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Cell Reports Methods Oct 2023Here, we present a standardized, "off-the-shelf" proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high...
Here, we present a standardized, "off-the-shelf" proteomics pipeline working in a single 96-well plate to achieve deep coverage of cellular proteomes with high throughput and scalability. This integrated pipeline streamlines a fully automated sample preparation platform, a data-independent acquisition (DIA) coupled with high-field asymmetric waveform ion mobility spectrometer (FAIMS) interface, and an optimized library-free DIA database search strategy. Our systematic evaluation of FAIMS-DIA showing single compensation voltage (CV) at -35 V not only yields the deepest proteome coverage but also best correlates with DIA without FAIMS. Our in-depth comparison of direct-DIA database search engines shows that Spectronaut outperforms others, providing the highest quantifiable proteins. Next, we apply three common DIA strategies in characterizing human induced pluripotent stem cell (iPSC)-derived neurons and show single-shot mass spectrometry (MS) using single-CV (-35 V)-FAIMS-DIA results in >9,000 quantifiable proteins with <10% missing values, as well as superior reproducibility and accuracy compared with other existing DIA methods.
Topics: Humans; Proteomics; Tandem Mass Spectrometry; Reproducibility of Results; Induced Pluripotent Stem Cells; Proteome
PubMed: 37729920
DOI: 10.1016/j.crmeth.2023.100593 -
Emerging Topics in Life Sciences May 2021Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In... (Review)
Review
Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In particular, protein post-translational signaling and protein-protein interactions combine to manipulate cellular responses and regulate plant homeostasis with precise temporal and spatial control. Understanding these proteomic networks are essential to addressing ongoing global crises, including those of food security, rising global temperatures, and the need for renewable materials and fuels. Technological advances in mass spectrometry-based proteomics are enabling investigations of unprecedented depth, and are increasingly being optimized for and applied to plant systems. This review highlights recent advances in plant proteomics, with an emphasis on spatially and temporally resolved analysis of post-translational modifications and protein interactions. It also details the necessity for generation of a comprehensive plant cell atlas while highlighting recent accomplishments within the field.
Topics: Mass Spectrometry; Plants; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 33620075
DOI: 10.1042/ETLS20200270 -
Genomics, Proteomics & Bioinformatics Oct 2021In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in... (Review)
Review
In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.
Topics: Proteome; Proteomics; Reproducibility of Results; Tandem Mass Spectrometry
PubMed: 35007772
DOI: 10.1016/j.gpb.2021.08.012 -
Nucleic Acids Research Jan 2022Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric...
Proteome-pI 2.0 is an update of an online database containing predicted isoelectric points and pKa dissociation constants of proteins and peptides. The isoelectric point-the pH at which a particular molecule carries no net electrical charge-is an important parameter for many analytical biochemistry and proteomics techniques. Additionally, it can be obtained directly from the pKa values of individual charged residues of the protein. The Proteome-pI 2.0 database includes data for over 61 million protein sequences from 20 115 proteomes (three to four times more than the previous release). The isoelectric point for proteins is predicted by 21 methods, whereas pKa values are inferred by one method. To facilitate bottom-up proteomics analysis, individual proteomes were digested in silico with the five most commonly used proteases (trypsin, chymotrypsin, trypsin + LysC, LysN, ArgC), and the peptides' isoelectric point and molecular weights were calculated. The database enables the retrieval of virtual 2D-PAGE plots and customized fractions of a proteome based on the isoelectric point and molecular weight. In addition, isoelectric points for proteins in NCBI non-redundant (nr), UniProt, SwissProt, and Protein Data Bank are available in both CSV and FASTA formats. The database can be accessed at http://isoelectricpointdb2.org.
Topics: Amino Acid Sequence; Computational Biology; Databases, Protein; Electrophoresis, Gel, Two-Dimensional; Isoelectric Point; Molecular Weight; Peptides; Proteome; Proteomics
PubMed: 34718696
DOI: 10.1093/nar/gkab944 -
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 -
Expert Review of Proteomics Jun 2021Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast... (Review)
Review
INTRODUCTION
Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples.
AREAS COVERED
We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented.
EXPERT OPINION
The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.
Topics: Extracellular Matrix; Fibroblasts; Humans; Mass Spectrometry; Proteome; Proteomics
PubMed: 34129411
DOI: 10.1080/14789450.2021.1941893 -
Analytical Chemistry Jul 2022Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample...
Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.
Topics: Digestion; Peptides; Proteome; Proteomics; Water
PubMed: 35817413
DOI: 10.1021/acs.analchem.1c05487 -
Electrophoresis Dec 2012While neurovascular diseases such as ischemic and hemorrhagic stroke are the leading causes of disability in the world, the repertoire of therapeutic interventions has... (Review)
Review
While neurovascular diseases such as ischemic and hemorrhagic stroke are the leading causes of disability in the world, the repertoire of therapeutic interventions has remained remarkably limited. There is a dire need to develop new diagnostic, prognostic, and therapeutic options. The study of proteomics is particularly enticing for cerebrovascular diseases such as stroke, which most likely involve multiple gene interactions resulting in a wide range of clinical phenotypes. Currently, rapidly progressing neuroproteomic techniques have been employed in clinical and translational research to help identify biologically relevant pathways, to understand cerebrovascular pathophysiology, and to develop novel therapeutics and diagnostics. Future integration of proteomic with genomic, transcriptomic, and metabolomic studies will add new perspectives to better understand the complexities of neurovascular injury. Here, we review cerebrovascular proteomics research in both preclinical (animal, cell culture) and clinical (blood, urine, cerebrospinal fluid, microdialyates, tissue) studies. We will also discuss the rewards, challenges, and future directions for the application of proteomics technology to the study of various disease phenotypes. To capture the dynamic range of cerebrovascular injury and repair with a translational targeted and discovery approach, we emphasize the importance of complementing innovative proteomic technology with existing molecular biology models in preclinical studies, and the need to advance pharmacoproteomics to directly probe clinical physiology and gauge therapeutic efficacy at the bedside.
Topics: Animals; Biomarkers; Cerebrovascular Disorders; Humans; Mass Spectrometry; Nerve Tissue Proteins; Proteome; Proteomics
PubMed: 23161401
DOI: 10.1002/elps.201200481 -
Molecular & Cellular Proteomics : MCP Sep 2023Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and...
Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population of incompletely differentiated immune cells. They are known to suppress T cell activity and are implicated in multiple chronic diseases, which make them an attractive cell population for drug discovery. Here, we characterized the baseline proteomes and phospho-proteomes of mouse MDSC differentiated from a progenitor cell line to a depth of 7000 proteins and phosphorylation sites. We also validated the cellular system for drug discovery by recapitulating and identifying known and novel molecular responses to the well-studied MDSC drugs entinostat and mocetinostat. We established a high-throughput drug screening platform using a MDSC/T cell coculture system and assessed the effects of ∼21,000 small molecule compounds on T cell proliferation and IFN-γ secretion to identify novel MDSC modulator. The most promising candidates were validated in a human MDSC system, and subsequent proteomic experiments showed significant upregulation of several proteins associated with the reduction of reactive oxygen species (ROS). Proteome-wide solvent-induced protein stability assays identified Acyp1 and Cd74 as potential targets, and the ROS-reducing drug phenotype was validated by measuring ROS levels in cells in response to compound, suggesting a potential mode of action. We anticipate that the data and chemical tools developed in this study will be valuable for further research on MDSC and related drug discovery.
Topics: Mice; Humans; Animals; Myeloid-Derived Suppressor Cells; High-Throughput Screening Assays; Proteome; Proteomics; Reactive Oxygen Species
PubMed: 37586548
DOI: 10.1016/j.mcpro.2023.100632 -
Molecular Systems Biology Jul 2021While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of...
While informative, protein amounts and physical protein associations do not provide a full picture of protein function. This Commentary highlights the potential of structural and stability proteomic technologies to derive new insights in biology and medicine.
Topics: Biophysics; Proteome; Proteomics
PubMed: 34293219
DOI: 10.15252/msb.202110442