bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2024–11–03
twenty papers selected by
Giovanny Rodríguez Blanco, Uniklinikum Graz
  1. Carafe enables high quality in silico spectral library generation for data-independent acquisition proteomics.
  2. Simultaneous metabolomics and lipidomics analysis based on 4in1 online analysis system reveal metabolic signatures in atherosclerotic mice.
  3. Hybrid Quadrupole Mass Filter-Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics.
  4. Optimizing SureQuant for Targeted Peptide Quantification: a Technical Comparison with PRM and SWATH-MS Methods.
  5. Modeling compound lipid homeostasis using stable isotope tracing.
  6. Annotating full-scan MS data using tandem MS libraries.
  7. Quantifying mitochondrial protein import by mePRODmt proteomics.
  8. MSFragger-DDA+ Enhances Peptide Identification Sensitivity with Full Isolation Window Search.
  9. Simultaneous Quantification of Carboxylate Enantiomers in Multiple Human Matrices with the Hydrazide-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.
  10. Single-Cell Lipidomics: An Automated and Accessible Microfluidic Workflow Validated by Capillary Sampling.
  11. A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.
  12. Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.
  13. Advances and challenges in non-targeted analysis: An insight into sample preparation and detection by liquid chromatography-mass spectrometry.
  14. Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry.
  15. The Circulating Proteome─Technological Developments, Current Challenges, and Future Trends.
  16. Recent Advancements in Proteomic Sample Preparation from Recombinant Chinese Hamster Ovary Cells.
  17. Understanding LC/MS-Based Metabolomics: A Detailed Reference for Natural Product Analysis.
  18. SEMQuant: Extending Sipros-Ensemble with Match-Between-Runs for Comprehensive Quantitative Metaproteomics.
  19. Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
  20. Analytical Tools for HCP Detection, Identification, and Quantitation.
  1. bioRxiv. 2024 Oct 18. pii: 2024.10.15.618504. [Epub ahead of print]
    Carafe enables high quality in silico spectral library generation for data-independent acquisition proteomics.
    Bo Wen, Chris Hsu, Wen-Feng Zeng, Michael Riffle, Alexis Chang, Miranda Mudge, Brook Nunn, Matthew D Berg, Judit Villén, Michael J MacCoss, William S Noble.
      Data-independent acquisition (DIA)-based mass spectrometry is becoming an increasingly popular mass spectrometry acquisition strategy for carrying out quantitative proteomics experiments. Most of the popular DIA search engines make use of in silico generated spectral libraries. However, the generation of high-quality spectral libraries for DIA data analysis remains a challenge, particularly because most such libraries are generated directly from data-dependent acquisition (DDA) data or are from in silico prediction using models trained on DDA data. In this study, we developed Carafe, a tool that generates high-quality experiment-specific in silico spectral libraries by training deep learning models directly on DIA data. We demonstrate the performance of Carafe on a wide range of DIA datasets, where we observe improved fragment ion intensity prediction and peptide detection relative to existing pretrained DDA models.
    DOI:  https://doi.org/10.1101/2024.10.15.618504
  2. Talanta. 2024 Oct 28. pii: S0039-9140(24)01488-7. [Epub ahead of print]283 127109
    Simultaneous metabolomics and lipidomics analysis based on 4in1 online analysis system reveal metabolic signatures in atherosclerotic mice.
    Meng Ding, Luwei Zheng, Xiaolin Hua, Mengxuan Chen, Qisheng Zhong, Taohong Huang, Ping Li, Hua Yang.
      Developing efficient and comprehensive analysis methods for metabolomics and lipidomics in the biological tissues and body fluids is essential for understanding the disease mechanisms. Although various two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) methods have been proposed to expand metabolite coverage, achieving higher efficiency in integrated metabolomics and lipidomics studies remains a technical challenge. In this work, a novel 4in1 online analysis system with excellent reproducibility and mass accuracy was constructed for metabolomics and lipidomics study in various biological samples from atherosclerotic mice. This system enabled the simultaneous detection in both positive and negative ion modes with extensive polarity separation in a single analytical run. Using the 4in1 online analysis system, we identified distinct but complementary metabolic signatures associated with atherosclerosis in different biological samples. Specifically, a total of 230 and 170 differential metabolites or lipids were detected in mice plasma samples and aortic tissue samples, respectively, including glycerophospholipids, sphingolipids, fatty acyls, glycerolipids, carboxylic acids, and pyrimidine nucleosides. Additionally, atherosclerosis-related metabolic pathways involved in biosynthesis of unsaturated fatty acids, sphingolipid metabolism, cholesterol metabolism, glycerophospholipid metabolism, and choline metabolism further revealed. These findings demonstrate that the novel 4in1 online analysis system is a faithful, stable and powerful tool for comprehensive metabolomics and lipidomics studies in complex biological matrices.
    Keywords:  4in1; Atherosclerosis; Lipidomics; Metabolomics; Quadrupole time-of-flight mass spectrometry; Two-dimensional liquid chromatography
    DOI:  https://doi.org/10.1016/j.talanta.2024.127109
  3. J Proteome Res. 2024 Oct 30.
    Hybrid Quadrupole Mass Filter-Radial Ejection Linear Ion Trap and Intelligent Data Acquisition Enable Highly Multiplex Targeted Proteomics.
    Philip M Remes, Cristina C Jacob, Lilian R Heil, Nicholas Shulman, Brendan X MacLean, Michael J MacCoss.
      Targeted mass spectrometry (MS) methods are powerful tools for the selective and sensitive analysis of peptides identified in global discovery experiments. Selected reaction monitoring (SRM) is the most widely accepted clinical MS method due to its reliability and performance. However, SRM and parallel reaction monitoring (PRM) are limited in throughput and are typically used for assays with around 100 targets or fewer. Here we introduce a new MS platform featuring a quadrupole mass filter, collision cell, and linear ion trap architecture, capable of targeting 5000-8000 peptides per hour. This high multiplexing capability is facilitated by acquisition rates of 70-100 Hz and real-time chromatogram alignment. We present a Skyline external software tool for building targeted methods based on data-independent acquisition chromatogram libraries or unscheduled analysis of heavy labeled standards. Our platform demonstrates ∼10× lower limits of quantitation (LOQs) than traditional SRM on a triple quadrupole instrument for highly multiplexed assays, due to parallel product ion accumulation. Finally, we explore how analytical figures of merit vary with method duration and the number of analytes, providing insights into optimizing assay performance.
    Keywords:  MRM; PRM; SRM; Stellar; Thermo; high-throughput; hybrid quadrupole mass filter; linear ion trap; peptide; quantitation; targeted
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00599
  4. Anal Chem. 2024 Oct 28.
    Optimizing SureQuant for Targeted Peptide Quantification: a Technical Comparison with PRM and SWATH-MS Methods.
    Minia Antelo-Varela, Dirk Bumann, Alexander Schmidt.
      Bacterial infections are a major threat to human health worldwide. A better understanding of the properties and physiology of bacterial pathogens in human tissues is required to develop urgently needed novel control strategies. Mass spectrometry-based proteomics could yield such data, but identifying and quantifying scarce bacterial proteins against a preponderance of human proteins is challenging. Here, we explored the recently introduced SureQuant method for highly sensitive targeted mass spectrometry. Using a major human pathogen, the Gram-positive bacteria Staphylococcus aureus, as an example, we evaluated several parameters, including the number of targets and intensity thresholds, for optimal qualitative and quantitative protein analysis. By comparison, we found that SureQuant achieved the same quantitative performance as standard parallel reaction monitoring while allowing accurate and precise quantification of up to 400 targets. SureQuant also surpassed the sensitivity and quantification capabilities of global data-independent acquisition methods. Finally, to facilitate method development, we provide optimized MS parameters for the sensitive quantification of different peptide panel sizes. This study provides a foundation for the broader application of SureQuant in the analysis of clinical specimens containing trace amounts of bacterial proteins as well as other studies requiring ultrasensitive detection of low-abundant proteins.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03622
  5. bioRxiv. 2024 Oct 17. pii: 2024.10.16.618599. [Epub ahead of print]
    Modeling compound lipid homeostasis using stable isotope tracing.
    Karl Wessendorf-Rodriguez, Maureen L Ruchhoeft, Ethan L Ashley, Hector M Galvez, Christopher W Murray, Grace McGregor, Shrikaar Kambhampati, Reuben J Shaw, Christian M Metallo.
      Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors, highlighting the molecular detail revealed by CL-MFA.
    DOI:  https://doi.org/10.1101/2024.10.16.618599
  6. bioRxiv. 2024 Oct 17. pii: 2024.10.14.618269. [Epub ahead of print]
    Annotating full-scan MS data using tandem MS libraries.
    Shipei Xing, Vincent Charron-Lamoureux, Yasin El Abiead, Pieter C Dorrestein.
      Full-scan mass spectrometry (MS) data from both liquid chromatography (LC) and MS imaging capture multiple ion forms, including their in-source fragments. Here we leverage such fragments to structurally annotate full-scan data from LC-MS or MS imaging by matching against peak intensity scaled tandem MS spectral libraries using precursor-tolerant reverse match scoring. Applied to inflammatory bowel disease and imaging datasets, we show the approach facilitates re-analyses of data in public repositories.
    DOI:  https://doi.org/10.1101/2024.10.14.618269
  7. Methods Enzymol. 2024 ;pii: S0076-6879(24)00347-1. [Epub ahead of print]706 449-474
    Quantifying mitochondrial protein import by mePRODmt proteomics.
    Süleyman Bozkurt, Bhavesh S Parmar, Christian Münch.
      Mitochondrial protein import is crucial for maintaining cellular health and homeostasis. Disruptions in this process have been linked to various diseases. Traditional methods for studying mitochondrial protein import predominantly focus on individual proteins and lack the dynamic resolution needed to fully appreciate the complexity of mitochondrial proteostasis and protein trafficking. To address these limitations, we developed a technique called mitochondria-specific multiplexed enhanced protein dynamics (mePRODmt). This method is a novel application of the mePROD methodology and utilizes pulsed stable isotope labeling with amino acids in cell culture (pSILAC)-based proteomics approach to study transient mitochondrial protein import. This chapter outlines the mePRODmt protocol, which includes the preparation of heavy SILAC-labeled peptides for boosting overall mitochondrial peptide signals (booster), SILAC labeling of cultured cells under experimental conditions, mitochondria isolation, sample preparation for multiplex proteomics using tandem mass tags (TMT) for isobaric labeling, recommended liquid chromatography-mass spectrometry (LC-MS) settings for reporter ion quantitation and a data analysis pipeline to analyze pSILAC-TMT data.
    Keywords:  Mass spectrometry; Mitochondria; Mitochondrial protein import; Proteomics; SILAC; TMT multiplex; Translation; mePROD; pSILAC
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.017
  8. bioRxiv. 2024 Oct 15. pii: 2024.10.12.618041. [Epub ahead of print]
    MSFragger-DDA+ Enhances Peptide Identification Sensitivity with Full Isolation Window Search.
    Fengchao Yu, Yamei Deng, Alexey I Nesvizhskii.
      Liquid chromatography-mass spectrometry (LC-MS) based proteomics, particularly in the bottom-up approach, relies on the digestion of proteins into peptides for subsequent separation and analysis. The most prevalent method for identifying peptides from data-dependent acquisition (DDA) mass spectrometry data is database search. Traditional tools typically focus on identifying a single peptide per tandem mass spectrum (MS2), often neglecting the frequent occurrence of peptide co-fragmentations leading to chimeric spectra. Here, we introduce MSFragger-DDA+, a novel database search algorithm that enhances peptide identification by detecting co-fragmented peptides with high sensitivity and speed. Utilizing MSFragger's fragment ion indexing algorithm, MSFragger-DDA+ performs a comprehensive search within the full isolation window for each MS2, followed by robust feature detection, filtering, and rescoring procedures to refine search results. Evaluation against established tools across diverse datasets demonstrated that, integrated within the FragPipe computational platform, MSFragger-DDA+ significantly increases identification sensitivity while maintaining stringent false discovery rate (FDR) control. It is also uniquely suited for wide-window acquisition (WWA) data. MSFragger-DDA+ provides an efficient and accurate solution for peptide identification, enhancing the detection of low-abundance co-fragmented peptides. Coupled with the FragPipe platform, MSFragger-DDA+ enables more comprehensive and accurate analysis of proteomics data.
    DOI:  https://doi.org/10.1101/2024.10.12.618041
  9. Anal Chem. 2024 Oct 30.
    Simultaneous Quantification of Carboxylate Enantiomers in Multiple Human Matrices with the Hydrazide-Assisted Ultrahigh-Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry.
    Yuting Sun, Qingyu Hu, Jiali Zuo, He Wang, Zhendong Guo, Yulan Wang, Huiru Tang.
      Many chiral carboxylic acids with α-amino, α-hydroxyl, and α-methyl groups are concurrently present in mammals establishing unique molecular phenotypes and multiple biological functions, especially host-microbiota symbiotic interactions. Their chirality-resolved simultaneous quantification is essential to reveal the biochemical details of physiology and pathophysiology, though challenging with their low abundances in some biological matrices and difficulty in enantiomer resolution. Here, we developed a method of the chirality-resolved metabolomics with sensitivity-enhanced quantitation via probe-promotion (Met-SeqPro) for analyzing these chiral carboxylic acids. We designed and synthesized a hydrazide-based novel chiral probe, (S)-benzoyl-proline-hydrazide (SBPH), to convert carboxylic acids into amide diastereomers to enhance their retention and chiral resolution on common C18 columns. Using the d5-SBPH-labeled enantiomers as internal standards, we then developed an optimized ultrahigh-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of 60 enantiomers of 30 chiral carboxylic acids in one run. This enantiomer-resolved method showed excellent sensitivity (LOD < 4 fmol-on-column), linearity (R2 > 0.992), precision (CV < 15%), accuracy (|RE| < 20%), and recovery (80-120%) in multiple biological matrices. With the method, we then quantified 60 chiral carboxylic acids in human urine, plasma, feces, and A549 cells to define their metabolomic phenotypes. This provides basic data for human phenomics and a promising tool for investigating the mammal-microbiome symbiotic interactions.
    DOI:  https://doi.org/10.1021/acs.analchem.4c04187
  10. Anal Chem. 2024 Oct 26.
    Single-Cell Lipidomics: An Automated and Accessible Microfluidic Workflow Validated by Capillary Sampling.
    Anastasia Kontiza, Johanna von Gerichten, Kyle D G Saunders, Matt Spick, Anthony D Whetton, Carla F Newman, Melanie J Bailey.
      We report the first demonstration of a microfluidics-based approach to measure lipids in single living cells using widely available liquid chromatography mass spectrometry (LC-MS) instrumentation. The method enables the rapid sorting of live cells into liquid chambers formed on standard Petri dishes and their subsequent dispensing into vials for analysis using LC-MS. This approach facilitates automated sampling, data acquisition, and analysis and carries the additional advantage of chromatographic separation, aimed at reducing matrix effects present in shotgun lipidomics approaches. We demonstrate that our method detects comparable numbers of features at around 200 lipids in populations of single cells versus established live single-cell capillary sampling methods and with greater throughput, albeit with the loss of spatial resolution. We also show the importance of optimization steps in addressing challenges from lipid contamination, especially in blanks, and demonstrate a 75% increase in the number of lipids identified. This work opens up a novel, accessible, and high-throughput way to obtain single-cell lipid profiles and also serves as an important validation of single-cell lipidomics through the use of different sampling methods.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03435
  11. bioRxiv. 2024 Oct 15. pii: 2024.10.11.617883. [Epub ahead of print]
    A Streamlined High-Throughput LC-MS Assay for Quantifying Peptide Degradation in Cell Culture.
    Samuel J Rozans, Abolfazl S Moghaddam, E Thomas Pashuck.
      Peptides are widely used in biomaterials due to their easy of synthesis, ability to signal cells, and modify the properties of biomaterials. A key benefit of using peptides is that they are natural substrates for cell-secreted enzymes, which creates the possibility of utilizing cell-secreted enzymes for tuning cell-material interactions. However, these enzymes can also induce unwanted degradation of bioactive peptides in biomaterials, or in peptide therapies. Liquid chromatography-mass spectrometry (LC-MS) is a widely used, powerful methodology that can separate complex mixtures of molecules and quantify numerous analytes within a single run. There are several challenges in using LC-MS for the multiplexed quantification of cell-induced peptide degradation, including the need for non-degradable internal standards and the identification of optimal sample storage conditions. Another problem is that cell culture media and biological samples typically contain both proteins and lipids that can accumulate on chromatography columns and degrade their performance. However, removing these constituents can be expensive, time consuming, and increases sample variability. Here we show that directly injecting samples onto the LC-MS without any purification enables rapid and accurate quantification of peptide concentration, and that hundreds of LC-MS runs can be done on a single column without a significantly diminish the ability to quantify the degradation of peptide libraries. We also show that column failure is evident when hydrophilic peptides fail to be retained on the column, and this can be easily identified using standard peptide mixtures for column benchmarking. In total, this work introduces a simple and effective method for simultaneously quantifying the degradation of dozens of peptides in cell culture. By providing a streamlined and cost-effective method for the direct quantification of peptide degradation in complex biological samples, this work enables more efficient assessment of peptide stability and functionality, facilitating the development of advanced biomaterials and peptide-based therapies.
    DOI:  https://doi.org/10.1101/2024.10.11.617883
  12. Nat Commun. 2024 Oct 30. 15(1): 9378
    Spatial proteomics of single cells and organelles on tissue slides using filter-aided expansion proteomics.
    Zhen Dong, Wenhao Jiang, Chunlong Wu, Ting Chen, Jiayi Chen, Xuan Ding, Shu Zheng, Kiryl D Piatkevich, Yi Zhu, Tiannan Guo.
      Hydrogel-based tissue expansion combined with mass spectrometry (MS) offers an emerging spatial proteomics approach. Here, we present a filter-aided expansion proteomics (FAXP) strategy for spatial proteomics analysis of archived formalin-fixed paraffin-embedded (FFPE) specimens. Compared to our previous ProteomEx method, FAXP employed a customized tip device to enhance both the stability and throughput of sample preparation, thus guaranteeing the reproducibility and robustness of the workflow. FAXP achieved a 14.5-fold increase in volumetric resolution. It generated over 8 times higher peptide yield and a 255% rise in protein identifications while reducing sample preparation time by 50%. We also demonstrated the applicability of FAXP using human colorectal FFPE tissue samples. Furthermore, for the first time, we achieved bona fide single-subcellular proteomics under image guidance by integrating FAXP with laser capture microdissection.
    DOI:  https://doi.org/10.1038/s41467-024-53683-7
  13. J Chromatogr A. 2024 Oct 19. pii: S0021-9673(24)00833-1. [Epub ahead of print]1737 465459
    Advances and challenges in non-targeted analysis: An insight into sample preparation and detection by liquid chromatography-mass spectrometry.
    Vivek Mandal, Jinal Ajabiya, Nasir Khan, Rakesh K Tekade, Pinaki Sengupta.
      Unknown impurities, metabolites and harmful pollutants present in pharmaceutical products, biological and environmental samples, respectively are of high concern in terms of their detection and quantification. The targeted analysis aims to quantify known chemical entities, but it lacks the ability to identify unknown components present in a sample. Non-targeted analysis is an analytical approach that can be made applicable to various disciplines of science to effectively search for unknown chemical, biological, or environmental entities that can answer various baffling mysteries of research. It employs various high-end analytical techniques that can specifically screen out multiple unknown compounds from complex mixtures. Non-targeted analysis is also applicable for complex studies such as metabolomics to search unidentified metabolites of new chemical entities. This review critically discusses the current advancements in non-targeted analysis related to the analysis of pharmaceutical, biological, and environmental samples. Various steps like sample collection, handling, preparation, extraction, its analysis using advanced techniques like high-resolution mass spectrometry, liquid chromatography mass spectrometry, and lastly interpretation of the huge amounts of complex data obtained upon analysis of complex matrices have been discussed broadly in this article. Besides the advantages of non-targeted analysis over targeted analysis, limitations, bioinformatics tools, sources of error, and research gaps have been critically analyzed.
    Keywords:  Bio-informatics tools; Bioanalysis; LC-MS/MS; Non-targeted analysis; Sample preparation
    DOI:  https://doi.org/10.1016/j.chroma.2024.465459
  14. Anal Bioanal Chem. 2024 Oct 25.
    Development and application of a dual isotopic labeling method for enhanced detection and quantification of stimulants in urine samples using high-resolution mass spectrometry.
    Yang Wang, Wanli Li, Xiaojun Deng.
      Given the critical nature of anti-doping efforts, the detection of stimulant substances is shifting from accurate qualitative analysis to precise quantitative analysis. Additionally, the use of liquid chromatography-high-resolution mass spectrometry (LC-HRMS) in detecting stimulants is becoming more widespread. However, the lack of isotope-labeled internal standards is causing increasing issues with quantitative accuracy. Furthermore, challenges such as the mass spectrometric response of small molecules and the separation of isomers present additional difficulties. We have developed a quantitative method for stimulant substances containing amine or phenol hydroxyl groups, using a dual-label derivatization system. This method offers a new perspective for analyzing and detecting low molecular weight substances, isomers, or those with poor LC-MS response, and proposes a solution to the problem of missing isotope-labeled internal standards. Methodological validation has shown that this approach has promising application potential.
    Keywords:  Dual isotopic labeling; High-resolution mass spectrometry (HRMS); LC–MS response enhancement; Quantitative analysis; Stimulant detection
    DOI:  https://doi.org/10.1007/s00216-024-05612-2
  15. J Proteome Res. 2024 Oct 31.
    The Circulating Proteome─Technological Developments, Current Challenges, and Future Trends.
    Philipp E Geyer, Daniel Hornburg, Maria Pernemalm, Stefanie M Hauck, Krishnan K Palaniappan, Vincent Albrecht, Laura F Dagley, Robert L Moritz, Xiaobo Yu, Fredrik Edfors, Yves Vandenbrouck, Johannes B Mueller-Reif, Zhi Sun, Virginie Brun, Sara Ahadi, Gilbert S Omenn, Eric W Deutsch, Jochen M Schwenk.
      Recent improvements in proteomics technologies have fundamentally altered our capacities to characterize human biology. There is an ever-growing interest in using these novel methods for studying the circulating proteome, as blood offers an accessible window into human health. However, every methodological innovation and analytical progress calls for reassessing our existing approaches and routines to ensure that the new data will add value to the greater biomedical research community and avoid previous errors. As representatives of HUPO's Human Plasma Proteome Project (HPPP), we present our 2024 survey of the current progress in our community, including the latest build of the Human Plasma Proteome PeptideAtlas that now comprises 4608 proteins detected in 113 data sets. We then discuss the updates of established proteomics methods, emerging technologies, and investigations of proteoforms, protein networks, extracellualr vesicles, circulating antibodies and microsamples. Finally, we provide a prospective view of using the current and emerging proteomics tools in studies of circulating proteins.
    Keywords:  PTM; PeptideAtlas; affinity; biomarker discovery; blood; extracellular vesicle; mass spectrometry; microsampling; plasma; serum
    DOI:  https://doi.org/10.1021/acs.jproteome.4c00586
  16. Methods Mol Biol. 2025 ;2853 139-154
    Recent Advancements in Proteomic Sample Preparation from Recombinant Chinese Hamster Ovary Cells.
    Michael Henry, Paula Meleady.
      Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and post-translationally modify recombinant proteins that are compatible with human use. Proteomics, along with other "omic platforms," are being used to understand the biology of CHO cells with the ultimate aim to enhance CHO cell factories for more efficient production of biopharmaceuticals. Liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis has become the standard technique for profiling proteomes. There are three stages to a typical bottom-up approach, namely, sample preparation, LC-MS/MS, and data analysis. Although there have been major advances in LC-MS/MS instrumentation and data analysis tools, sample preparation is still the crucial stage that affects the overall efficiency of any proteomic study. In this chapter, we present a comparison of a number of widely used sample preparation methods for proteomic applications, including in-solution digestion and commercially available device-based kits. All methods performed well; however, each method has inherent advantages and disadvantages.
    Keywords:  Chinese hamster ovary cells; Filter-aided sample preparation; Mass spectrometry; Proteomics; Sample preparation for proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-4104-0_10
  17. Proteomics Clin Appl. 2024 Oct 30. e202400048
    Understanding LC/MS-Based Metabolomics: A Detailed Reference for Natural Product Analysis.
    Jyotirmay Sarkar, Rajveer Singh, Shivani Chandel.
      Liquid chromatography, when used in conjunction with mass spectrometry (LC/MS), is a powerful tool for conducting accurate and reproducible investigations of numerous metabolites in natural products (NPs). LC/MS has gained prominence in metabolomic research due to its high throughput, the availability of multiple ionization techniques and its ability to provide comprehensive metabolite coverage. This unique method can significantly influence various scientific domains. This review offers a comprehensive overview of the current state of LC/MS-based metabolomics in the investigation of NPs. This review provides a thorough overview of the state of the art in LC/MS-based metabolomics for the investigation of NPs. It covers the principles of LC/MS, various aspects of LC/MS-based metabolomics such as sample preparation, LC modes, method development, ionization techniques and data pre-processing. Moreover, it presents the applications of LC/MS-based metabolomics in numerous fields of NPs research such as including biomarker discovery, the agricultural research, food analysis, the study of marine NPs and microbiological research. Additionally, this review discusses the challenges and limitations of LC/MS-based metabolomics, as well as emerging trends and developments in this field.
    Keywords:  data pre‐processing; liquid chromatography; mass spectrometry; metabolomics; natural products method development
    DOI:  https://doi.org/10.1002/prca.202400048
  18. Bioinform Res Appl. 2024 Jul;14956 102-115
    SEMQuant: Extending Sipros-Ensemble with Match-Between-Runs for Comprehensive Quantitative Metaproteomics.
    Bailu Zhang, Shichao Feng, Manushi Parajuli, Yi Xiong, Chongle Pan, Xuan Guo.
      Metaproteomics, utilizing high-throughput LC-MS, offers a profound understanding of microbial communities. Quantitative metaproteomics further enriches this understanding by measuring relative protein abundance and revealing dynamic changes under different conditions. However, the challenge of missing peptide quantification persists in metaproteomics analysis, particularly in data-dependent acquisition mode, where high-intensity precursors for MS2 scans are selected. To tackle this issue, the match-between-runs (MBR) technique is used to transfer peptides between LC-MS runs. Inspired by the benefits of MBR and the need for streamlined metaproteomics data analysis, we developed SEMQuant, an end-to-end software integrating Sipros-Ensemble's robust peptide identifications with IonQuant's MBR function. The experiments show that SEMQuant consistently obtains the highest or second highest number of quantified proteins with notable precision and accuracy. This demonstrates SEMQuant's effectiveness in conducting comprehensive and accurate quantitative metaproteomics analyses across diverse datasets and highlights its potential to propel advancements in microbial community studies. SEMQuant is freely available under the GNU GPL license at https://github.com/Biocomputing-Research-Group/SEMQuant.
    Keywords:  Label-Free Quantification; Mass Spectrometry; Match-Between-Runs; Metaproteomics
    DOI:  https://doi.org/10.1007/978-981-97-5087-0_9
  19. Methods Enzymol. 2024 ;pii: S0076-6879(24)00361-6. [Epub ahead of print]706 215-242
    Analysis of mitochondrial targeting signal cleavage and protein processing by mass spectrometry.
    Fabian Stockert, Henrique Baeta, Pitter F Huesgen.
      The majority of mitochondrial proteins are encoded in the nucleus, synthesized in the cytosol and imported into mitochondria mediated by an N-terminal mitochondrial targeting sequences (MTS). After import, the MTS is cleaved off by the mitochondrial processing peptidase (MPP) and subsets of the imported proteins are further processed by the aminopeptidase intermediate cleaving peptidase 55 (ICP55), the mitochondrial intermediate peptidase (MIP), octapeptidyl aminopeptidase 1 (Oct1) or other proteolytic enzymes. Mutations that impair the mitochondrial processing machinery or mitochondrial protein degradation result in rare but severe human diseases. In addition, aging and various stress conditions are associated with altered proteolysis of mitochondrial proteins. Enrichment of protein terminal peptides in combination with mass spectrometry-based identification and quantification has become the method of choice to study proteolytic processing. Here, we describe an updated step-by-step protocol for the enrichment of N-terminal peptides by Hypersensitive Undecanal-mediated Enrichment of N-Terminal peptides (HUNTER). We describe analysis of mass spectrometry data acquired for HUNTER samples and present a suite of dedicated Python and R scripts for HUNTER quality control, classification of the enriched peptides, annotation of mitochondrial processing sites and quantitative evaluation. The scripts are freely available at https://github.com/FabianStockert/mito_annotation.
    Keywords:  Data analysis; Degradomics; Mass spectrometry; Mitochondria; N-terminome; Peptide quantification; Positional annotation; Protein N-termini; Proteolytic processing
    DOI:  https://doi.org/10.1016/bs.mie.2024.07.025
  20. Methods Mol Biol. 2025 ;2853 235-248
    Analytical Tools for HCP Detection, Identification, and Quantitation.
    Faiza Kanwal, Michael Henry, Paula Meleady.
      Identifying and quantifying host cell proteins (HCPs) are crucial in developing and manufacturing biopharmaceutical products. ELISA has been used widely for identifying and quantifying HCPs in biopharmaceutical products. Liquid chromatography-mass spectrometry (LC-MS) has recently emerged as an orthogonal tool for HCP analysis, providing comprehensive and quantitative information on individual HCPs. This chapter explores the advancements in LC-MS/MS methodologies for HCP analysis, including sample preparation, data acquisition modes, and data analysis strategies. It also discusses the challenges of HCP identification and quantitation, and recent innovations to overcome these limitations. The application of LC-MS/MS methods in HCP profiling holds significant promise for improving process control and ensuring product safety in biopharmaceutical manufacturing.
    Keywords:  Biopharmaceutical manufacturing; Data acquisition modes; Data analysis strategies; Host cell proteins; LC-MS/MS; Sample preparation
    DOI:  https://doi.org/10.1007/978-1-0716-4104-0_15
This page is being maintained by Thomas Krichel. It was last updated on 2024‒12‒09 at 5:53.