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Journal of Proteome Research Jul 2023Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are...
Mass spectrometry is unmatched in its versatility for studying practically any aspect of the proteome. Because the foundations of mass spectrometry-based proteomics are complex and span multiple scientific fields, proteomics can be perceived as having a high barrier to entry. This tutorial is intended to be an accessible illustrated guide to the technical details of a relatively simple quantitative proteomic experiment. An attempt is made to explain the relevant concepts to those with limited knowledge of mass spectrometry and a basic understanding of proteins. An experimental overview is provided, from the beginning of sample preparation to the analysis of protein group quantities, with explanations of how the data are acquired, processed, and analyzed. A selection of advanced topics is briefly surveyed and works for further reading are cited. To conclude, a brief discussion of the future of proteomics is given, considering next-generation protein sequencing technologies that may complement mass spectrometry to create a fruitful future for proteomics.
Topics: Proteomics; Mass Spectrometry; Proteome; Specimen Handling
PubMed: 37260118
DOI: 10.1021/acs.jproteome.2c00838 -
Journal of Proteomics Oct 2018The enormous diversity of proteoforms produces tremendous complexity within cellular proteomes, facilitates intricate networks of molecular interactions, and constitutes... (Review)
Review
The enormous diversity of proteoforms produces tremendous complexity within cellular proteomes, facilitates intricate networks of molecular interactions, and constitutes a formidable analytical challenge for biomedical researchers. Currently, quantitative whole-proteome profiling often relies on non-targeted liquid chromatography-mass spectrometry (LC-MS), which samples proteoforms broadly, but can suffer from lower accuracy, sensitivity, and reproducibility compared with targeted LC-MS. Recent advances in bottom-up proteomics using targeted LC-MS have enabled previously unachievable identification and quantification of target proteins and posttranslational modifications within complex samples. Consequently, targeted LC-MS is rapidly advancing biomedical research, especially systems biology research in diverse areas that include proteogenomics, interactomics, kinomics, and biological pathway modeling. With the recent development of targeted LC-MS assays for nearly the entire human proteome, targeted LC-MS is positioned to enable quantitative proteomic profiling of unprecedented quality and accessibility to support fundamental and clinical research. Here we review recent applications of bottom-up proteomics using targeted LC-MS for systems biology research. SIGNIFICANCE: Advances in targeted proteomics are rapidly advancing systems biology research. Recent applications include systems-level investigations focused on posttranslational modifications (such as phosphoproteomics), protein conformation, protein-protein interaction, kinomics, proteogenomics, and metabolic and signaling pathways. Notably, absolute quantification of metabolic and signaling pathway proteins has enabled accurate pathway modeling and engineering. Integration of targeted proteomics with other technologies, such as RNA-seq, has facilitated diverse research such as the identification of hundreds of "missing" human proteins (genes and transcripts that appear to encode proteins but direct experimental evidence was lacking).
Topics: Animals; Biomedical Research; Gene Expression Profiling; Humans; Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics; Signal Transduction; Systems Biology
PubMed: 29452276
DOI: 10.1016/j.jprot.2018.02.008 -
EMBO Molecular Medicine Nov 2019Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few...
Plasma and serum are rich sources of information regarding an individual's health state, and protein tests inform medical decision making. Despite major investments, few new biomarkers have reached the clinic. Mass spectrometry (MS)-based proteomics now allows highly specific and quantitative readout of the plasma proteome. Here, we employ Plasma Proteome Profiling to define quality marker panels to assess plasma samples and the likelihood that suggested biomarkers are instead artifacts related to sample handling and processing. We acquire deep reference proteomes of erythrocytes, platelets, plasma, and whole blood of 20 individuals (> 6,000 proteins), and compare serum and plasma proteomes. Based on spike-in experiments, we determine sample quality-associated proteins, many of which have been reported as biomarker candidates as revealed by a comprehensive literature survey. We provide sample preparation guidelines and an online resource ( www.plasmaproteomeprofiling.org) to assess overall sample-related bias in clinical studies and to prevent costly miss-assignment of biomarker candidates.
Topics: Bias; Biomarkers; Female; Germany; Healthy Volunteers; Humans; Male; Plasma; Proteome; Proteomics; Specimen Handling
PubMed: 31566909
DOI: 10.15252/emmm.201910427 -
Nature Methods Mar 2023In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying... (Review)
Review
In the last decade, single-cell RNA sequencing routinely performed on large numbers of single cells has greatly advanced our understanding of the underlying heterogeneity of complex biological systems. Technological advances have also enabled protein measurements, further contributing to the elucidation of cell types and states present in complex tissues. Recently, there have been independent advances in mass spectrometric techniques bringing us one step closer to characterizing single-cell proteomes. Here we discuss the challenges of detecting proteins in single cells by both mass spectrometry and sequencing-based methods. We review the state of the art for these techniques and propose that there is a space for technological advancements and complementary approaches that maximize the advantages of both classes of technologies.
Topics: Proteomics; Mass Spectrometry; Proteome; High-Throughput Nucleotide Sequencing
PubMed: 36864196
DOI: 10.1038/s41592-023-01791-5 -
Molecular Systems Biology Mar 2022Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular...
Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.
Topics: Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics; Workflow
PubMed: 35226415
DOI: 10.15252/msb.202110798 -
Annual Review of Plant Biology May 2022Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it... (Review)
Review
Proteins are intimately involved in executing and controlling virtually all cellular processes. To understand the molecular mechanisms that underlie plant phenotypes, it is essential to investigate protein expression, interactions, and modifications, to name a few. The proteome is highly dynamic in time and space, and a plethora of protein modifications, protein interactions, and network constellations are at play under specific conditions and developmental stages. Analysis of proteomes aims to characterize the entire protein complement of a particular cell type, tissue, or organism-a challenging task, given the dynamic nature of the proteome. Modern mass spectrometry-based proteomics technology can be used to address this complexity at a system-wide scale by the global identification and quantification of thousands of proteins. In this review, we present current methods and technologies employed in mass spectrometry-based proteomics and provide examples of dynamic changes in the plant proteome elucidated by proteomic approaches.
Topics: Mass Spectrometry; Plants; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35138880
DOI: 10.1146/annurev-arplant-102620-031308 -
International Journal of Molecular... Jan 2017n/a.
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Topics: Plant Proteins; Plants; Proteome; Proteomics; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 28054969
DOI: 10.3390/ijms18010088 -
Annual Review of Genomics and Human... Aug 2022Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological... (Review)
Review
Proteins are the molecular effectors of the information encoded in the genome. Proteomics aims at understanding the molecular functions of proteins in their biological context. In contrast to transcriptomics and genomics, the study of proteomes provides deeper insight into the dynamic regulatory layers encoded at the protein level, such as posttranslational modifications, subcellular localization, cell signaling, and protein-protein interactions. Currently, mass spectrometry (MS)-based proteomics is the technology of choice for studying proteomes at a system-wide scale, contributing to clinical biomarker discovery and fundamental molecular biology. MS technologies are continuously being developed to fulfill the requirements of speed, resolution, and quantitative accuracy, enabling the acquisition of comprehensive proteomes. In this review, we present how MS technology and acquisition methods have evolved to meet the requirements of cutting-edge proteomics research, which is describing the human proteome and its dynamic posttranslational modifications with unprecedented depth. Finally, we provide a perspective on studying proteomes at single-cell resolution.
Topics: Genome; Humans; Mass Spectrometry; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 35440146
DOI: 10.1146/annurev-genom-112921-024948 -
Annual Review of Virology Sep 2021The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any... (Review)
Review
The abundance, localization, modifications, and protein-protein interactions of many host cell and virus proteins can change dynamically throughout the course of any viral infection. Studying these changes is critical for a comprehensive understanding of how viruses replicate and cause disease, as well as for the development of antiviral therapeutics and vaccines. Previously, we developed a mass spectrometry-based technique called quantitative temporal viromics (QTV), which employs isobaric tandem mass tags (TMTs) to allow precise comparative quantification of host and virus proteomes through a whole time course of infection. In this review, we discuss the utility and applications of QTV, exemplified by numerous studies that have since used proteomics with a variety of quantitative techniques to study virus infection through time.
Topics: Mass Spectrometry; Proteome; Proteomics; Viral Proteins; Viruses
PubMed: 34129369
DOI: 10.1146/annurev-virology-091919-104458 -
Methods in Molecular Biology (Clifton,... 2022Plasma and serum are rich sources of proteins that are commonly used for clinical proteome profiling and biomarkers discovery. However, high-throughput plasma proteome...
Plasma and serum are rich sources of proteins that are commonly used for clinical proteome profiling and biomarkers discovery. However, high-throughput plasma proteome profiling and quantitative analysis using mass spectrometry are challenging because of the large dynamic range of protein abundance and complexity. To overcome these challenges, we developed a convenient high-throughput workflow of depleted plasma using the 4D-Proteomics feature of the Bruker timsTOF Pro mass spectrometer with data-dependent (PASEF) and data-independent acquisition (diaPASEF) method that can potentially be used in a clinical proteome profiling and biomarker discoveries. This workflow is robust, optimal for high throughput, high proteome depth, and is reproducible. In our sample preparation steps, we used immuno-depletion steps to remove high-abundance plasma proteins, and without any further cleanup steps, we can use depleted plasma samples directly for enzymatic digestion. Immuno-depletion steps and 4D-Proteomics features of timsTOF Pro increase the plasma proteome depth, and accuracy with the identification of >800 protein groups.
Topics: Biomarkers; Blood Proteins; Plasma; Proteome; Proteomics
PubMed: 36127608
DOI: 10.1007/978-1-0716-2565-1_36