rapid growth and proliferation, with amino acids serving as essential nutrients critical for protein synthesis, energy metabolism, nucleotide production, and redox balance. The increased reliance of tumor cells on specific amino acids represents a promising therapeutic target. This review provides an in-depth analysis of the biological roles of amino acids in cancer, identifies vulnerabilities associated with amino acid dependency, and discusses strategies to leverage these weaknesses for enhanced cancer treatment. We explore the mechanisms governing amino acid uptake, utilization, and metabolism in tumor cells, as well as their interactions with the tumor microenvironment. Additionally, the review addresses the challenges and prospects of targeting amino acid metabolism in cancer therapy, including issues of resistance, the complexity of metabolic pathways, and the potential for personalized treatment approaches.

Keywords:  amino acid; amino acid metabolism; cancer metabolism; cancer therapy; metabolism

DOI:  https://doi.org/10.3389/fonc.2025.1582116
Metabolomics. 2025 Jun 14. 21(4): 79

Untargeted analysis of hydrophilic metabolites using enhanced LC-MS separation with a pentafluoro phenylpropyl-functionalized column and prediction-based MS/MS spectrum annotation.

Masaru Sato, Kazutaka Ikeda.

   INTRODUCTION: Wide-targeted metabolome analysis has been applied in studies on the biology of mammals, plants, and microorganisms. However, there are still issues regarding both analytical and informatics technologies for establishing an untargeted and comprehensive analysis of hydrophilic primary and secondary metabolites.
OBJECTIVES: This study aimed to develop an improved chromatographic method for analyzing hydrophilic metabolites and an annotation method for these diverse metabolites.
METHODS: We investigated the performance of a pentafluoro phenylpropyl-functionalized column (PFP column) for the comprehensive analysis of hydrophilic metabolites by liquid chromatography-mass spectrometry (LC-MS). Peaks were annotated using MS/MS spectral similarity searches of the predicted and experimental MS/MS spectra in metabolite structure databases.
RESULTS: The improved retention and peak shapes of the standard compounds were obtained using LC-MS analysis with a PFP column. The mobile phases comprised water with 0.1% formic acid and methanol with 0.1% formic acid and 10 mM ammonium formate. From the annotation results of the 48 standard compounds, the chemical structures were correctly annotated in 54% of the compounds. However, over 70% of the compounds were annotated as biologically relevant based on the natural product classification. When these methods were applied to the analysis of tomato fruits, 658 and 458 peaks were detected and annotated in the positive and negative ion analyses, respectively.
CONCLUSION: Metabolome analysis combined with LC-MS analysis and annotation can contribute to the comprehensive analysis of hydrophilic metabolites.

Keywords:  Hydrophilic metabolite; LC-MS analysis; Metabolite annotation; Untargeted metabolome analysis

DOI:  https://doi.org/10.1007/s11306-025-02272-w
Metabolomics. 2025 Jun 19. 21(4): 84

Untargeted and semi-targeted metabolomics approach for profiling small intestinal and fecal metabolome using high-resolution mass spectrometry.

Alexandre Tronel, Morgane Roger-Margueritat, Caroline Plazy, Valérie Cunin, Ipsita Mohanty, Pieter C Dorrestein, Thomas Soranzo, Audrey Le Gouellec.

   INTRODUCTION: The gut microbiome is a complex ecosystem stratified that varies along different sections of the gut. It comprises a wide array of metabolites originating from both food, host, and microbes. Microbially-derived metabolites, such as bile acids, short-chain fatty acids, and indole derivatives, are of significant interest due to their direct interactions with host physiology and regulating function. Most current studies on the gut microbiome focus on fecal samples, which do not fully represent the upper parts of the gut due to its stratification. To collect microbiome samples from the proximal gut microbiome, endoscopic methods or new non-invasive medical devices can be used.
OBJECTIVES: To enable comprehensive profiling of the gut metabolome and analyze key metabolites, we developed a combined approach combining untargeted and semi-targeted metabolomics using a Q-Exactive Plus Orbitrap mass spectrometer.
METHODS: Initially, we selected 49 metabolites of interest for the gut metabolome based on four distinct criteria. We validated these metabolites through repeatability and linearity tests and created a compound database using the software TraceFinder (ThermoFisher Scientific). For untargeted metabolomics, we established a workflow for the annotation and discovery of molecules.
RESULTS: Finally, 37 metabolites were validated for semi-targeted metabolomics, and we conducted a proof of concept on small intestinal and fecal samples form a clinical trial (NCT05477069).
CONCLUSION: Our combined approach, facilitated by molecular networking, demonstrated the potential to discover new metabolites.

Keywords:  Combined approach; LC-MS/MS; Metabolites; Microbiome; Small intestine

DOI:  https://doi.org/10.1007/s11306-025-02288-2
Anal Chem. 2025 Jun 20.

Quantification of Endogenous Steroids and Hormonal Contraceptives in Human Plasma via Surrogate Calibration and UHPLC-MS/MS.

Min Su, Bernhard Drotleff, Tamara Janker, Zoé Bürger, Ann-Christin S Kimmig, Birgit Derntl, Michael Lämmerhofer.

Quantifying endogenous and exogenous steroids at low concentrations in biological matrices remains a major analytical challenge. Immunoassay-based diagnostics are limited by cross-reactivity, particularly at low levels, prompting a shift toward (ultra)high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC-MS/MS) for clinical applications. A key limitation for endogenous hormone quantification is the absence of a true blank matrix for external calibration. To address this, we developed a surrogate calibration method employing 1,2-dimethylimidazole-5-sulfonyl chloride (DMIS) derivatization for estrogens, enabling sensitive and selective quantification alongside nonderivatized steroids. Stable isotope-labeled surrogate calibrants and internal standards were used to achieve matrix-matched quantification within a clinically relevant range. Parallelism between analytes and surrogate calibrants was systematically verified in plasma across multiple calibration levels. The method was further optimized through the use of narrow-bore UHPLC columns and refined chromatographic conditions to enhance sensitivity and resolution for a broad analyte panel. Combined with efficient protein precipitation and 96-well plate-based solid-phase extraction, the developed assay achieves pg/mL-level quantification in human plasma with high precision and accuracy. This integrated approach uniquely combines surrogate calibration for endogenous steroids with external calibration for exogenous contraceptives, including sensitive DMIS-based derivatization for estrogens, enabling comprehensive hormonal profiling in a single run. Beyond its analytical scope, the method outlines a structured validation strategy, which is aligned with regulatory principles, and may therefore serve as a practical reference for future LC-MS/MS assays employing surrogate calibration.

DOI: https://doi.org/10.1021/acs.analchem.5c01912
Metabolomics. 2025 Jun 14. 21(4): 81

Faecal amino acids stability: investigating optimal sampling conditions for analysis.

Roza C M Opperman, Eva Vermeer, Sofie Bosch, Tim G J de Meij, Nanne K H de Boer, Eduard A Struys.

   INTRODUCTION: Faecal amino acids are promising non-invasive diagnostic biomarkers, but stability remains unclear.
OBJECTIVES: This study examined whether storage conditions, sampling site within the sample, freeze-thaw cycles, and the OMNImet®·GUT device affect faecal amino acid concentrations.
METHODS: Faecal samples from three donors underwent various pre-analytical conditions. Amino acids were analysed through targeted liquid chromatography-tandem mass spectrometry.
RESULTS: Most amino acids remained stable across sampling sites and freeze-thaw cycles. Storage at -20 °C preserved integrity, whereas 4 °C and 20 °C storage led to variations. The OMNImet®·GUT device stabilised some amino acids but showed inconsistencies.
CONCLUSION: Pre-analytical conditions influence faecal amino acid concentrations. Standardisation is essential for biomarker reliability.

Keywords:  Faecal amino acids; LC-MS/MS; Pre-analytical conditions; Sample stability; Targeted metabolomics

DOI:  https://doi.org/10.1007/s11306-025-02279-3
RSC Chem Biol. 2025 Jun 12.

Quantitative neuropeptide analysis by mass spectrometry: advancing methodologies for biological discovery.

Angel Erbey Ibarra, Wenxin Wu, Haoran Zhang, Lingjun Li.

Neuropeptides are critical endogenous signaling molecules involved in a wide range of biological processes, including neurotransmission, hormonal regulation, immune responses, and stress management. Despite their importance, the field of neuropeptide research has been historically hampered by significant technical challenges. These include their low abundance in biological systems, diverse and complex post-translational modifications, dynamic expression patterns, and susceptibility to degradation. As such, traditional proteomics approaches often fall short of accurately characterizing neuropeptides, underscoring the need for specialized methodologies to unlock their biological and translational potential. This review evaluates state-of-the-art quantitative mass spectrometry (MS)-based peptidomics, emphasizing their impact on neuropeptide analysis. We highlight how strategies in label-free and label-based quantitation, tandem MS acquisition, and mass spectrometry imaging provide unprecedented sensitivity and throughput for capturing the landscape of neuropeptides and their modifications. Importantly, the review bridges technological innovation with practical applications, highlighting how these approaches have been utilized to uncover novel neuropeptides and elucidate their roles in systems biology and disease pathways.

DOI: https://doi.org/10.1039/d5cb00082c
Methods Mol Biol. 2025 ;2921 319-329

Activity-Based Profiling of Reactive Nucleophilic Cysteine Sites from Tissue Proteomes for Drug Discovery Applications.

Hasini Kalpage, An Chi, Smaranda Bodea.

  Identifying engaged nucleophilic sites in a proteome-wide manner has become a powerful means to leverage activity-based protein profiling toward assessing the target landscape of covalent compounds ranging from fragments to approved drugs. In this chapter, we provide a detailed protocol for reactive cysteine profiling from tissue lysates using a low-input high throughput-compatible workflow based on multiplexing via TMT-16pro. We apply this method to a representative biological context, human heart tissue lysates treated with highly reactive and promiscuous electrophilic scout fragments. We detect over 11,000 cysteine sites and multiple engaged ligandable sites specific to each scout fragment, revealing the quantitative accuracy of this method. Due to the low input material requirements, this protocol can be applied to many different contexts relevant to drug discovery, including the profiling of tissues from animal models after in vivo treatment with covalent drugs.

Keywords:  Covalent small molecule; Drug discovery; High-throughput; Mass spectrometry; TMTpro; Tissue proteome

DOI:  https://doi.org/10.1007/978-1-0716-4502-4_17
J Proteome Res. 2025 Jun 14.

Screening of Protein Carbonylation Sites in Human Serum by Ion Mobility Mass Spectrometry.

Juan C Rojas Echeverri, Sanja Milkovska-Stamenova, Ulf Wagner, Ralf Hoffmann.

  Excessive oxidative stress, associated with various diseases, can induce protein carbonylation-nonenzymatic modifications involving aldehyde or keto group formation. These modifications are structurally diverse and low in abundance, which complicates their detection and quantitation. Here, we developed a strategy to identify and quantify protein carbonylation in human serum proteins from 39 rheumatoid arthritis patients and 29 healthy donors. Reactive carbonyl groups were derivatized with an aldehyde reactive probe (ARP), digested with trypsin, enriched via avidin affinity chromatography, and analyzed using RP-HPLC-ESI-IMS-MS/MS. Ion mobility spectrometry (IMS) was applied in both data-dependent (DDA) and data-independent acquisition (DIA) modes. DDA generated spectral libraries of ARP-derivatized peptides (ARP-peptides), which enabled peptide-centric detection in DIA data. We manually confirmed 86 ARP-peptides, with 93.8% of peak areas showing signal-to-background ratios >3. Among the 32 unique carbonylation sites, 28 were on human serum albumin, with hotspots at Cys58, Lys214, Lys219, Lys223, Lys456, Lys543, Lys549, and Lys565. Six previously unreported species were identified using IMS, DIA, ARP-reporter ions, and de novo sequencing. The ARP-peptides were quantified with ≥ 75% intrabatch reproducibility (coefficient of variation <20%). Similar modification levels were observed in both groups, suggesting basal, disease-independent carbonylation in abundant serum proteins.

Keywords:  DDA; DIA; aldehyde reactive probe (ARP); carbonylation; human serum; ion mobility spectrometry

DOI:  https://doi.org/10.1021/acs.jproteome.5c00093