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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 -
Methods in Molecular Biology (Clifton,... 2023Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental...
Mass spectrometry-based proteomics provide a powerful tool for plant research, allowing global detection of steady-state levels of proteins under a given experimental setup. Here, we provide an optimized protocol for proteomic profiling using tandem mass tag (TMT) labeling followed by liquid chromatography-mass spectrometry (LC-MS/MS) to quantitate phosphopeptides and non-phosphopeptides from the same samples. The outlined protocol comprises a series of successive steps, namely, SDS (sodium dodecyl sulfate) protein extraction, protein precipitation, digestion, TMT labeling, phosphopeptide enrichment, high pH reversed-phase fractionation, LC-MS/MS analysis, protein identification, and data analysis. Our proteome-scale protocol requires 0.1 mg protein per sample and allows for the reliable and accurate quantification of more than 8000 proteins in Arabidopsis plant samples across multiple conditions, including low abundant peptides.
Topics: Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Phosphopeptides; Proteome
PubMed: 36413327
DOI: 10.1007/978-1-0716-2784-6_22 -
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 -
Journal of Agricultural and Food... Aug 2022Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and...
Barley is one of the key cereal grains for malting and brewing industries. However, climate variability and unprecedented weather events can impact barley yield and end-product quality. The genetic background and environmental conditions are key factors in defining the barley proteome content and malting characteristics. Here, we measure the barley proteome and malting characteristics of three barley lines grown in Western Australia, differing in genetic background and growing location, by applying liquid chromatography-mass spectrometry (LC-MS). Using data-dependent acquisition LC-MS, 1571 proteins were detected with high confidence. Quantitative data acquired using sequential window acquisition of all theoretical (SWATH) MS on barley samples resulted in quantitation of 920 proteins. Multivariate analyses revealed that the barley lines' genetics and their growing locations are strongly correlated between proteins and desired traits such as the malt yield. Linking meteorological data with proteomic measurements revealed how high-temperature stress in northern regions affects seed temperature tolerance during malting, resulting in a higher malt yield. Our results show the impact of environmental conditions on the barley proteome and malt characteristics; these findings have the potential to expedite breeding programs and malt quality prediction.
Topics: Hordeum; Phenotype; Plant Breeding; Proteome; Proteomics
PubMed: 35981222
DOI: 10.1021/acs.jafc.2c03816 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2019Detailed investigation of the neural crest cell proteome has lingered behind that of the transcriptome due to the challenge of obtaining sufficient starting material for...
Detailed investigation of the neural crest cell proteome has lingered behind that of the transcriptome due to the challenge of obtaining sufficient starting material for subsequent proteomic investigation. Compounded by the complexity of protein abundance, large number of posttranslational modifications, and the stochastic nature of proteomics approaches, little data so far exists describing the true neural crest cell proteome. However, recent advances in instrument sensitivity and recovery of material during sample preparation have alleviated many of these problems and make proteomics analysis an underutilized tool to study neural crest cell biology. Here we present a quantitative proteomics protocol for deep analysis of both whole proteome and posttranslational modifications and a separate protocol for ultrasensitive proteomic profiling from submicrogram amounts of protein.
Topics: Animals; Humans; Neural Crest; Protein Processing, Post-Translational; Proteome; Proteomics
PubMed: 30977072
DOI: 10.1007/978-1-4939-9412-0_12 -
Methods in Molecular Biology (Clifton,... 2022Protein glycosylation is increasingly recognized as a common class of modifications within microbial species that can shape protein functions and the proteome at large....
Protein glycosylation is increasingly recognized as a common class of modifications within microbial species that can shape protein functions and the proteome at large. Due to this, there is an increasing need for robust analytical methods, which allow for the identification and characterization of microbial glycopeptides from proteome samples in a high-throughput manner. Using affinity-based enrichment (either hydrophilicity or antibody-based approaches) glycopeptides can easily be separated from non-glycosylated peptides and analyzed using mass spectrometry. By utilizing multiple mass spectrometry fragmentation approaches and open searching-based bioinformatic techniques, novel glycopeptides can be identified and characterized without prior knowledge of the glycans used for glycosylation. Using these approaches, glycopeptides within samples can rapidly be identified as well as quantified to understand how glycosylation changes in response to stimuli or how changes in glycosylation systems impact the glycoproteome. This chapter outlines a set of robust protocols for the initial preparation, enrichment, and analysis of microbial glycopeptides for both qualitative and quantitative glycoproteomic studies. Using these approaches, glycosylation events can be easily identified by researchers without the need for extensive manual analysis of proteomic datasets.
Topics: Glycopeptides; Glycosylation; Mass Spectrometry; Proteome; Proteomics
PubMed: 35612741
DOI: 10.1007/978-1-0716-2124-0_11 -
Journal of Proteome Research Dec 2022Isobaric labeling-based proteomics is widely applied in deep proteome quantification. Among the platforms for isobaric labeled proteomic data analysis, the commercial...
Isobaric labeling-based proteomics is widely applied in deep proteome quantification. Among the platforms for isobaric labeled proteomic data analysis, the commercial software Proteome Discoverer (PD) is widely used, incorporating the search engine CHIMERYS, while FragPipe (FP) is relatively new, free for noncommercial purposes, and integrates the engine MSFragger. Here, we compared PD and FP over three public proteomic data sets labeled using 6plex, 10plex, and 16plex tandem mass tags. Our results showed the protein abundances generated by the two software are highly correlated. PD quantified more proteins (10.02%, 15.44%, 8.19%) than FP with comparable NA ratios (0.00% vs. 0.00%, 0.85% vs. 0.38%, and 11.74% vs. 10.52%) in the three data sets. Using the 16plex data set, PD and FP outputs showed high consistency in quantifying technical replicates, batch effects, and functional enrichment in differentially expressed proteins. However, FP saved 93.93%, 96.65%, and 96.41% of processing time compared to PD for analyzing the three data sets, respectively. In conclusion, while PD is a well-maintained commercial software integrating various additional functions and can quantify more proteins, FP is freely available and achieves similar output with a shorter computational time. Our results will guide users in choosing the most suitable quantification software for their needs.
Topics: Proteome; Proteomics; Tandem Mass Spectrometry; Software
PubMed: 36315902
DOI: 10.1021/acs.jproteome.2c00390 -
Proteomics Dec 2022As the common and significant chemical modifications, post-translational modifications (PTMs) play a key role in the functional proteome. Affected by the signal... (Review)
Review
As the common and significant chemical modifications, post-translational modifications (PTMs) play a key role in the functional proteome. Affected by the signal interference, low concentration, and insufficient ionization efficiency of impurities, the direct detection of PTMs by mass spectrometry (MS) still faces many challenges. Therefore, sample preparation and enrichment are an indispensable link before MS analysis of PTMs in proteomics. The rapid development of functionalized materials with diverse morphologies and compositions provides an avenue for sample preparation and enrichment for PTMs analysis. In this review, we summarize recent advances in the application of novel functionalized materials in sample preparation for phosphoproteomes and glycoproteomes analysis. In addition, this review specifically discusses the design and preparation of functionalized materials based on different enrichment mechanisms, and proposes research directions and potential challenges for proteomic PTMs research.
Topics: Proteomics; Phosphorylation; Glycosylation; Proteome; Protein Processing, Post-Translational
PubMed: 36100958
DOI: 10.1002/pmic.202200070