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Biotechnology and Bioengineering Oct 2023Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such... (Review)
Review
Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such as those coupled to gas chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.
Topics: Gas Chromatography-Mass Spectrometry; Fermentation; Mass Spectrometry; Metabolomics; Metabolome
PubMed: 37272489
DOI: 10.1002/bit.28450 -
STAR Protocols Sep 2023Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows...
Tandem mass tags data-dependent acquisition (TMT-DDA) as well as data-independent acquisition-based label-free quantification (LFQ-DIA) have become the leading workflows to achieve deep proteome and phosphoproteome profiles. We present a modular pipeline for TMT-DDA and LFQ-DIA that integrates steps to perform scalable phosphoproteome profiling, including protein lysate extraction, clean-up, digestion, phosphopeptide enrichment, and TMT-labeling. We also detail peptide and/or phosphopeptide fractionation and pre-mass spectrometry desalting and provide researchers guidance on choosing the best workflow based on sample number and input. For complete details on the use and execution of this protocol, please refer to Koenig et al. and Martínez-Val et al..
Topics: Phosphopeptides; Proteome; Proteomics; Mass Spectrometry; Workflow
PubMed: 37659085
DOI: 10.1016/j.xpro.2023.102536 -
Frontiers in Immunology 2023Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for...
INTRODUCTION
Mass spectrometry-based immunopeptidomics is the only unbiased method to identify naturally presented HLA ligands, which is an indispensable prerequisite for characterizing novel tumor antigens for immunotherapeutic approaches. In recent years, improvements based on devices and methodology have been made to optimize sensitivity and throughput in immunopeptidomics. However, developments in ligand isolation, mass spectrometric analysis, and subsequent data processing can have a marked impact on the quality and quantity of immunopeptidomics data.
METHODS
In this work, we compared the immunopeptidome composition in terms of peptide yields, spectra quality, hydrophobicity, retention time, and immunogenicity of two established immunoprecipitation methods (column-based and 96-well-based) using cell lines as well as primary solid and hematological tumor samples.
RESULTS
Although, we identified comparable overall peptide yields, large proportions of method-exclusive peptides were detected with significantly higher hydrophobicity for the column-based method with potential implications for the identification of immunogenic tumor antigens. We showed that column preparation does not lose hydrophilic peptides in the hydrophilic washing step. In contrast, an additional 50% acetonitrile elution could partially regain lost hydrophobic peptides during 96-well preparation, suggesting a reduction of the bias towards the column-based method but not completely equalizing it.
DISCUSSION
Together, this work showed how different immunoprecipitation methods and their adaptions can impact the peptide repertoire of immunopeptidomic analysis and therefore the identification of potential tumor-associated antigens.
Topics: Humans; Peptides; Neoplasms; Mass Spectrometry; Antigens, Neoplasm; Immunoprecipitation
PubMed: 37545538
DOI: 10.3389/fimmu.2023.1219720 -
Mass Spectrometry Reviews 2024Exploring the chemical content of individual cells not only reveals underlying cell-to-cell chemical heterogeneity but is also a key component in understanding how cells... (Review)
Review
Exploring the chemical content of individual cells not only reveals underlying cell-to-cell chemical heterogeneity but is also a key component in understanding how cells combine to form emergent properties of cellular networks and tissues. Recent technological advances in many analytical techniques including mass spectrometry (MS) have improved instrumental limits of detection and laser/ion probe dimensions, allowing the analysis of micron and submicron sized areas. In the case of MS, these improvements combined with MS's broad analyte detection capabilities have enabled the rise of single-cell and single-organelle chemical characterization. As the chemical coverage and throughput of single-cell measurements increase, more advanced statistical and data analysis methods have aided in data visualization and interpretation. This review focuses on secondary ion MS and matrix-assisted laser desorption/ionization MS approaches for single-cell and single-organelle characterization, which is followed by advances in mass spectral data visualization and analysis.
Topics: Single-Cell Analysis; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Humans; Animals; Organelles; Spectrometry, Mass, Secondary Ion; Mass Spectrometry
PubMed: 37010120
DOI: 10.1002/mas.21841 -
Electrophoresis Dec 2023Single-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides... (Review)
Review
Single-cell heterogeneity in metabolism, drug resistance and disease type poses the need for analytical techniques for single-cell analysis. As the metabolome provides the closest view of the status quo in the cell, studying the metabolome at single-cell resolution may unravel said heterogeneity. A challenge in single-cell metabolome analysis is that metabolites cannot be amplified, so one needs to deal with picolitre volumes and a wide range of analyte concentrations. Due to high sensitivity and resolution, MS is preferred in single-cell metabolomics. Large numbers of cells need to be analysed for proper statistics; this requires high-throughput analysis, and hence automation of the analytical workflow. Significant advances in (micro)sampling methods, CE and ion mobility spectrometry have been made, some of which have been applied in high-throughput analyses. Microfluidics has enabled an automation of cell picking and metabolite extraction; image recognition has enabled automated cell identification. Many techniques have been used for data analysis, varying from conventional techniques to novel combinations of advanced chemometric approaches. Steps have been set in making data more findable, accessible, interoperable and reusable, but significant opportunities for improvement remain. Herein, advances in single-cell analysis workflows and data analysis are discussed, and recommendations are made based on the experimental goal.
Topics: Metabolomics; Mass Spectrometry; Metabolome; Specimen Handling; Single-Cell Analysis
PubMed: 37667867
DOI: 10.1002/elps.202300105 -
Methods in Molecular Biology (Clifton,... 2024The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of...
The diagnosis of alpha-1-antitrypsin (A1AT) deficiency is established by quantitation of protein concentration in serum (immunoassay) followed by determination of specific allelic variants by phenotyping (isoelectric focusing (IEF) gel electrophoresis) and/or allele-specific genotyping. Various phenotyping and genotyping methodologies are available, and each has their own advantages and disadvantages. As an alternative, mass spectrometry is emerging as a powerful tool in the identification and quantitation of proteins and peptides. The method described here, referred to as proteotyping, is a proteomic method using trypsin digestion and tandem mass spectrometry that detects the most common deficiency alleles, S and Z, associated with A1AT deficiency.This qualitative mass spectrometry method is based on the principle that the S and Z mutations lead to amino acid changes which result in a change in the mass of the A1AT protein. When the A1AT protein is proteolytically digested, multiple peptides are generated, two of which include the sites of the S and Z mutations, respectively. Peptides generated from wild-type A1AT (M alleles) differ in sequence and mass from peptides generated from the S and Z alleles at these two specific locations. The mass difference allows for differentiation of S and Z peptides, representing the deficiency alleles, from non-S and non-Z peptides, representing the wild-type alleles (M). Interpretation of the peptide patterns in conjunction with A1AT quantitation by immunoassay allows for an accurate assessment for the presence of deficiency alleles in the majority of patients.
Topics: Humans; Liquid Chromatography-Mass Spectrometry; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Alleles
PubMed: 38108970
DOI: 10.1007/978-1-0716-3605-3_9 -
Expert Opinion on Drug Discovery Mar 2024Ultra-high-throughput mass spectrometry, uHT-MS, is a technology that utilizes ionization and sample delivery technologies optimized to enable sampling from well plates... (Review)
Review
INTRODUCTION
Ultra-high-throughput mass spectrometry, uHT-MS, is a technology that utilizes ionization and sample delivery technologies optimized to enable sampling from well plates at > 1 sample per second. These technologies do not need a chromatographic separation step and can be utilized in a wide variety of assays to detect a broad range of analytes including small molecules, lipids, and proteins.
AREAS COVERED
This manuscript provides a brief historical review of high-throughput mass spectrometry and the recently developed technologies that have enabled uHT-MS. The report also provides examples and references on how uHT-MS has been used in biochemical and chemical assays, nuisance compound profiling, protein analysis and high throughput experimentation for chemical synthesis.
EXPERT OPINION
The fast analysis time provided by uHT-MS is transforming how biochemical and chemical assays are performed in drug discovery. The potential to associate phenotypic responses produced by 1000's of compound treatments with changes in endogenous metabolite and lipid signals is becoming feasible. With the augmentation of simple, fast, high-throughput sample preparation, the scope of uHT-MS usage will increase. However, it likely will not supplant LC-MS for analyses that require low detection limits from complex matrices or characterization of complex biotherapeutics such as antibody-drug conjugates.
Topics: Humans; Mass Spectrometry; Drug Discovery; Liquid Chromatography-Mass Spectrometry
PubMed: 38111363
DOI: 10.1080/17460441.2023.2293153 -
Journal of the American Society For... Sep 2023Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein-protein interactions (PPIs). Throughout the entire workflow, the... (Review)
Review
Cross-linking mass spectrometry (XL-MS) is widely used in the analysis of protein structure and protein-protein interactions (PPIs). Throughout the entire workflow, the utilization of cross-linkers and the interpretation of cross-linking data are the core steps. In recent years, the development of cross-linkers and analytical software mostly follow up on the classical models of non-cleavable cross-linkers such as BS3/DSS and MS-cleavable cross-linkers such as DSSO. Although such a paradigm promotes the maturity and robustness of the XL-MS field, it confines the innovation and flexibility of new cross-linkers and analytical software. This critical insight will discuss the classification, advantages, and disadvantages of existing data analysis search engines. Take the new platinum-based metal cross-linker as an example, potential pitfalls in characterization of cross-linked peptides using existing software are discussed. Finally, ideas on developing more flexible, comprehensive, and user-friendly cross-linkers and software tools are proposed.
Topics: Workflow; Peptides; Proteins; Mass Spectrometry; Software; Cross-Linking Reagents
PubMed: 37537999
DOI: 10.1021/jasms.3c00123 -
Current Opinion in Chemical Biology Aug 2023Single cell metabolomics is a rapidly advancing field of bio-analytical chemistry which aims to observe cellular biology with the greatest detail possible. Mass... (Review)
Review
Single cell metabolomics is a rapidly advancing field of bio-analytical chemistry which aims to observe cellular biology with the greatest detail possible. Mass spectrometry imaging and selective cell sampling (e.g. using nanocapillaries) are two common approaches within the field. Recent achievements such as observation of cell-cell interactions, lipids determining cell states and rapid phenotypic identification demonstrate the efficacy of these approaches and the momentum of the field. However, single cell metabolomics can only continue with the same impetus if the universal challenges to the field are met, such as the lack of strategies for standardisation and quantification, and lack of specificity/sensitivity. Mass spectrometry imaging and selective cell sampling come with unique advantages and challenges which, in many cases are complementary to each other. We propose here that the challenges specific to each approach could be ameliorated with collaboration between the two communities driving these approaches.
Topics: Lipids; Metabolomics; Mass Spectrometry
PubMed: 37224735
DOI: 10.1016/j.cbpa.2023.102327 -
Molecular & Cellular Proteomics : MCP Sep 2023The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a... (Review)
Review
The active release of proteins into the extracellular space and the proteolytic cleavage of cell surface proteins are key processes that coordinate and fine-tune a multitude of physiological functions. The entirety of proteins that fulfill these extracellular tasks are referred to as the secretome and are of special interest for the investigation of biomarkers of disease states and physiological processes related to cell-cell communication. LC-MS-based proteomics approaches are a valuable tool for the comprehensive and unbiased characterization of this important subproteome. This review discusses procedures, opportunities, and limitations of mass spectrometry-based secretomics to better understand and navigate the complex analytical landscape for studying protein secretion in biomedical science.
Topics: Mass Spectrometry; Proteomics; Membrane Proteins
PubMed: 37597723
DOI: 10.1016/j.mcpro.2023.100636