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Proteomics Apr 2024Due to their oftentimes ambiguous nature, phosphopeptide positional isomers can present challenges in bottom-up mass spectrometry-based workflows as search engine scores... (Review)
Review
Due to their oftentimes ambiguous nature, phosphopeptide positional isomers can present challenges in bottom-up mass spectrometry-based workflows as search engine scores alone are often not enough to confidently distinguish them. Additional scoring algorithms can remedy this by providing confidence metrics in addition to these search results, reducing ambiguity. Here we describe challenges to interpreting phosphoproteomics data and review several different approaches to determine sites of phosphorylation for both data-dependent and data-independent acquisition-based workflows. Finally, we discuss open questions regarding neutral losses, gas-phase rearrangement, and false localization rate estimation experienced by both types of acquisition workflows and best practices for managing ambiguity in phosphosite determination.
Topics: Phosphorylation; Mass Spectrometry; Algorithms; Search Engine; Phosphopeptides
PubMed: 37897210
DOI: 10.1002/pmic.202300088 -
Bioinformatics (Oxford, England) Aug 2023The widespread application of mass spectrometry (MS)-based proteomics in biomedical research increasingly requires robust, transparent, and streamlined solutions to...
SUMMARY
The widespread application of mass spectrometry (MS)-based proteomics in biomedical research increasingly requires robust, transparent, and streamlined solutions to extract statistically reliable insights. We have designed and implemented AlphaPeptStats, an inclusive Python package with currently with broad functionalities for normalization, imputation, visualization, and statistical analysis of label-free proteomics data. It modularly builds on the established stack of Python scientific libraries and is accompanied by a rigorous testing framework with 98% test coverage. It imports the output of a range of popular search engines. Data can be filtered and normalized according to user specifications. At its heart, AlphaPeptStats provides a wide range of robust statistical algorithms such as t-tests, analysis of variance, principal component analysis, hierarchical clustering, and multiple covariate analysis-all in an automatable manner. Data visualization capabilities include heat maps, volcano plots, and scatter plots in publication-ready format. AlphaPeptStats advances proteomic research through its robust tools that enable researchers to manually or automatically explore complex datasets to identify interesting patterns and outliers.
AVAILABILITY AND IMPLEMENTATION
AlphaPeptStats is implemented in Python and part of the AlphaPept framework. It is released under a permissive Apache license. The source code and one-click installers are freely available and on GitHub at https://github.com/MannLabs/alphapeptstats.
Topics: Proteomics; Software; Mass Spectrometry; Algorithms; Search Engine
PubMed: 37527012
DOI: 10.1093/bioinformatics/btad461 -
Chemical Communications (Cambridge,... Feb 2024Mass spectrometry imaging (MSI) has become increasingly utilized in the analysis of biological molecules. MSI grants the ability to spatially map thousands of molecules... (Review)
Review
Mass spectrometry imaging (MSI) has become increasingly utilized in the analysis of biological molecules. MSI grants the ability to spatially map thousands of molecules within one experimental run in a label-free manner. While MSI is considered by most to be a qualitative method, recent advancements in instrumentation, sample preparation, and development of standards has made quantitative MSI (qMSI) more common. In this feature article, we present a tailored review of recent advancements in qMSI of therapeutics and biomolecules such as lipids and peptides/proteins. We also provide detailed experimental considerations for conducting qMSI studies on biological samples, aiming to advance the methodology.
Topics: Mass Spectrometry; Proteins; Peptides; Diagnostic Imaging; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 38284765
DOI: 10.1039/d3cc05988j -
Molecules (Basel, Switzerland) May 2024Over the years, there has been notable progress in understanding the pathogenesis and treatment modalities of diabetes and its complications, including the application... (Review)
Review
Over the years, there has been notable progress in understanding the pathogenesis and treatment modalities of diabetes and its complications, including the application of metabolomics in the study of diabetes, capturing attention from researchers worldwide. Advanced mass spectrometry, including gas chromatography-tandem mass spectrometry (GC-MS/MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS), and ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS), etc., has significantly broadened the spectrum of detectable metabolites, even at lower concentrations. Advanced mass spectrometry has emerged as a powerful tool in diabetes research, particularly in the context of metabolomics. By leveraging the precision and sensitivity of advanced mass spectrometry techniques, researchers have unlocked a wealth of information within the metabolome. This technology has enabled the identification and quantification of potential biomarkers associated with diabetes and its complications, providing new ideas and methods for clinical diagnostics and metabolic studies. Moreover, it offers a less invasive, or even non-invasive, means of tracking disease progression, evaluating treatment efficacy, and understanding the underlying metabolic alterations in diabetes. This paper summarizes advanced mass spectrometry for the application of metabolomics in diabetes mellitus, gestational diabetes mellitus, diabetic peripheral neuropathy, diabetic retinopathy, diabetic nephropathy, diabetic encephalopathy, diabetic cardiomyopathy, and diabetic foot ulcers and organizes some of the potential biomarkers of the different complications with the aim of providing ideas and methods for subsequent in-depth metabolic research and searching for new ways of treating the disease.
Topics: Humans; Biomarkers; Metabolomics; Diabetes Mellitus; Diabetes Complications; Tandem Mass Spectrometry; Mass Spectrometry; Animals
PubMed: 38893405
DOI: 10.3390/molecules29112530 -
BioTechniques Apr 2024Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein-protein interactions when genome-wide, affinity-tagged cell collections are used. Such...
Immunoprecipitation (IP) coupled with mass spectrometry effectively maps protein-protein interactions when genome-wide, affinity-tagged cell collections are used. Such studies have recorded significant portions of the compositions of physiological protein complexes, providing draft 'interactomes'; yet many constituents of protein complexes still remain uncharted. This gap exists partly because high-throughput approaches cannot optimize each IP. A key challenge for IP optimization is stabilizing interactions during the transfer from cells to test tubes; failure to do so leads to the loss of genuine interactions during the IP and subsequent failure to detect. Our high-content screening method explores the relationship between chemical conditions and IP outcomes, enabling rapid empirical optimization of conditions for capturing target macromolecular assemblies.
Topics: Mass Spectrometry; Immunoprecipitation
PubMed: 38425263
DOI: 10.2144/btn-2023-0051 -
Journal of Proteome Research Sep 2023Missing values are a notable challenge when analyzing mass spectrometry-based proteomics data. While the field is still actively debating the best practices, the... (Review)
Review
Missing values are a notable challenge when analyzing mass spectrometry-based proteomics data. While the field is still actively debating the best practices, the challenge increased with the emergence of mass spectrometry-based single-cell proteomics and the dramatic increase in missing values. A popular approach to deal with missing values is to perform imputation. Imputation has several drawbacks for which alternatives exist, but currently, imputation is still a practical solution widely adopted in single-cell proteomics data analysis. This perspective discusses the advantages and drawbacks of imputation. We also highlight 5 main challenges linked to missing value management in single-cell proteomics. Future developments should aim to solve these challenges, whether it is through imputation or data modeling. The perspective concludes with recommendations for reporting missing values, for reporting methods that deal with missing values, and for proper encoding of missing values.
Topics: Proteomics; Mass Spectrometry; Single-Cell Analysis; Algorithms
PubMed: 37530557
DOI: 10.1021/acs.jproteome.3c00227 -
International Journal of Molecular... Dec 2023Metformin is the most prescribed glucose-lowering drug worldwide; globally, over 100 million patients are prescribed this drug annually. Some different action mechanisms...
Metformin is the most prescribed glucose-lowering drug worldwide; globally, over 100 million patients are prescribed this drug annually. Some different action mechanisms have been proposed for this drug, but, surprisingly, no metabolite of metformin has ever been described. It was considered interesting to investigate the possible reaction of metformin with glucose following the Maillard reaction pattern. The reaction was first performed in in vitro conditions, showing the formation of two adducts that originated by the condensation of the two molecular species with the losses of one or two water molecules. Their structures were investigated by liquid chromatography coupled with mass spectrometry (HPLC-MS), tandem mass spectrometry (MS/MS) and accurate mass measurements (HRMS). The species originated via the reaction of glucose and metformin and were called and , and some structural hypotheses were conducted. It is worth to emphasize that they were detected in urine samples from a diabetic patient treated with metformin and consequently they must be considered metabolites of the drug, which has never been identified before now. The glucose-related substructure of these compounds could reflect an improved transfer across cell membranes and, consequently, new hypotheses could be made about the biological targets of metformin.
Topics: Humans; Metformin; Tandem Mass Spectrometry; Liquid Chromatography-Mass Spectrometry; Cell Membrane; Glucose
PubMed: 38203351
DOI: 10.3390/ijms25010180 -
Methods in Molecular Biology (Clifton,... 2024Mass spectrometry is a high throughput technique widely used for metabolic fingerprinting of plant material. Among the diverse plant metabolites, pigments such as...
Mass spectrometry is a high throughput technique widely used for metabolic fingerprinting of plant material. Among the diverse plant metabolites, pigments such as anthocyanins play a determinant role in plant defence mechanisms, protecting them from biotic and abiotic stresses. Anthocyanins are phenolic water-soluble glycosides or acyl-glycosides of anthocyanidins which could be accurately detected and quantified through mass spectrometry. This chapter describes how to extract anthocyanins from higher plant materials and quantify them through a liquid chromatography-mass spectrometry (LC-MS) based method.
Topics: Anthocyanins; Tandem Mass Spectrometry; Glycosides; Liquid Chromatography-Mass Spectrometry; Phenols
PubMed: 38587741
DOI: 10.1007/978-1-0716-3826-2_10 -
Journal of the American Society For... Aug 2023Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups...
Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragments─similarly to that in ES+─along with y-NH/z, c, c-NH/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages -terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage -terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.
Topics: Tandem Mass Spectrometry; Citrulline; Spectrometry, Mass, Electrospray Ionization; Peptides; Anions; Ions
PubMed: 37414397
DOI: 10.1021/jasms.3c00044 -
Food Research International (Ottawa,... Sep 2023Food inspection covers a broad range of topics, including nutrient analysis, food pollutants, food auxiliary materials, additives, and food sensory identification. The... (Review)
Review
BACKGROUND
Food inspection covers a broad range of topics, including nutrient analysis, food pollutants, food auxiliary materials, additives, and food sensory identification. The foundation of diverse subjects like food science, nutrition, health research, and the food industry, as well as the desired reference for drafting trade and food legislation, makes food inspection highly significant. Because of their high efficiency, sensitivity, and accuracy, instrumental analysis methods have gradually replaced conventional analytical methods as the primary means of food hygiene inspection.
SCOPE AND APPROACH
Metabolomics-based analysis technology, such as nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and capillary electrophoresis-mass spectrometry (CE-MS), has become a widely used analytics platform. This research provides a bird's eye view of the application and future of metabolomics-related technologies in food inspection.
KEY FINDINGS AND CONCLUSIONS
We have provided a summary of the features and the application range of various metabolomics techniques, the strengths and weaknesses of different metabolomics platforms, and their implementation in specific inspection procedures. These procedures encompass the identification of endogenous metabolites, the detection of exogenous toxins and food additives, analysis of metabolite alterations during processing and storage, as well as the recognition of food adulteration. Despite the widespread utilization and significant contributions of metabolomics-based food inspection technologies, numerous challenges persist as the food industry advances and technology continues to improve. Thus, we anticipate addressing these potential issues in the future.
Topics: Humans; Food Inspection; Metabolomics; Gas Chromatography-Mass Spectrometry; Mass Spectrometry; Technology
PubMed: 37330829
DOI: 10.1016/j.foodres.2023.113071