bims-mascan Biomed News
on Mass spectrometry in cancer research
Issue of 2021–05–09
forty-two papers selected by
Giovanny Rodriguez Blanco, University of Edinburgh
  1. High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry.
  2. Ceramide Analysis by Multiple Linked-Scan Mass Spectrometry Using a Tandem Quadrupole Instrument.
  3. Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis.
  4. Mass Spectrometry-Based Lipidomics: An Overview.
  5. LipidSuite: interactive web server for lipidomics differential and enrichment analysis.
  6. Global Lipidomics Profiling by a High Resolution LC-MS Platform.
  7. Quantification of Plasma Oxylipins Using Solid-Phase Extraction and Reversed-Phase Liquid Chromatography-Triple Quadrupole Mass Spectrometry.
  8. Mass Spectrometry-Based Shotgun Lipidomics Using Charge-Switch Derivatization for Analysis of Complex Long-Chain Fatty Acids.
  9. Comprehensive Structural Characterization of Lipids by Coupling Paternò-Büchi Reaction and Tandem Mass Spectrometry.
  10. Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics.
  11. Shotgun Lipidomic Analysis of Leishmania Cells.
  12. Comprehensive Mouse Skin Ceramide Analysis on a Solid-Phase and TLC Separation with High-Resolution Mass Spectrometry Platform.
  13. Quo Vadis Caenorhabditis elegans Metabolomics-A Review of Current Methods and Applications to Explore Metabolism in the Nematode.
  14. Lipid metabolism in cancer: New perspectives and emerging mechanisms.
  15. Label-Free Proteomics of Quantity-Limited Samples Using Ion Mobility-Assisted Data-Independent Acquisition Mass Spectrometry.
  16. Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches.
  17. Mass Spectrometric Analysis of Meibomian Gland Lipids.
  18. High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.
  19. Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy.
  20. Application of SILAC Labeling in Phosphoproteomics Analysis.
  21. Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.
  22. Quantitative Approach Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectrometry.
  23. Mass Spectrometric Analysis of Bioactive Sphingolipids in Fungi.
  24. Analysis of differential neonatal lethality in cystathionine β-synthase deficient mouse models using metabolic profiling.
  25. Alteration in the Cerebrospinal Fluid Lipidome in Parkinson's Disease: A Post-Mortem Pilot Study.
  26. Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.
  27. Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics.
  28. Tandem Mass Tags for Comparative and Discovery Proteomics.
  29. Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison.
  30. Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data.
  31. Targeting lipid metabolism is an emerging strategy to enhance the efficacy of anti-HER2 therapies in HER2-positive breast cancer.
  32. Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in the Ischemic Rat Brain Section: A Practical Approach.
  33. 2nSILAC for Quantitative Proteomics of Prototrophic Baker's Yeast.
  34. Quantitative Mass Spectrometry-Based Proteomics: An Overview.
  35. Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue.
  36. Proteomic approaches to assist in diagnosis and prognosis of oral cancer.
  37. Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).
  38. Establishing a Custom-Fit Data-Independent Acquisition Method for Label-Free Proteomics.
  39. Urine metabolomic biomarkers for prediction of isolated fetal congenital heart defect.
  40. The influence of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer.
  41. Mechanisms of Metabolic Reprogramming in Cancer Cells Supporting Enhanced Growth and Proliferation.
  42. Advancing Semen Evaluation Using Lipidomics.
  1. Methods Mol Biol. 2021 ;2228 205-224
    High-Throughput Profiling of Proteome and Posttranslational Modifications by 16-Plex TMT Labeling and Mass Spectrometry.
    Kaiwen Yu, Zhen Wang, Zhiping Wu, Haiyan Tan, Ashutosh Mishra, Junmin Peng.
      Mass spectrometry (MS)-based proteomic profiling of whole proteome and protein posttranslational modifications (PTMs) is a powerful technology to measure the dynamics of proteome with high throughput and deep coverage. The reproducibility of quantification benefits not only from the fascinating developments in high-performance liquid chromatography (LC) and high-resolution MS with enhanced scan rates but also from the invention of multiplexed isotopic labeling strategies, such as the tandem mass tags (TMT). In this chapter, we introduce a 16-plex TMT-LC/LC-MS/MS protocol for proteomic profiling of biological and clinical samples. The protocol includes protein extraction, enzymatic digestion, PTM peptide enrichment, TMT labeling, and two-dimensional reverse-phase liquid chromatography fractionation coupled with tandem mass spectrometry (MS/MS) analysis, followed by computational data processing. In general, more than 10,000 proteins and tens of thousands of PTM sites (e.g., phosphorylation and ubiquitination) can be confidently quantified. This protocol provides a general protein measurement tool, enabling the dissection of protein dysregulation in any biological samples and human diseases.
    Keywords:  Database; Liquid chromatography; Mass spectrometry; Phosphorylation; Posttranslational modifications; Proteome; Proteomics; Tandem mass tag; Ubiquitin; Ubiquitination
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_15
  2. Methods Mol Biol. 2021 ;2306 123-137
    Ceramide Analysis by Multiple Linked-Scan Mass Spectrometry Using a Tandem Quadrupole Instrument.
    Fong-Fu Hsu.
      Ceramides are a special class of sphingolipids and play a central role in sphingolipid metabolism, and have diverse structures. In this book chapter, tandem quadrupole mass spectrometric approaches applying multiple linked scannings including various constant neutral loss scan (NLS) and precursor ion scan (PIS), the unique applicable feature of a triple-stage quadrupole (TSQ) instrument for analysis of ceramides desorbed as [M-H]- and [M+Li]+ ions are described. These multiple dimensional tandem mass spectrometric approaches are fully adapted to the conventional shotgun lipidomics workflow with minimal or without prior chromatographic separation to profile ceramide molecules, and thus detection of a whole class of ceramide or various specific ceramide subclasses in crude lipid extract can be achieved. With addition of internal standard(s), semi-quantitation of ceramide in the lipid extract of biological origin is possible. Examples have shown promise in ceramide profiling of several whole lipid extracts from porcine brain, the model Dictyostelium Discoideum cells for cancer study, and skin.
    Keywords:  Brain ceramides; Epidermal ceramides; Linked scan; Mass spectrometry; Multiple dimensional tandem mass spectrometry; Neutral loss scan; Precursor ion scan
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_9
  3. Methods Mol Biol. 2021 ;2306 61-75
    Chemical Derivatization-Aided High Resolution Mass Spectrometry for Shotgun Lipidome Analysis.
    Vinzenz Hofferek, Huaqi Su, Gavin E Reid.
      Chemical derivatization coupled with nano-electrospray ionization (nESI) and ultra-high resolution accurate mass spectrometry (UHRAMS) is an established approach to overcome isobaric and isomeric mass interference limitations, and improve the analytical performance, of direct-infusion (i.e., "shotgun") lipidome analysis strategies for "sum composition" level identification and quantification of individual lipid species from within complex mixtures. Here, we describe a protocol for sequential functional group selective derivatization of aminophospholipids and O-alk-1'-enyl (i.e., plasmalogen) lipids, that when integrated into a shotgun lipidomics workflow involving deuterium-labeled internal lipid standard addition, monophasic lipid extraction, and nESI-UHRAMS analysis, enables the routine identification and quantification of >500 individual lipid species at the "sum composition" level, across four lipid categories and from >30 lipid classes and subclasses.
    Keywords:  Chemical derivatization; Deuterated lipid internal standard; Direct infusion; High resolution mass spectrometry; Lipidomics; Semi-quantitative analysis
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_5
  4. Methods Mol Biol. 2021 ;2306 1-10
    Mass Spectrometry-Based Lipidomics: An Overview.
    Fong-Fu Hsu.
      Over the last few decades, MS-based lipidomics has emerged as a powerful tool to study lipids in biological systems. This success is driven by the constant demand for complete and reliable data. The improvement of MS-based lipidomics will continue to be dependent on the advances in the technology of mass spectrometry and related techniques including separation and bioinformatics, and more importantly, on gaining insight into the knowledge of lipid chemistry essential to develop methodology for lipid analysis. It is hoped that the protocols in this book, collected from experts in their fields, can offer the beginner and the advanced user alike, useful tips toward successful lipidomic analysis.
    Keywords:  Overview; Shotgun lipidomics; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_1
  5. Nucleic Acids Res. 2021 May 05. pii: gkab327. [Epub ahead of print]
    LipidSuite: interactive web server for lipidomics differential and enrichment analysis.
    Ahmed Mohamed, Michelle M Hill.
      Advances in mass spectrometry enabled high throughput profiling of lipids but differential analysis and biological interpretation of lipidomics datasets remains challenging. To overcome this barrier, we present LipidSuite, an end-to-end differential lipidomics data analysis server. LipidSuite offers a step-by-step workflow for preprocessing, exploration, differential analysis and enrichment analysis of untargeted and targeted lipidomics. Three lipidomics data formats are accepted for upload: mwTab file from Metabolomics Workbench, Skyline CSV Export, and a numerical matrix. Experimental variables to be used in analysis are uploaded in a separate file. Conventional lipid names are automatically parsed to enable lipid class and chain length analyses. Users can interactively explore data, choose subsets based on sample types or lipid classes or characteristics, and conduct univariate, multivariate and unsupervised analyses. For complex experimental designs and clinical cohorts, LipidSuite offers confounding variables adjustment. Finally, data tables and plots can be both interactively viewed or downloaded for publication or reports. Overall, we anticipate this free, user-friendly webserver to facilitate differential lipidomics data analysis and re-analysis, and fully harness biological interpretation from lipidomics datasets. LipidSuite is freely available at http://suite.lipidr.org.
    DOI:  https://doi.org/10.1093/nar/gkab327
  6. Methods Mol Biol. 2021 ;2306 39-51
    Global Lipidomics Profiling by a High Resolution LC-MS Platform.
    Thomas Züllig, Martin Trötzmüller, Harald C Köfeler.
      Lipidomics is the determination of big lipid assemblies by mass spectrometry. When using chromatography coupled high resolution mass spectrometry, lipids can be identified by exact mass, fragment spectra, and retention time. This protocol covers lipid extraction, LC-MS data acquisition by Orbitrap mass spectrometry and data processing by Lipid Data Analyzer, a custom developed open source software.
    Keywords:  Chromatography; LC-MS; Lipid data analyzer; Lipidomics; Lipids; Mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_3
  7. Methods Mol Biol. 2021 ;2306 171-186
    Quantification of Plasma Oxylipins Using Solid-Phase Extraction and Reversed-Phase Liquid Chromatography-Triple Quadrupole Mass Spectrometry.
    Guan-Yuan Chen, Qibin Zhang.
      Oxylipins are an important class of bioactive lipids derived from polyunsaturated fatty acids. They can be both pro- and anti-inflammatory and function as important mediators in various pathological conditions. However, comprehensive analysis of oxylipins still remains a challenge because of their low abundance in plasma and the dominance of structurally similar isomeric species. Herein, we describe a simple and rapid method to comprehensively analyze oxylipins in blood plasma, which utilizes solid-phase extraction in 96-well format for efficient sample cleanup. Separation and detection of more than 130 oxylipins is accomplished by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring in negative-ion mode. The absolute concentrations of oxylipins in human plasma are determined using the calibration curves constructed from internal standards. Detailed methods and precautions are presented for a successful adoption of this method in analytical laboratory.
    Keywords:  Human plasma; LC-MS/MS; Lipid mediators; MRM; Oxylipins; SPE
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_12
  8. Methods Mol Biol. 2021 ;2306 93-103
    Mass Spectrometry-Based Shotgun Lipidomics Using Charge-Switch Derivatization for Analysis of Complex Long-Chain Fatty Acids.
    Cheryl Frankfater, Fong-Fu Hsu.
      Charge-switch derivatization to convert long-chain fatty acids (LCFAs) to their N-(4-aminomethylphenyl) pyridinium (AMPP) derivatives (FA-AMPP derivative) drastically increases their sensitivity (>102) detected by electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI). Lipidomic analyses of the FA-AMPP derivatives by ESI combined with CID tandem mass spectrometry (MS2), or by MALDI-TOF/TOF affords unambiguous structural characterization of LCFAs, including many unusual microbial LCFAs that contain various functional groups such as methyl, hydroxyl, cyclopropyl, and double bond(s). The ease of preparation of the FA-AMPP derivatives, the tremendous gain in sensitivity after derivatization, and more importantly, the readily recognizable product ion spectra that contain rich structurally informative fragment ions for locating functional groups make this method one of the most powerful techniques for LCFA identification and quantification.
    Keywords:  Charge conversion; Long-chain fatty acids; MALDI-TOF/TOF; Mass spectrometry; Microbial lipids; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_7
  9. Methods Mol Biol. 2021 ;2306 53-60
    Comprehensive Structural Characterization of Lipids by Coupling Paternò-Büchi Reaction and Tandem Mass Spectrometry.
    Qingyuan Hu, Yu Xia, Xiaoxiao Ma.
      Since the invention of soft ionization methods, in particular electrospray ionization (ESI), mass spectrometry (MS) has become the method of choice for both qualitative and quantitative analysis of lipids from complex samples. A large number of lipids can be readily detected from a single mass spectrum free from molecular fragmentation that may complicate spectral interpretation. This has been the driving force for MS to play a predominant role in lipidomics. However, elucidation of the detailed lipid structures, especially the location of carbon-carbon double bond (C=C), remains challenging for MS-based lipid analysis workflows. Here we describe the coupling of photochemical derivatization of C=C via Paternò-Büchi (PB) reaction with tandem mass spectrometry (MS/MS) to identify C=C locations in unsaturated lipids and quantify lipid C=C location isomers. The PB reaction can be conducted online in ~30 s, which transforms a C=C into the oxetane ring structure. Subjecting PB products of lipids to MS/MS leads to the formation of abundant C=C-specific fragment ions upon low energy collision-induced dissociation.
    Keywords:  Carbon-carbon double bond; Lipid; Photochemical derivation; Structural elucidation; Tandem mass spectrometry
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_4
  10. Methods Mol Biol. 2021 ;2228 353-384
    Detailed Method for Performing the ExSTA Approach in Quantitative Bottom-Up Plasma Proteomics.
    Andrew J Percy, Christoph H Borchers.
      The use of stable isotope-labeled standards (SIS) is an analytically valid means of quantifying proteins in biological samples. The nature of the labeled standards and their point of insertion in a bottom-up proteomic workflow can vary, with quantification methods utilizing curves in analytically sound practices. A promising quantification strategy for low sample amounts is external standard addition (ExSTA). In ExSTA, multipoint calibration curves are generated in buffer using serially diluted natural (NAT) peptides and a fixed concentration of SIS peptides. Equal concentrations of SIS peptides are spiked into experimental sample digests, with all digests (control and experimental) subjected to solid-phase extraction prior to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Endogenous peptide concentrations are then determined using the regression equation of the standard curves. Given the benefits of ExSTA in large-scale analysis, a detailed protocol is provided herein for quantifying a multiplexed panel of 125 high-to-moderate abundance proteins in undepleted and non-enriched human plasma samples. The procedural details and recommendations for successfully executing all phases of this quantification approach are described. As the proteins have been putatively correlated with various noncommunicable diseases, quantifying these by ExSTA in large-scale studies should help rapidly and precisely assess their true biomarker efficacy.
    Keywords:  Forward curve; Human plasma; Protein; Proteomics; Quantification; Stable isotope-labeled standard; Standard curve
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_25
  11. Methods Mol Biol. 2021 ;2306 215-225
    Shotgun Lipidomic Analysis of Leishmania Cells.
    Kai Zhang, Fong-Fu Hsu.
      To understand the interplay of lipids between Leishmania promastigotes, amastigotes, and vertebrate host cells, a robust method for cultivating Leishmania parasites, lipid extraction, and shotgun lipidomic analysis with loop injection is described. This book chapter provides the step-by-step workflow to guide readers from sample preparation to the global lipid analysis by multiple stage mass spectrometry with high resolution and tandem quadrupole mass spectrometric approaches toward studying the metabolomic roles that lipids may play in Leishmania parasite infections.
    Keywords:  Glycerophospholipids; High-resolution mass spectrometry; Leishmania lipids; Sphingolipids
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_14
  12. Methods Mol Biol. 2021 ;2306 139-155
    Comprehensive Mouse Skin Ceramide Analysis on a Solid-Phase and TLC Separation with High-Resolution Mass Spectrometry Platform.
    Meei-Hua Lin, Jeffrey H Miner, Fong-Fu Hsu.
      Lipidomic analyses by mass spectrometry (MS) of epidermal ceramides, a large family of lipids crucial to the permeability barrier of the skin, have been reported previously. To ensure the accuracy of lipid identification, we describe here the isolation of mouse newborn epidermal lipids followed by fractionation with solid-phase extraction columns, and lipidomic analyses by high-resolution MS for structural identification. We also describe here the employment of thin layer chromatography, an old but useful tool, in facilitating the structural characterization of the epidermal lipid species by MS.
    Keywords:  Epidermal lipids; Mass spectrometry; Protein-bound lipids; Solid-phase extraction; Thin layer chromatography
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_10
  13. Metabolites. 2021 Apr 29. pii: 284. [Epub ahead of print]11(5):
    Quo Vadis Caenorhabditis elegans Metabolomics-A Review of Current Methods and Applications to Explore Metabolism in the Nematode.
    Liesa Salzer, Michael Witting.
      Metabolomics and lipidomics recently gained interest in the model organism Caenorhabditis elegans (C. elegans). The fast development, easy cultivation and existing forward and reverse genetic tools make the small nematode an ideal organism for metabolic investigations in development, aging, different disease models, infection, or toxicology research. The conducted type of analysis is strongly depending on the biological question and requires different analytical approaches. Metabolomic analyses in C. elegans have been performed using nuclear magnetic resonance (NMR) spectroscopy, direct infusion mass spectrometry (DI-MS), gas-chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) or combinations of them. In this review we provide general information on the employed techniques and their advantages and disadvantages in regard to C. elegans metabolomics. Additionally, we reviewed different fields of application, e.g., longevity, starvation, aging, development or metabolism of secondary metabolites such as ascarosides or maradolipids. We also summarised applied bioinformatic tools that recently have been used for the evaluation of metabolomics or lipidomics data from C. elegans. Lastly, we curated metabolites and lipids from the reviewed literature, enabling a prototypic collection which serves as basis for a future C. elegans specific metabolome database.
    Keywords:  Caenorhabditis elegans; NMR; lipidomics; mass-spectrometry; metabolomics
    DOI:  https://doi.org/10.3390/metabo11050284
  14. Dev Cell. 2021 May 03. pii: S1534-5807(21)00322-1. [Epub ahead of print]
    Lipid metabolism in cancer: New perspectives and emerging mechanisms.
    Lindsay A Broadfield, Antonino Alejandro Pane, Ali Talebi, Johannes V Swinnen, Sarah-Maria Fendt.
      Tumors undergo metabolic transformations to sustain uncontrolled proliferation, avoid cell death, and seed in secondary organs. An increased focus on cancer lipid metabolism has unveiled a number of mechanisms that promote tumor growth and survival, many of which are independent of classical cellular bioenergetics. These mechanisms include modulation of ferroptotic-mediated cell death, support during tumor metastasis, and interactions with the cells of the tumor microenvironment. As such, targeting lipid metabolism for anti-cancer therapies is attractive, with recent work on small-molecule inhibitors identifying compounds to target lipid metabolism. Here, we discuss these topics and identify open questions.
    Keywords:  cancer; immunometabolism; lipids; metabolism; metastasis; tumor microenvironment
    DOI:  https://doi.org/10.1016/j.devcel.2021.04.013
  15. Methods Mol Biol. 2021 ;2228 327-339
    Label-Free Proteomics of Quantity-Limited Samples Using Ion Mobility-Assisted Data-Independent Acquisition Mass Spectrometry.
    Ute Distler, Malte Sielaff, Stefan Tenzer.
      Over the past two decades, unbiased data-independent acquisition (DIA) approaches have gained increasing popularity in the bottom-up proteomics field. Here, we describe an ion mobility separation enhanced DIA workflow for large-scale label-free quantitative proteomics studies where starting material is limited. We set a special focus on the single pot solid-phase-enhanced sample preparation (SP3) protocol, which is well suited for the processing of quantity-limited samples.
    Keywords:  Bottom-up proteomics; Data-independent acquisition; Ion mobility separation; Label-free quantification; Quantitative proteomics; Single pot solid-phase-enhanced sample preparation (SP3)
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_23
  16. Methods Mol Biol. 2021 ;2228 225-235
    Quantification and Identification of Post-Translational Modifications Using Modern Proteomics Approaches.
    Anja Holtz, Nathan Basisty, Birgit Schilling.
      Post-translational modifications (PTMs) occur dynamically, allowing cells to quickly respond to changes in the environment. Lysine residues can be targeted by several modifications including acylations (acetylation, succinylation, malonylation, glutarylation, and others), methylation, ubiquitination, and other modifications. One of the most efficient methods for the identification of post-translational modifications is utilizing immunoaffinity enrichment followed by high-resolution mass spectrometry. This workflow can be coupled with comprehensive data-independent acquisition (DIA) mass spectrometry to be a high-throughput, label-free PTM quantification approach. Below we describe a detailed protocol to process tissue by homogenization and proteolytically digest proteins, followed by immunoaffinity enrichment of lysine-acetylated peptides to identify and quantify relative changes of acetylation comparing different conditions.
    Keywords:  Acetylation; Data-independent acquisition; Mass spectrometry; Post-translational modifications; Quantification
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_16
  17. Methods Mol Biol. 2021 ;2306 157-170
    Mass Spectrometric Analysis of Meibomian Gland Lipids.
    Jianzhong Chen.
      The precorneal tear film keeps the eye surface moist and helps to maintain normal eye function. The outermost lipid layer of the tear film, which attenuates tear film evaporation, contains meibum secreted from the meibomian gland. Most meibum lipids are neutral, including wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs), along with some polar lipids including free fatty acids (FFAs), O-acyl-ω-hydroxy fatty acids (OAHFAs), and trace phospholipids. Detection of neutral lipids by mass spectrometry (MS) is challenging due to interference from impurities, particularly when working with minute-volume meibum samples. Here, we describe procedures for sample preparation and MS analysis of these elusive meibum lipids that can be used to examine dry eye disease mechanisms. Because the method described here minimizes impurity peaks for lipids generally, neutral and otherwise, it may be applied to high-sensitivity analysis of other biological samples.
    Keywords:  Cholesteryl esters; Diesters; Dry eye; Fatty acids; O-acyl-ω-hydroxy fatty acids; Ocular surface; Phospholipids; Plasticizer; Shotgun lipidomics; Wax esters
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_11
  18. Methods Mol Biol. 2021 ;2306 11-37
    High Confidence Shotgun Lipidomics Using Structurally Selective Ion Mobility-Mass Spectrometry.
    Bailey S Rose, Katrina L Leaptrot, Rachel A Harris, Stacy D Sherrod, Jody C May, John A McLean.
      Ion mobility (IM) is a gas phase separation strategy that can either supplement or serve as a high-throughput alternative to liquid chromatography (LC) in shotgun lipidomics. Incorporating the IM dimension in untargeted lipidomics workflows can help resolve isomeric lipids, and the collision cross section (CCS) values obtained from the IM measurements can provide an additional molecular descriptor to increase lipid identification confidence. This chapter provides a broad overview of an untargeted ion mobility-mass spectrometry (IM-MS) workflow using a commercial drift tube ion mobility-quadrupole-time-of-flight mass spectrometer (IM-QTOF) for high confidence lipidomics.
    Keywords:  Collision cross section; Drift tube ion mobility spectrometry; Ion mobility spectrometry; Lipidomics; Tandem MS/MS
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_2
  19. Methods Mol Biol. 2021 ;2228 185-203
    Relative Quantification of Phosphorylated and Glycosylated Peptides from the Same Sample Using Isobaric Chemical Labelling with a Two-Step Enrichment Strategy.
    Ivan Silbern, Pan Fang, Yanlong Ji, Lenz Christof, Henning Urlaub, Kuan-Ting Pan.
      Post-translational modifications (PTMs) are essential for the regulation of all cellular processes. The interplay of various PTMs on a single protein or different proteins comprises a complexity that we are far from understanding in its entirety. Reliable strategies for the enrichment and accurate quantification of PTMs are needed to study as many PTMs on proteins as possible. In this protocol we present a liquid chromatography-tandem mass spectrometry (LC/MS/MS)-based workflow that enables the enrichment and quantification of phosphorylated and N-glycosylated peptides from the same sample. After extraction and digestion of proteins, we label the peptides with stable isotope-coded tandem mass tags (TMTs) and enrich N-glycopeptides and phosphopeptides by using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) and titanium dioxide (TiO2) beads, respectively. Labelled and enriched N-glycopeptides and phosphopeptides are further separated by high pH (basic) reversed-phase chromatography and analyzed by LC/MS/MS. The enrichment strategies, together with quantification of two different PTM types from the same sample, allow investigation of the interplay of those two PTMs, which are important for signal transduction inside the cell (phosphorylation), as well as for messaging between cells through decoration of the cellular surface (glycosylation).
    Keywords:  Isobaric labelling; N-Glycosylation; Phosphorylation; Post-translational modifications; TMT; TiO2; ZIC-HILIC
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_14
  20. Methods Mol Biol. 2021 ;2228 167-183
    Application of SILAC Labeling in Phosphoproteomics Analysis.
    Markus Stepath, Thilo Bracht.
      The analysis of disease-related changes in the phosphorylation status of cellular signal transduction networks is of major interest to biomedical researchers. Mass spectrometry-based proteomics allows the analysis of phosphorylation in a global manner. However, several technical challenges need to be addressed when the phosphorylation of proteins is analyzed. Low-abundant phosphopeptides need to be enriched before analysis, thereby introducing additional steps in sample preparation. Consequently, the applied quantification strategies should be robust towards elaborate sampling handling, rendering label-based quantification strategies the methods of choice in many experiments. Here, we present a protocol for SILAC labeling and the subsequent isolation of phosphopeptides using TiO2 affinity chromatography. We outline the corresponding LC-MS/MS analysis and the essential steps of data processing.
    Keywords:  Phosphoproteomics; Phosphorylation; Quantification; SILAC; Signaling network dynamics
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_13
  21. Methods Mol Biol. 2021 ;2306 77-91
    Quantitative Analysis of Polyphosphoinositide, Bis(monoacylglycero)phosphate, and Phosphatidylglycerol Species by Shotgun Lipidomics After Methylation.
    Meixia Pan, Chao Qin, Xianlin Han.
      Phospholipids play important roles in biological process even at a very low level. For example, bis(monoacylglycerol)phosphate (BMP) is involved in the pathogenesis of lysosomal storage diseases, and polyphosphoinositides (PPI) play critical roles in cellular signaling and functions. Phosphatidylglycerol (PG), a structural isomer of BMP, mediates lipid-protein and lipid-lipid interactions, and inhibits platelet activating factor and phosphatidylcholine transferring. However, due to their low abundance, the analysis of these phospholipids from biological samples is technically challenging. Therefore, the cellular function and metabolism of these phospholipids are still elusive. This chapter overviews a novel method of shotgun lipidomics after methylation with trimethylsilyl-diazomethane (TMS-D) for accurate and comprehensive analysis of these phospholipid species in biological samples. Firstly, a modified Bligh and Dyer procedure is performed to extract tissue lipids for PPI analysis, whereas modified methyl-tert-butylether (MTBE) extraction and modified Folch extraction methods are described to extract tissue lipids for PPI analysis. Secondly, TMS-D methylation is performed to derivatize PG/BMP and PPI, respectively. Then, we described the shotgun lipidomics strategies that can be used as cost-effective and relatively high-throughput methods to determine BMP, PG, and PPI species and isomers with different phosphate position(s) and fatty acyl chains. The described method of shotgun lipidomics after methylation achieves feasible and reliable quantitative analysis of low-abundance lipid classes. The application of this novel method should enable us to reveal the metabolism and functions of these phospholipids in healthy and disease states.
    Keywords:  Bis(monoacylglycerol)phosphate; Mass spectrometry; Methylation; Phosphatidylglycerol; Polyphosphoinositide; Shotgun lipidomics
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_6
  22. Methods Mol Biol. 2021 ;2228 159-166
    Quantitative Approach Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-ToF) Mass Spectrometry.
    Brooke A Dilmetz, Peter Hoffmann, Mark R Condina.
      Quantitation using mass spectrometry (MS) is a routine approach for multiple analytes, including small molecules and peptides. Electrospray-based MS platforms are typically employed, as they provide highly reproducible outputs for batch processing of multiple samples. Quantitation using matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry, while less commonly adopted, offers the ability to monitor analytes at significantly higher throughput and lower cost compared with ESI MS. Achieving accurate quantitation using this approach requires the development of appropriate sample preparation, spiking of appropriate internal standards, and acquisition to minimize spot-to-spot variability. Here we describe the preparation of samples for accurate quantitation using MALDI-ToF MS. The methodology presented shows the ability to quantitate perfluorooctanesulfonic acid (PFOS) from contaminated water.
    Keywords:  MALDI-ToF; Monoclonal antibodies; PFOS; Quantitation
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_12
  23. Methods Mol Biol. 2021 ;2306 239-255
    Mass Spectrometric Analysis of Bioactive Sphingolipids in Fungi.
    Ashutosh Singh, Maurizio Del Poeta.
      As biomolecules, sphingolipids represent a broad spectrum of structures ranging from simple long chain bases to complex glycosphingolipids. While several different mass spectrometry based approaches have been proven to be useful in qualitative and quantitative analysis of sphingolipids, we find that electrospray ionization tandem mass spectrometry (ESI-MS/MS) in the multiple-reaction monitoring (MRM) mode using a triple quadrupole instrument, coupled to high-performance liquid chromatography (HPLC), is the most suitable approach for the analysis. In this chapter, we describe the method in a step-by-step manner towards the targeted analysis of sphingolipids in fungi. With optimized HPLC separation and instrument settings, this MRM approach affords detection of many sphingolipid species simultaneously with good sensitivity.
    Keywords:  High-performance liquid chromatography; Mass spectrometry; Multiple-reaction monitoring; Sphingolipids
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_16
  24. FASEB J. 2021 Jun;35(6): e21629
    Analysis of differential neonatal lethality in cystathionine β-synthase deficient mouse models using metabolic profiling.
    Sapna Gupta, Liqun Wang, Michael J Slifker, Kathy Q Cai, Kenneth N Maclean, Brandi Wasek, Teodoro Bottiglieri, Warren D Kruger.
      Cystathionine beta-synthase (CBS) is a key enzyme of the trans-sulfuration pathway that converts homocysteine to cystathionine. Loss of CBS activity due to mutation results in CBS deficiency, an inborn error of metabolism characterized by extreme elevation of plasma total homocysteine (tHcy). C57BL6 mice containing either a homozygous null mutation in the cystathionine β-synthase (Cbs-/- ) gene or an inactive human CBS protein (Tg-G307S Cbs-/- ) are born in mendelian numbers, but the vast majority die between 18 and 21 days of age due to liver failure. However, adult Cbs null mice that express a hypomorphic allele of human CBS as a transgene (Tg-I278T Cbs-/- ) show almost no neonatal lethality despite having serum tHcy levels similar to mice with no CBS activity. Here, we characterize liver and serum metabolites in neonatal Cbs+/- , Tg-G307S Cbs-/- , and Tg-I278T Cbs-/- mice at 6, 10, and 17 days of age to understand this difference. In serum, we observe similar elevations in tHcy in both Tg-G307S Cbs-/- and Tg-I278T Cbs-/- compared to control animals, but methionine is much more severely elevated in Tg-G307S Cbs-/- mice. Large scale metabolomic analysis of liver tissue confirms that both methionine and methionine-sulfoxide are significantly more elevated in Tg-G307S Cbs-/- animals, along with significant differences in several other metabolites including hexoses, amino acids, other amines, lipids, and carboxylic acids. Our data are consistent with a model that the neonatal lethality observed in CBS-null mice is driven by excess methionine resulting in increased stress on a variety of related pathways including the urea cycle, TCA cycle, gluconeogenesis, and phosphatidylcholine biosynthesis.
    Keywords:  Homocysteine; inborn error; metabolomics; methionine; mouse models
    DOI:  https://doi.org/10.1096/fj.202100302R
  25. Biomedicines. 2021 Apr 29. pii: 491. [Epub ahead of print]9(5):
    Alteration in the Cerebrospinal Fluid Lipidome in Parkinson's Disease: A Post-Mortem Pilot Study.
    Joaquín Fernández-Irigoyen, Paz Cartas-Cejudo, Marta Iruarrizaga-Lejarreta, Enrique Santamaría.
      Lipid metabolism is clearly associated to Parkinson's disease (PD). Although lipid homeostasis has been widely studied in multiple animal and cellular models, as well as in blood derived from PD individuals, the cerebrospinal fluid (CSF) lipidomic profile in PD remains largely unexplored. In this study, we characterized the post-mortem CSF lipidomic imbalance between neurologically intact controls (n = 10) and PD subjects (n = 20). The combination of dual extraction with ultra-performance liquid chromatography-electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC-ESI-qToF-MS/MS) allowed for the monitoring of 257 lipid species across all samples. Complementary multivariate and univariate data analysis identified that glycerolipids (mono-, di-, and triacylglycerides), saturated and mono/polyunsaturated fatty acids, primary fatty amides, glycerophospholipids (phosphatidylcholines, phosphatidylethanolamines), sphingolipids (ceramides, sphingomyelins), N-acylethanolamines and sterol lipids (cholesteryl esters, steroids) were significantly increased in the CSF of PD compared to the control group. Interestingly, CSF lipid dyshomeostasis differed depending on neuropathological staging and disease duration. These results, despite the limitation of being obtained in a small population, suggest extensive CSF lipid remodeling in PD, shedding new light on the deployment of CSF lipidomics as a promising tool to identify potential lipid markers as well as discriminatory lipid species between PD and other atypical parkinsonisms.
    Keywords:  Parkinson’s disease; cerebrospinal fluid; lipidomics; lipids; mass-spectrometry
    DOI:  https://doi.org/10.3390/biomedicines9050491
  26. Methods Mol Biol. 2021 ;2306 227-238
    Characterization of the Uncommon Lipid Families in Corynebacterium glutamicum by Mass Spectrometry.
    Raju V V Tatituri, Fong-Fu Hsu.
      This book chapter provides readers the step-by-step instruction for cell growth, lipid isolation, and lipid analysis to obtain the lipidome of Corynebacterium glutamicum (C. glutamicum) in the genus Corynebacterium, a biotechnologically important bacterium. We separate the lipid families by preparative HPLC with an analytical C-8 column, followed by linear ion-trap multiple stage mass spectrometry (LIT MSn) with high-resolution mass measurement to define the structures of cytidine diphosphate diacylglycerol (CDP-DAG), glucuronosyl diacylglycerol (GlcA-DAG), α-D-mannopyranosyl-(1 → 4)-α-D-glucuronyl diacylglycerol (Man-GlcA-DAG), 1-mycolyl-2-acyl-phosphatidylglycerol (MA-PG), and acyl trehalose monomycolate (acyl-TMM) whose structures have been previously mis-assigned or not defined by mass spectrometric means. We also define the structures of mycolic acid, phosphatidylglycerol, phosphatidylinositol, cardiolipin, trehalose dimycolate lipids in the cell wall. The similarity of the lipidome to that in the Mycobacterium genera is consistent with the notion that Corynebacterium and Mycobacterium are gram-positive bacteria belonging to the suborder Corynebacterineae.
    Keywords:  CDP-diacylglycerol; Lipidomics; Microbial lipids; Multiple stage linear ion-trap mass spectrometry; Mycolic acid-containing PG
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_15
  27. Front Genet. 2021 ;12 635971
    Three-in-One Simultaneous Extraction of Proteins, Metabolites and Lipids for Multi-Omics.
    Jianing Kang, Lisa David, Yangyang Li, Jing Cang, Sixue Chen.
      Elucidation of complex molecular networks requires integrative analysis of molecular features and changes at different levels of information flow and regulation. Accordingly, high throughput functional genomics tools such as transcriptomics, proteomics, metabolomics, and lipidomics have emerged to provide system-wide investigations. Unfortunately, analysis of different types of biomolecules requires specific sample extraction procedures in combination with specific analytical instrumentation. The most efficient extraction protocols often only cover a restricted type of biomolecules due to their different physicochemical properties. Therefore, several sets/aliquots of samples are needed for extracting different molecules. Here we adapted a biphasic fractionation method to extract proteins, metabolites, and lipids from the same sample (3-in-1) for liquid chromatography-tandem mass spectrometry (LC-MS/MS) multi-omics. To demonstrate utility of the improved method, we used bacteria-primed Arabidopsis leaves to generate multi-omics datasets from the same sample. In total, we were able to analyze 1849 proteins, 1967 metabolites, and 424 lipid species in single samples. The molecules cover a wide range of biological and molecular processes, and allow quantitative analyses of different molecules and pathways. Our results have shown the clear advantages of the multi-omics method, including sample conservation, high reproducibility, and tight correlation between different types of biomolecules.
    Keywords:  3-in-1 method; Arabidopsis; disease; lipidomics; metabolomics; multi-omics; proteomics
    DOI:  https://doi.org/10.3389/fgene.2021.635971
  28. Methods Mol Biol. 2021 ;2228 117-131
    Tandem Mass Tags for Comparative and Discovery Proteomics.
    Oliver Pagel, Laxmikanth Kollipara, Albert Sickmann.
      Relative or comparative proteomics provides valuable insights about the altered protein abundances across different biological samples in a single (labeled) or series (label-free) of LC-MS measurement(s). Chemical labeling of peptides using isobaric mass tags for identification and quantification of different proteomes simultaneously has become a routine in the so-called discovery proteomics in the past decade. One of the earliest isobaric tags-based technologies is TMT (tandem mass tags), which relies on the comparison of the unique "reporter ions" intensities for relative peptide/protein quantification. This differential labeling approach has evolved over time with respect to its multiplexing capability, i.e., from just 2 samples (TMTduplex) to 10 samples (TMT10plex) and a nowadays of up to 16 samples (TMTpro 16plex). Here, we describe a straightforward protocol to perform relatively deep proteome quantitative analyses using TMT10plex.
    Keywords:  LC–MS/MS; Multiplexing; Relative quantitative proteomics; TMT
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_9
  29. Methods Mol Biol. 2021 ;2228 293-306
    Quantitative MS Workflow for a High-Quality Secretome Analysis by a Quantitative Secretome-Proteome Comparison.
    Gereon Poschmann, Nina Prescher, Kai Stühler.
      Cells secrete proteins to communicate with their environment. Therefore, it is interesting to characterize the proteins which are released from cells under certain experimental conditions the so-called secretome. Here, often proteins from conditioned medium of cultured cells are analyzed, but these additionally might include also contaminating proteins of serum that have not been sufficiently removed or proteins from dying cells. To provide high-quality secretome data and minimize potential contaminants, we describe a quantitative comparison of conditioned medium and the cellular proteome. The described workflow comprises cell cultivation, sample preparation, and final data analysis which is based on the comparison of data from label-free mass spectrometric quantification of proteins from the conditioned medium with corresponding cellular proteomes enabling the detection of bona fide secreted proteins.
    Keywords:  Contaminants; Quantitative MS; Secretome
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_21
  30. Methods Mol Biol. 2021 ;2228 1-20
    Important Issues in Planning a Proteomics Experiment: Statistical Considerations of Quantitative Proteomic Data.
    Karin Schork, Katharina Podwojski, Michael Turewicz, Christian Stephan, Martin Eisenacher.
      Mass spectrometry is frequently used in quantitative proteomics to detect differentially regulated proteins. A very important but unfortunately oftentimes neglected part in detecting differential proteins is the statistical analysis. Data from proteomics experiments are usually high-dimensional and hence require profound statistical methods. It is especially important to already correctly design a proteomic experiment before it is conducted in the laboratory. Only this can ensure that the statistical analysis is capable of detecting truly differential proteins afterward. This chapter thus covers aspects of both statistical planning as well as the actual analysis of quantitative proteomic experiments.
    Keywords:  Data preprocessing; Experimental design; Fold change; Multiple testing; Normalization; Sample size calculation; Statistical hypothesis test; Volcano plot
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_1
  31. Cancer Lett. 2021 May 04. pii: S0304-3835(21)00189-0. [Epub ahead of print]
    Targeting lipid metabolism is an emerging strategy to enhance the efficacy of anti-HER2 therapies in HER2-positive breast cancer.
    Francesca Ligorio, Ilaria Pellegrini, Lorenzo Castagnoli, Andrea Vingiani, Riccardo Lobefaro, Emma Zattarin, Marzia Santamaria, Serenella M Pupa, Giancarlo Pruneri, Filippo de Braud, Claudio Vernieri.
      De novo or acquired resistance of cancer cells to currently available Human Epidermal Growth Factor Receptor 2 (HER2) inhibitors represents a clinical challenge. Several resistance mechanisms have been identified in recent years, with lipid metabolism reprogramming, a well-established hallmark of cancer, representing the last frontier of preclinical and clinical research in this field. Fatty Acid Synthase (FASN), the key enzyme required for fatty acids (FAs) biosynthesis, is frequently overexpressed/activated in HER2-positive (HER2+) breast cancer (BC), and it crucially sustains HER2+ BC cell growth, proliferation and survival. After the synthesis of new, selective and well tolerated FASN inhibitors, clinical trials have been initiated to test if these compounds are able to re-sensitize cancer cells with acquired resistance to HER2 inhibition. More recently, the upregulation of FA uptake by cancer cells has emerged as a potentially new and targetable mechanism of resistance to anti-HER2 therapies in HER2+ BC, thus opening a new era in the field of targeting metabolic reprogramming in clinical setting. Here, we review the available preclinical and clinical evidence supporting the inhibition of FA biosynthesis and uptake in combination with anti-HER2 therapies in patients with HER2+ BC, and we discuss ongoing clinical trials that are investigating these combination approaches.
    Keywords:  Anti-HER2 resistance; CD36; FASN; HER2-Positive breast cancer; Metabolic reprogramming
    DOI:  https://doi.org/10.1016/j.canlet.2021.04.023
  32. Methods Mol Biol. 2021 ;2306 299-311
    Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Lipids in the Ischemic Rat Brain Section: A Practical Approach.
    Hay-Yan J Wang.
      Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) of lipids is considered one of the shotgun lipidomic techniques that explores the in situ distribution of lipids in tissue sections. To successfully perform this task, knowledge and experience in the conventional cryosection, tissue section collection and handling, and mass spectrometry data analysis and signal processing are needed. A MALDI-MSI protocol covering from the fresh organ collection, cryosection and tissue processing, matrix application, MSI data acquisition, to the final MSI display of lipid distribution in the brain section of an ischemic stroke rat is described to exemplify this technique. Due to the multidisciplinary nature of this approach, plenty of preparation, practice, and familiarization to several critical steps ahead of the engagement with actual biological samples are needed to ensure a successful MALDI-MSI presentation of lipids in situ.
    Keywords:  Brain tissue lipids; Frozen section; Ischemic stroke; Matrix sublimation; Matrix-assisted laser desorption/ionization-mass spectrometry imaging; Tissue handling and preparation; Tissue section drip-wash
    DOI:  https://doi.org/10.1007/978-1-0716-1410-5_20
  33. Methods Mol Biol. 2021 ;2228 253-270
    2nSILAC for Quantitative Proteomics of Prototrophic Baker's Yeast.
    Stefan Dannenmaier, Silke Oeljeklaus, Bettina Warscheid.
      Stable isotope labeling by amino acids in cell culture (SILAC) combined with high-resolution mass spectrometry is a quantitative strategy for the comparative analysis of (sub)proteomes. It is based on the metabolic incorporation of stable isotope-coded amino acids during growth of cells or organisms. Here, complete labeling of proteins with the amino acid(s) selected for incorporation needs to be guaranteed to enable accurate quantification on a proteomic scale. Wild-type strains of baker's yeast (Saccharomyces cerevisiae ), which is a widely accepted and well-studied eukaryotic model organism, are generally able to synthesize all amino acids on their own (i.e., prototrophic). To render them amenable to SILAC, auxotrophies are introduced by genetic manipulations. We addressed this limitation by developing a generic strategy for complete "native" labeling of prototrophic S. cerevisiae with isotope-coded arginine and lysine, referred to as "2nSILAC". It allows for directly using and screening several genome-wide yeast mutant collections that are easily accessible to the scientific community for functional proteomic studies but are based on prototrophic variants of S. cerevisiae.
    Keywords:  2nSILAC; Mass spectrometry; Mitochondria; Native SILAC; Prototroph; Quantitative proteome analysis; SILAC; Yeast
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_18
  34. Methods Mol Biol. 2021 ;2228 85-116
    Quantitative Mass Spectrometry-Based Proteomics: An Overview.
    Svitlana Rozanova, Katalin Barkovits, Miroslav Nikolov, Carla Schmidt, Henning Urlaub, Katrin Marcus.
      In recent decades, mass spectrometry has moved more than ever before into the front line of protein-centered research. After being established at the qualitative level, the more challenging question of quantification of proteins and peptides using mass spectrometry has become a focus for further development. In this chapter, we discuss and review actual strategies and problems of the methods for the quantitative analysis of peptides, proteins, and finally proteomes by mass spectrometry. The common themes, the differences, and the potential pitfalls of the main approaches are presented in order to provide a survey of the emerging field of quantitative, mass spectrometry-based proteomics.
    Keywords:  Absolute quantification; Chemical labeling; Isotope labeling; Label-free; Mass spectrometry; Metabolic labeling; Proteomics; Relative quantification
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_8
  35. Methods Mol Biol. 2021 ;2228 283-292
    Application of Label-Free Proteomics for Quantitative Analysis of Urothelial Carcinoma and Cystitis Tissue.
    Kathrin E Witzke, Frederik Großerueschkamp, Klaus Gerwert, Barbara Sitek.
      A label-free approach based on a highly reproducible and stable workflow allows for quantitative proteome analysis . Due to advantages compared to labeling methods, the label-free approach has the potential to measure unlimited samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes a new sample preparation technique depending on automatic annotation and tissue isolation via FTIR-guided laser microdissection, in-solution digestion, LC-MS/MS analyses, data evaluation by means of Proteome Discoverer and Progenesis software, and verification of differential proteins. We successfully applied this workflow in a proteomics study analyzing human cystitis and high-grade urothelial carcinoma tissue regarding the identification of a diagnostic tissue biomarker. The differential analysis of only 1 mm2 of isolated tissue cells led to 74 significantly differentially abundant proteins.
    Keywords:  AHNAK2; Bladder cancer; FTIR imaging; Label-free proteomics; Laser microdissection; Urothelial cell carcinoma (UCC)
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_20
  36. Expert Rev Proteomics. 2021 May 04.
    Proteomic approaches to assist in diagnosis and prognosis of oral cancer.
    Jamile de Oliveira Sá, Luciana Daniele Trino, Ana Karina Oliveira, Ariane Fidelis Busso Lopes, Daniela Campos Granato, Ana Gabriela Costa Normando, Erison Santos, Leandro Xavier Neves, Carolina Moretto Carnielli, Adriana Franco Paes Leme.
      Introduction: Oral squamous cell carcinoma (OSCC) ranks in the top ten leading causes of cancer worldwide, with 5-year survival rate of about 50%, high lymph node metastasis, and relapse rates. The OSCC diagnosis, prognosis, and treatment are mostly based on the clinical TNM classification. There is an urgent need for the discovery of biomarkers and therapeutic targets to assist in the clinical decision-making process.Areas covered: We summarize proteomic studies of the OSCC tumor, immune microenvironment, potential liquid biopsy sites, and post-translational modifications trying to retrieve information in the discovery and verification or (pre)validation phases. The search strategy was based on the combination of Mesh terms and expert refinement.Expert Opinion: Untargeted combined with targeted proteomics are strategies that provide reliable and reproducible quantitation of proteins and are the methods of choice of many groups worldwide. Undoubtedly, proteomics has been contributing to the understanding of the OSCC progression and uncovers potential candidates as biomarker or therapeutic targets. Nevertheless, none of these targets are available in the clinical practice yet. The scientific community needs to overcome the limitations investing in robust experimental designs to strengthen the value of the findings, leveraging the translation of the knowledge, and further supporting clinical decisions.
    Keywords:  biomarkers; liquid biopsies; mass spectrometry; oral cancer; peptidomics; post-translational modifications; proteomics; tumor microenvironment
    DOI:  https://doi.org/10.1080/14789450.2021.1924685
  37. Methods Mol Biol. 2021 ;2228 145-157
    Targeted Protein Quantification Using Parallel Reaction Monitoring (PRM).
    Katalin Barkovits, Weiqiang Chen, Michael Kohl, Thilo Bracht.
      Targeted proteomics represents an efficient method to quantify proteins of interest with high sensitivity and accuracy. Targeted approaches were first established for triple quadrupole instruments, but the emergence of hybrid instruments allowing for high-resolution and accurate-mass measurements of MS/MS fragment ions enabled the development of parallel reaction monitoring (PRM). In PRM analysis, specific peptides are measured as representatives of proteins in complex samples, with the full product ion spectra being acquired, allowing for identification and quantification of the peptides. Ideally, corresponding stable isotope-labeled peptides are spiked into the analyzed samples to account for technical variation and enhance the precision. Here, we describe the development of a PRM assay including the selection of appropriate peptides that fulfill the criteria to serve as unique surrogates of the targeted proteins. We depict the sequential steps of method development and the generation of calibration curves. Furthermore, we present the open-access tool CalibraCurve for the determination of the linear concentration ranges and limits of quantification (LOQ).
    Keywords:  Calibration curve; Limit of quantification; Parallel reaction monitoring; Stable isotope-labeled synthetic peptides; Targeted proteomics
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_11
  38. Methods Mol Biol. 2021 ;2228 307-325
    Establishing a Custom-Fit Data-Independent Acquisition Method for Label-Free Proteomics.
    Britta Eggers, Martin Eisenacher, Katrin Marcus, Julian Uszkoreit.
      Data-independent acquisition (DIA) has recently developed as a powerful tool to enhance the quantification of peptides and proteins within a variety of sample types, by overcoming the stochastic nature of classical data-dependent approaches, as well as by enabling the identification of all peptides detected in a mass spectrometric event. Here, we describe a workflow for the establishment of a sample-fitting DIA method using Spectronaut Pulsar X (Biognosys, Switzerland).
    Keywords:  Data-independent acquisition; Label-free quantification; Spectral library generation
    DOI:  https://doi.org/10.1007/978-1-0716-1024-4_22
  39. J Matern Fetal Neonatal Med. 2021 May 04. 1-8
    Urine metabolomic biomarkers for prediction of isolated fetal congenital heart defect.
    Perry Friedman, Ali Yilmaz, Zafer Ugur, Faryal Jafar, Amy Whitten, Ilyas Ustun, Onur Turkoglu, Stewart Graham, Ray Bahado Singh.
       OBJECTIVE: To identify maternal second and third trimester urine metabolomic biomarkers for the detection of fetal congenital heart defects (CHDs).
    STUDY DESIGN: This was a prospective study. Metabolomic analysis of randomly collected maternal urine was performed, comparing pregnancies with isolated, non-syndromic CHDs versus unaffected controls. Mass spectrometry (liquid chromatography and direct injection and tandem mass spectrometry, LC-MS-MS) as well as nuclear magnetic resonance spectrometry, 1H NMR, were used to perform the analyses between 14 0/7 and 37 0/7 weeks gestation. A total of 36 CHD cases and 41 controls were compared. Predictive algorithms using urine markers alone or combined with, clinical and ultrasound (US) (four-chamber view) predictors were developed and compared.
    RESULTS: A total of 222 metabolites were identified, of which 16 were overlapping between the two platforms. Twenty-three metabolite concentrations were found in significantly altered in CHD gestations on univariate analysis. The concentration of methionine was most significantly altered. A predictive algorithm combining metabolites (histamine, choline, glucose, formate, methionine, and carnitine) plus US four-chamber view achieved an AUC = 0.894; 95% CI, 0814-0.973 with a sensitivity of 83.8% and specificity of 87.8%. Enrichment pathway analysis identified several lipid related pathways that are dysregulated in CHD, including phospholipid biosynthesis, phosphatidylcholine biosynthesis, phosphatidylethanolamine biosynthesis, and fatty acid metabolism. This could be consistent with the increased risk of CHD in diabetic pregnancies.
    CONCLUSIONS: We report a novel, noninvasive approach, based on the analysis of maternal urine for isolated CHD detection. Further, the dysregulation of lipid- and folate metabolism appears to support prior data on the mechanism of CHD.
    Keywords:  Metabolomics; congenital heart defect; mass spectrometry; metabolites; nuclear magnetic resonance
    DOI:  https://doi.org/10.1080/14767058.2021.1914572
  40. Prostate. 2021 May 05.
    The influence of low-carbohydrate diets on the metabolic response to androgen-deprivation therapy in prostate cancer.
    Jen-Tsan Chi, Pao-Hwa Lin, Vladimir Tolstikov, Taofik Oyekunle, Gloria C G Alvarado, Adela Ramirez-Torres, Emily Y Chen, Valerie Bussberg, Bo Chi, Bennett Greenwood, Rangaprasad Sarangarajan, Niven R Narain, Michael A Kiebish, Stephen J Freedland.
       BACKGROUND: Prostate cancer (PC) is the second most lethal cancer for men. For metastatic PC, standard first-line treatment is androgen deprivation therapy (ADT). While effective, ADT has many metabolic side effects. Previously, we found in serum metabolome analysis that ADT reduced androsterone sulfate, 3-hydroxybutyric acid, acyl-carnitines but increased serum glucose. Since ADT reduced ketogenesis, we speculate that low-carbohydrate diets (LCD) may reverse many ADT-induced metabolic abnormalities in animals and humans.
    METHODS: In a multicenter trial of patients with PC initiating ADT randomized to no diet change (control) or LCD, we previously showed that LCD intervention led to significant weight loss, reduced fat mass, improved insulin resistance, and lipid profiles. To determine whether and how LCD affects ADT-induced metabolic changes, we analyzed serum metabolites after 3-, and 6-months of ADT on LCD versus control.
    RESULTS: We found androsterone sulfate was most consistently reduced by ADT and was slightly further reduced in the LCD arm. Contrastingly, LCD intervention increased 3-hydroxybutyric acid and various acyl-carnitines, counteracting their reduction during ADT. LCD also reversed the ADT-reduced lactic acid, alanine, and S-adenosyl methionine (SAM), elevating glycolysis metabolites and alanine. While the degree of androsterone reduction by ADT was strongly correlated with glucose and indole-3-carboxaldehyde, LCD disrupted such correlations.
    CONCLUSIONS: Together, LCD intervention significantly reversed many ADT-induced metabolic changes while slightly enhancing androgen reduction. Future research is needed to confirm these findings and determine whether LCD can mitigate ADT-linked comorbidities and possibly delaying disease progression by further lowering androgens.
    Keywords:  3-formyl indole; 3-hydroxybutyric acid; ADT; androgen sulfate; indole-3-carboxaldehyde; ketogenesis; low carbohydrate diet; metabolomics; prostate cancer
    DOI:  https://doi.org/10.1002/pros.24136
  41. Cells. 2021 Apr 29. pii: 1056. [Epub ahead of print]10(5):
    Mechanisms of Metabolic Reprogramming in Cancer Cells Supporting Enhanced Growth and Proliferation.
    Chelsea Schiliro, Bonnie L Firestein.
      Cancer cells alter metabolic processes to sustain their characteristic uncontrolled growth and proliferation. These metabolic alterations include (1) a shift from oxidative phosphorylation to aerobic glycolysis to support the increased need for ATP, (2) increased glutaminolysis for NADPH regeneration, (3) altered flux through the pentose phosphate pathway and the tricarboxylic acid cycle for macromolecule generation, (4) increased lipid uptake, lipogenesis, and cholesterol synthesis, (5) upregulation of one-carbon metabolism for the production of ATP, NADH/NADPH, nucleotides, and glutathione, (6) altered amino acid metabolism, (7) metabolism-based regulation of apoptosis, and (8) the utilization of alternative substrates, such as lactate and acetate. Altered metabolic flux in cancer is controlled by tumor-host cell interactions, key oncogenes, tumor suppressors, and other regulatory molecules, including non-coding RNAs. Changes to metabolic pathways in cancer are dynamic, exhibit plasticity, and are often dependent on the type of tumor and the tumor microenvironment, leading in a shift of thought from the Warburg Effect and the "reverse Warburg Effect" to metabolic plasticity. Understanding the complex nature of altered flux through these multiple pathways in cancer cells can support the development of new therapies.
    Keywords:  Warburg Effect; aerobic glycolysis; cancer; one-carbon metabolism; oxidative phosphorylation; pentose phosphate pathway
    DOI:  https://doi.org/10.3390/cells10051056
  42. Front Vet Sci. 2021 ;8 601794
    Advancing Semen Evaluation Using Lipidomics.
    Holly C Evans, Thu T N Dinh, Madison L Hardcastle, Alicia A Gilmore, Muhammet R Ugur, Mustafa Hitit, Frank Dean Jousan, Molly C Nicodemus, Erdogan Memili.
      Developing a deeper understanding of biological components of sperm is essential to improving cryopreservation techniques and reproductive technologies. To fully ascertain the functional determinants of fertility, lipidomic methods have come to the forefront. Lipidomics is the study of the lipid profile (lipidome) within a cell, tissue, or organism and provides a quantitative analysis of the lipid content in that sample. Sperm cells are composed of various lipids, each with their unique contribution to the overall function of the cell. Lipidomics has already been used to find new and exciting information regarding the fatty acid content of sperm cells from different species. While the applications of lipidomics are rapidly evolving, gaps in the knowledge base remain unresolved. Current limitations of lipidomics studies include the number of available samples to analyze and the total amount of cells within those samples needed to detect changes in the lipid profiles across different subjects. The information obtained through lipidomics research is essential to systems and cellular biology. This review provides a concise analysis of the most recent developments in lipidomic research. This scientific resource is important because these developments can be used to not only combat the reproductive challenges faced when using cryopreserved semen and artificial reproductive technologies in livestock such as cattle, but also other mammals, such as humans or endangered species.
    Keywords:  biomarkers; cryotolerance; fatty acids; lipids; livestock
    DOI:  https://doi.org/10.3389/fvets.2021.601794
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