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Journal of Chromatography. A Nov 2017Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics,... (Review)
Review
Microscale separation (e.g., liquid chromatography or capillary electrophoresis) coupled with mass spectrometry (MS) has become the primary tool for advanced proteomics, an indispensable technology for gaining understanding of complex biological processes. In recent decades significant advances have been achieved in MS-based proteomics. However, the current proteomics platforms still face an analytical challenge in overall sensitivity towards nanoproteomics applications for starting materials of less than 1μg total proteins (e.g., cellular heterogeneity in tissue pathologies). Herein, we review recent advances in microscale separation techniques and integrated sample processing strategies that improve the overall sensitivity and proteome coverage of the proteomics workflow, and their contributions towards nanoproteomics applications.
Topics: Chromatography, Liquid; Electrophoresis, Capillary; Mass Spectrometry; Proteome; Proteomics
PubMed: 28765000
DOI: 10.1016/j.chroma.2017.07.055 -
Proteomics. Clinical Applications Feb 2015Owing to recent advances in proteomics analytical methods and bioinformatics capabilities there is a growing trend toward using these capabilities for the development of... (Review)
Review
Owing to recent advances in proteomics analytical methods and bioinformatics capabilities there is a growing trend toward using these capabilities for the development of drugs to treat human disease, including target and drug evaluation, understanding mechanisms of drug action, and biomarker discovery. Currently, the genetic sequences of many major organisms are available, which have helped greatly in characterizing proteomes in model animal systems and humans. Through proteomics, global profiles of different disease states can be characterized (e.g. changes in types and relative levels as well as changes in PTMs such as glycosylation or phosphorylation). Although intracellular proteomics can provide a broad overview of physiology of cells and tissues, it has been difficult to quantify the low abundance proteins which can be important for understanding the diseased states and treatment progression. For this reason, there is increasing interest in coupling comparative proteomics methods with subcellular fractionation and enrichment techniques for membranes, nucleus, phosphoproteome, glycoproteome as well as low abundance serum proteins. In this review, we will provide examples of where the utilization of different proteomics-coupled enrichment techniques has aided target and biomarker discovery, understanding the drug targeting mechanism, and mAb discovery. Taken together, these improvements will help to provide a better understanding of the pathophysiology of various diseases including cancer, autoimmunity, inflammation, cardiovascular disease, and neurological conditions, and in the design and development of better medicines for treating these afflictions.
Topics: Animals; Chromatography, Affinity; Disease; Humans; Proteins; Proteome; Proteomics
PubMed: 25523641
DOI: 10.1002/prca.201400097 -
Physiological Research 2014Teeth have been a focus of interest for many centuries--due to medical problems with them. They are the hardest part of the human body and are composed of three... (Review)
Review
Teeth have been a focus of interest for many centuries--due to medical problems with them. They are the hardest part of the human body and are composed of three mineralized parts--enamel, dentin and cementum, together with the soft pulp. However, saliva also has a significant impact on tooth quality. Proteomic research of human teeth is now accelerating, and it includes all parts of the tooth. Some methodological problems still need to be overcome in this research field--mainly connected with calcified tissues. This review will provide an overview of the current state of research with focus on the individual parts of the tooth and pellicle layer as well as saliva. These proteomic results can help not only stomatology in terms of early diagnosis, identifying risk factors, and systematic control.
Topics: Gene Expression Profiling; Humans; Proteome; Proteomics; Saliva; Tissue Distribution; Tooth
PubMed: 24564654
DOI: 10.33549/physiolres.932702 -
In Vivo (Athens, Greece) 2017Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a... (Review)
Review
Despite the explosive increase in the use of Assisted Reproductive Technologies (ART) over the last 30 years, their success rates remain suboptimal. Proteomics is a rapidly-evolving technology-driven science that has already been widely applied in the exploration of human reproduction and fertility, providing useful insights into its physiology and leading to the identification of numerous proteins that may be potential biomarkers and/or treatment targets of a successful ART pregnancy. Here we present a brief overview of the techniques used in proteomic analyses and attempt a comprehensive presentation of recent data from mass spectrometry-based proteomic studies in humans, regarding all components of ARTs, including the male and female gamete, the derived zygote and embryo, the endometrium and, finally, the ART offspring both pre- and postnatally.
Topics: Animals; Biomarkers; Humans; Proteome; Proteomics; Reproduction; Reproductive Techniques, Assisted
PubMed: 28438852
DOI: 10.21873/invivo.11056 -
Biochimica Et Biophysica Acta. Proteins... Jul 2021Single-cell analysis came to change the way we look at cell populations. RNA sequencing of single cells allowed us to appreciate the diversity of cell types in the human... (Review)
Review
Single-cell analysis came to change the way we look at cell populations. RNA sequencing of single cells allowed us to appreciate the diversity of cell types in the human brain in an unprecedented manner and its power to reveal cell-type specific changes in cell populations has just begun to be explored. In this context, looking at the proteome of single cells promises to bring functional information and contribute to completing the picture. The potential of single cell proteome, in developing a better understanding of the intricate connections between the very diverse cell populations in the brain, is huge. Whereas early approaches to address single-cell proteome have identified hundreds of proteins, today, techniques combining isobaric labelling and LC-MS can lead to the identification of thousands of proteins. In this review, we describe methods which have been used to identify and quantify proteins from single cells and propose that the application of isobaric labeling and label-free quantitative proteomics approach for single-cell analysis is ready to provide useful information for the neurobiology field.
Topics: Animals; Chromatography, Liquid; Humans; Neurobiology; Proteome; Proteomics; Single-Cell Analysis; Tandem Mass Spectrometry
PubMed: 33845200
DOI: 10.1016/j.bbapap.2021.140658 -
Molecular & Cellular Proteomics : MCP Sep 2023Data-independent acquisition (DIA) mass spectrometry-based proteomics generates reproducible proteome data. The complex processing of the DIA data has led to the...
Data-independent acquisition (DIA) mass spectrometry-based proteomics generates reproducible proteome data. The complex processing of the DIA data has led to the development of multiple data analysis tools. In this study, we assessed the performance of five tools (OpenSWATH, EncyclopeDIA, Skyline, DIA-NN, and Spectronaut) using six DIA datasets obtained from TripleTOF, Orbitrap, and TimsTOF Pro instruments. By comparing identification and quantification metrics and examining shared and unique cross-tool identifications, we evaluated both library-based and library-free approaches. Our findings indicate that library-free approaches outperformed library-based methods when the spectral library had limited comprehensiveness. However, our results also suggest that constructing a comprehensive library still offers benefits for most DIA analyses. This study provides comprehensive guidance for DIA data analysis tools, benefiting both experienced and novice users of DIA-mass spectrometry technology.
Topics: Mass Spectrometry; Proteomics; Proteome; Gene Library; Data Analysis
PubMed: 37481071
DOI: 10.1016/j.mcpro.2023.100623 -
Methods in Molecular Biology (Clifton,... 2022Top-down proteomics methods have a distinct advantage over bottom-up methods in that they analyze intact proteins rather than digested peptides which can result in loss...
Top-down proteomics methods have a distinct advantage over bottom-up methods in that they analyze intact proteins rather than digested peptides which can result in loss of information regarding the intact protein. However, the analysis of intact proteins using top-down proteomics methods has been impeded by the low resolution of typical separation approaches applied in bottom-up proteomics studies. To increase the coverage of intact proteomes, orthogonal, two-dimensional separation techniques have been developed to improve the separation efficiency; in this chapter, we describe a two-dimensional HPLC separation technique that utilizes a high-pH mobile phase in the first dimension followed by a low-pH mobile phase in the second dimension. This two-dimensional pH-based HPLC approach demonstrates increased separation efficiency of intact proteins and increased proteome coverage when compared to one-dimensional HPLC in the analysis of larger and lower abundance proteoforms.
Topics: Chromatography, High Pressure Liquid; Peptides; Proteome; Proteomics; Tandem Mass Spectrometry
PubMed: 35657585
DOI: 10.1007/978-1-0716-2325-1_4 -
Proteomics Oct 2022Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine... (Review)
Review
Mass spectrometry (MS) has emerged at the forefront of quantitative proteomic techniques. Liquid chromatography-mass spectrometry (LC-MS) can be used to determine abundances of proteins and peptides in complex biological samples. Several methods have been developed and adapted for accurate quantification based on chemical isotopic labeling. Among various chemical isotopic labeling techniques, isobaric tagging approaches rely on the analysis of peptides from MS2-based quantification rather than MS1-based quantification. In this review, we will provide an overview of several isobaric tags along with some recent developments including complementary ion tags, improvements in sensitive quantitation of analytes with lower abundance, strategies to increase multiplexing capabilities, and targeted analysis strategies. We will also discuss limitations of isobaric tags and approaches to alleviate these restrictions through bioinformatic tools and data acquisition methods. This review will highlight several applications of isobaric tags, including biomarker discovery and validation, thermal proteome profiling, cross-linking for structural investigations, single-cell analysis, top-down proteomics, along with applications to different molecules including neuropeptides, glycans, metabolites, and lipids, while providing considerations and evaluations to each application.
Topics: Proteomics; Proteome; Tandem Mass Spectrometry; Isotope Labeling; Peptides; Biomarkers; Lipids
PubMed: 35687565
DOI: 10.1002/pmic.202100256 -
Nature Communications Aug 2022Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein...
Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.
Topics: Esters; Nuclear Proteins; Proteome; Proteomics; Transcription Factors; Ubiquitin-Protein Ligases
PubMed: 35987950
DOI: 10.1038/s41467-022-32689-z -
Cell Systems Nov 2023Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell...
Spatial proteomics combining microscopy-based cell phenotyping with ultrasensitive mass-spectrometry-based proteomics is an emerging and powerful concept to study cell function and heterogeneity in (patho)physiology. However, optimized workflows that preserve morphological information for phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected tissue are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low-input archival material. Benchmarking in murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions. Applied to human tonsil, we profiled 146 microregions including T and B lymphocyte niches and quantified cell-type-specific markers, cytokines, and transcription factors. These data also highlighted proteome dynamics within activated germinal centers, illuminating sites undergoing B cell proliferation and somatic hypermutation. This approach has broad implications in biomedicine, including early disease profiling and drug target and biomarker discovery. A record of this paper's transparent peer review process is included in the supplemental information.
Topics: Humans; Animals; Mice; Proteome; Proteomics; Mass Spectrometry
PubMed: 37909047
DOI: 10.1016/j.cels.2023.10.003