bims-mascan 2025-08-24 papers

bims-mascan

Biomed News

on Mass spectrometry in cancer research

Issue of 2025–08–24
seven papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz

Detection and quantitation of small proteins using mass spectrometry.
Scheduled data-dependent acquisition MS provides enhanced identification and sensitivity in clinical lipidomics.
Comparative Proteomic Analysis of Capillary and Micropillar Array Columns Using DIA: Insights from the TKO Yeast Standard.
Untargeted lipidomic deep-profiling of human plasma via high-performance aza-Prilezhaev aziridination-LC-MS: Unraveling CC isomeric signatures of Charcot-Marie-Tooth disease.
Analytical greenness metrics for metabolomics.
The natural stable isotope index for de novo structural elucidation of phytomolecules with geographical and biological tracing.
Effect of ammonium fluoride as a mobile phase additive on quantitativity of fatty acids in electrospray ionization-liquid chromatography-tandem mass spectrometry.

Mol Cell Proteomics. 2025 Aug 14. pii: S1535-9476(25)00151-3. [Epub ahead of print] 101052

Detection and quantitation of small proteins using mass spectrometry.

Pedro H C Franco, Rilee Zeinert, Jakob Meier-Credo, Gisela Storz, Julian D Langer.

Small proteins or microproteins, despite long being ignored, have important roles in the regulation of larger protein complexes, metabolic pathways and gene expression. However, these proteins remain underrepresented in proteomics studies due to low detection efficiency in traditional mass spectrometry workflows. Their inherent characteristics often lead to depletion during sample preparation and a low detection efficiency in liquid chromatography-mass spectrometry (LC-MS). To improve detection and quantitation, we took advantage of the large set of documented small proteins in Escherichia coli and systematically compared and optimized different sample preparation and LC-MS approaches. We evaluated different top-down and bottom-up approaches, including data-dependent acquisition (DDA), data-independent acquisition (DIA) and parallel reaction monitoring (PRM). Our data highlight the benefit of top-down proteomics for identifying new small proteins and bottom-up preparation coupled with PRM acquisition for quantitation of protein levels in different growth conditions. Our systematic comparison can serve as a guideline for MS detection of small proteins in all organisms.

DOI: https://doi.org/10.1016/j.mcpro.2025.101052
Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00820-7. [Epub ahead of print]1371 344426

Scheduled data-dependent acquisition MS provides enhanced identification and sensitivity in clinical lipidomics.

Sander Johannes Thorbjørnsen Guttorm, Steven Ray Haakon Wilson, Erik Koldberg Amundsen, Helge Rootwelt, Katja Benedikte Prestø Elgstøen.

   BACKGROUND: Lipidomics can provide critical insight into metabolic changes in health and disease, but faces challenges in sensitivity, lipid coverage, and annotation accuracy. To address these limitations, we optimized a liquid chromatography-mass spectrometry (LC-MS) method combining scheduled data-dependent acquisition (SDDA) and C30 column-based separations, aimed at improving global lipidomics for clinical diagnostics.
RESULTS: Compared to conventional DDA and Intelligent Data Acquisition (AcquireX), SDDA demonstrated a 2-fold increase in number of lipids annotated, with a 2-fold higher annotation confidence (Grade A and B) of those lipids compared to DDA. The repeatability and analytical robustness of the method were thoroughly evaluated across different clinical blood matrices, i.e. serum, EDTA-plasma, and dried blood spots (DBS). Serum provided the highest repeatability and lipid coverage, with more than 2000 lipid species annotated. A proof-of-concept study assessing postprandial lipidomic changes in response to intake of a long-chain triglyceride fat emulsion was used to demonstrate the method's applicability in clinical lipidomics. The method detected significant changes in the levels of various lipids, including triacylglycerols, diacylglycerols, bile acids, phosphatidylethanolamines, and lyso-phosphatidylethanolamines, following lipid ingestion.
SIGNIFICANCE AND NOVELTY: The optimized lipidomics method (C30-SDDA) enhances lipid coverage and annotation confidence, proving valuable for studying metabolic alterations and biomarker discovery using blood matrices. These findings underscore the clinical potential of this method for advancing diagnostics and personalized medicine.

Keywords:  C30 chromatography; Clinical lipidomics; Global lipidomics; LC-MS; Scheduled DDA

DOI:  https://doi.org/10.1016/j.aca.2025.344426
J Proteome Res. 2025 Aug 19.

Comparative Proteomic Analysis of Capillary and Micropillar Array Columns Using DIA: Insights from the TKO Yeast Standard.

Tian Zhang, Ting-Yu Wei, Joao A Paulo.

  The effective separation of complex peptide mixtures is a cornerstone of mass spectrometry-based proteomics analysis as it enhances the accuracy and depth of proteomic analyses. Here, we compare data sets collected of whole-cell tryptic peptides, which were fractionated by either conventional flame-pulled, C18 packed-bed microcapillary columns or a microfabricated pillar array column (μPAC). Sixteen samples that included four different yeast strains (Δmet6, Δpfk2, Δura2, and wildtype) were analyzed in quadruplicate using data-independent acquisition. Each column enabled the quantification of >4700 proteins, with >95.4% overlap between column formats. The μPAC showed higher MS1 and MS2 signal intensities while maintaining similar peptide characteristics as the capillary column. The capillary column favored slightly longer and more hydrophobic peptides. Both columns achieved high data completeness at the protein level (>95%) and reproducible quantification, with μPAC offering slight improvements. Principal component and correlation analyses confirmed the capture of yeast strain-specific differences, with hierarchical clustering prioritizing strain over column effects. Protein quantification validated gene knockouts in both column formats, demonstrating similar accuracy of quantification. These findings highlight the μPAC as a standardized and robust alternative to capillary columns in proteomic analysis.

Keywords:  Accucore; TKO; ascend; data-independent acquisition; μPAC

DOI:  https://doi.org/10.1021/acs.jproteome.5c00465
Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00822-0. [Epub ahead of print]1371 344428

Untargeted lipidomic deep-profiling of human plasma via high-performance aza-Prilezhaev aziridination-LC-MS: Unraveling CC isomeric signatures of Charcot-Marie-Tooth disease.

Yuling Li, Weiran Zhao, Xiang Xiang, Yunlei Hu, Xiaoqiang Zhang, Wenjian Sun, Xiaoxuan Liu.

   BACKGROUND: Perturbed lipid metabolism in cellular environment plays an essential role in the progression of peripheral neuropathy, such as Charcot-Marie-Tooth disease type 1A (CMT1A). Current deep-profiling of lipidome still has certain limitations for high-throughput analysis of clinical samples, such as low sensitivity, extensive requirement of instrument modification, technician-dependent pretreatment and lack of method-adapted data-processing software.
RESULTS: Herein, a new generation aza-Prilezhaev aziridination (APA) reagent has been designed for coupling with high-resolution liquid chromatography mass spectrometry (LC-MS) to conduct comprehensive untargeted deep lipidomics of CMT1A disease. It facilitates large-scale lipid structural annotations over a wide polarity range in a single run with high-sensitivity. With the developed method, an extensive library containing information on retention time, m/z, and MS/MS down to CC location levels for 393 unsaturated lipids of CMT1A was created. Significant disease-related differences in CC isomer profiles were observed from the comparison of plasma between CMT1A patients (N = 9) and controls (N = 8). Specifically, 40 different lipids were identified, predominantly comprising n-6, n-7, and n-9 CC positional isomers.
SIGNIFICANCE: This innovative methodology would offer substantial potential to enhance diagnosis and therapeutic strategies for CMT1A and other diseases based on lipid isomerization information.

Keywords:  Automatic data-processing; Aziridination; CC location; Charcot-Marie-Tooth disease; Lipidomics; Liquid chromatography mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2025.344428
Metabolomics. 2025 Aug 19. 21(5): 121

Analytical greenness metrics for metabolomics.

Ren-Qi Wang, Yun Wang, Juan-Na Song, Huai-Dong Yu, Xi-Zhi Niu, Elize Smit.

   BACKGROUND: Metabolomics is rapidly evolving, addressing analytical chemistry challenges in the qualification and quantitation of metabolites in extremely complex samples. Targeted metabolomics involves the extraction and analysis of target compounds, often present at extremely low concentrations, whilst untargeted metabolomics requires the use of sophisticated analytical techniques to deal with the simultaneous identification or quantitation of hundreds of compounds. Given the high energy consumption and excessive amounts of waste generated by metabolomics studies, greenness metrics are essential to account for sustainable development.
AIM OF REVIEW: To determine the applicability of the Analytical GREEnness calculator (AGREE) in evaluating the analytical greenness of metabolomics methods. Specifically, the analytical protocols of 16 state-of-art metabolomics studies, including nine targeted and seven untargeted metabolomics studies, are fully dissected, and detailed greenness parameters for each procedure are rationally estimated.
KEY SCIENTIFIC CONCEPTS OF REVIEW: The calculated AGREE metrics unequivocally show the main weaknesses of greenness in current research, and guidelines for sustainable practices in metabolomics are provided. The results indicate that offline sample preparation and the lack of automation and miniaturization are key areas that must be addressed to make metabolomics more sustainable. Important aspects that should be considered include the complexity of sample preparation procedures, the use of toxic reagents and derivatizing agents, the amount of waste generated, and sample throughput.

Keywords:  Green analytical chemistry; Greenness metrics; Mass spectrometry; Metabolomics; NMR

DOI:  https://doi.org/10.1007/s11306-025-02323-2
Anal Chim Acta. 2025 Oct 15. pii: S0003-2670(25)00816-5. [Epub ahead of print]1371 344422

The natural stable isotope index for de novo structural elucidation of phytomolecules with geographical and biological tracing.

Xiaojing Yan, Xin Zhou, Mingyang Gong, Weidan Li, Mingyue Zhang, Tianci Huo, Xinyu Jiang, Xiaoyuan Hu, Xingchen Huang, Linhui Liu, Xue Feng, Qisong Zhang, Hongying Zhong.

  Not like the sequencing of genome and proteome that deals with the linear combination of base-pairs or amino acids, identification of unknown natural products has been always challenging due to diverse structures and various isomers. Searching fragment ions generated by mass spectrometers against databases has been the well-recognized approach. Because current searching algorithms generally compare unit masses and associated absolute intensities of ions, long lists of candidates with close similarity matching scores make it very difficult to achieve unambiguous structural identification. By using phytomolecules present in Allium sativum L. as examples, we demonstrated a natural stable isotope index (nSII) calculator for de novo structural elucidation along with geographical and biological tracing. The proposed nSII calculator provides a tool for mass spectrometric analysis of intramolecular- and compound-specific natural stable isotope ratios that are correlated with the activation or deactivation of metabolic pathways. It offers experimental evidences for the elucidation of elemental compositions, relationships among fragment ions and structural isoforms. Based on nSII calculator, a weighted mean index (wMI) has been established for the comprehensive examination of structural candidates, which inspects both mass spectrometric intensities and natural stable isotopes. Specific structural motifs that contain elements with specific isotopes are identified with unique nSII values, wMI values and accurate masses. Importantly, the nSII values are calculated as intensity ratios of heavy isotopes over light isotopes, which decreases interferences of signal fluctuations. Experimental results demonstrate that nSII and wMI values are useful to reject invalid candidates from the long lists of simple similarity matching reports, while other valid candidates may still be further confirmed with other techniques in addition to mass spectrometry. It should provide a label-free mass spectrometric approach for geographical and biological tracing of environment-metabolism interactions and enzyme-specific isotope effects if an isotopic ratio mass spectrometer (IR MS) can be combined.

Keywords:  Biological tracing; De novo structural elucidation; Environmental tracing; Mass spectrometry; Natural stable isotope index

DOI:  https://doi.org/10.1016/j.aca.2025.344422
Anal Sci. 2025 Aug 18.

Effect of ammonium fluoride as a mobile phase additive on quantitativity of fatty acids in electrospray ionization-liquid chromatography-tandem mass spectrometry.

Kohei Kawabata, Haruka Muraoka, Yuya Ohtsuki, Soo Takasu, Yukihiro Esaka, Akira Tokumura, Hiroyuki Nishi.

  Fatty acids (FAs) are essential molecules in biological systems and have crucial roles for fundamental components of cellular membranes, energy stores and mediators for cellular functions. The growing importance of FAs has also paid attention to analytical methods for the determination of FA contents in various samples accurately. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis is a powerful tool due to less time for sample preparation and analysis. This analytical approach enables to quantify levels of FA and other lipid classes in various samples simultaneously without derivatization. In the present study, the effects of ammonium salts on the sensitivity of FAs in LC-MS/MS analysis were evaluated. First, authors performed the comparison of FA analysis with different mobile phases, which contain ammonium formate, ammonium fluoride, ammonium acetate or ammonium carbonate. Interestingly, the mobile phase containing ammonium fluoride enhanced the sensitivity of targeted FA species more than other ammonium salts. Second, the difference of quantativity of FA species was examined. It was indicated that both carbon chain length and the number of double bonds affect ionization of FA species. Finally, the method of FA analysis based on ammonium fluoride was applied to FA profiling of lotus root. It is indicated that mobile phase containing ammonium fluoride enabled to evaluate FA contents with highly sensitivity in LC-MS/MS analysis.

Keywords:  Ammonium fluoride; Fatty acid; Liquid chromatography tandem mass spectrometry

DOI:  https://doi.org/10.1007/s44211-025-00832-7