Detection Of Protein Post-translational Modifications Research Articles

Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore.

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

Multiplex Identification of Post-Translational Modifications at Point-of-Care by Deep Learning-Assisted Hydrogel Sensors.

Multiplex detection of protein post-translational modifications (PTMs), especially at point-of-care, is of great significance in cancer diagnosis. Herein, we report a machine learning-assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely-related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real-time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early-stage diagnosis of protein-related diseases.

Multiplex Identification of Post‐Translational Modifications at Point‐of‐Care by Deep Learning‐Assisted Hydrogel Sensors

AbstractMultiplex detection of protein post‐translational modifications (PTMs), especially at point‐of‐care, is of great significance in cancer diagnosis. Herein, we report a machine learning‐assisted photonic crystal hydrogel (PCH) sensor for multiplex detection of PTMs. With closely‐related PCH sensors microfabricated on a single chip, our design achieved not only rapid screening of PTMs at specific protein sites by using only naked eyes/cellphone, but also the feasibility of real‐time monitoring of phosphorylation reactions. By taking advantage of multiplex sensor chips and a neural network algorithm, accurate prediction of PTMs by both their types and concentrations was enabled. This approach was ultimately used to detect and differentiate up/down regulation of different phosphorylation sites within the same protein in live mammalian cells. Our developed method thus holds potential for POC identification of various PTMs in early‐stage diagnosis of protein‐related diseases.

Data-independent acquisition proteomics methods for analyzing post-translational modifications.

Protein post-translational modifications (PTMs) increase the functional diversity of the cellular proteome. Accurate and high throughput identification and quantification of protein PTMs is a key task in proteomics research. Recent advancements in data-independent acquisition (DIA) mass spectrometry (MS) technology have achieved deep coverage and accurate quantification of proteins and PTMs. This review provides an overview of DIA data processing methods that cover three aspects of PTMs analysis, that is, detection of PTMs, site localization, and characterization of complex modification moieties, such as glycosylation. In addition, a survey of deep learning methods that boost DIA-based PTMs analysis is presented, including in silico spectral library generation, as well as feature scoring and error rate control. The limitations and future directions of DIA methods for PTMs analysis are also discussed. Novel data analysis methods will take advantage of advanced MS instrumentation techniques to empower DIA MS for in-depth and accurate PTMs measurements.

Versatile derivatization for GC-MS and LC-MS: alkylation with trialkyloxonium tetrafluoroborates for inorganic anions, chemical warfare agent degradation products, organic acids, and proteomic analysis.

Analytical chemists resort to derivatization for improving the detection performance of certain categories of analytes. Within this context, alkylation reactions are regarded as an important asset for many methods based on GC-MS and LC-MS. Trialkyloxonium tetrafluoroborates (R[Formula: see text][BF4]-) are powerful alkylating agents with ionic liquid properties: they are nonvolatile salts soluble in water which are easier and safer to handle with respect to common alkylating agents like diazomethane. R[Formula: see text][BF4]- can perform the alkylation in both organic and aqueous media at pH conditions ranging from acidic to alkaline. Recent analytical applications of trialkyloxonium derivatizations include the high-precision determination of inorganic anions in complex matrices, the qualitative confirmation of chemical warfare agent degradation products in soils, the profiling of carboxylic acids in urine, and the detection of protein post-translational modifications induced by carbon dioxide. The common denominator for all methods presented can be found in the simplicity of the alkylation protocol which, in most of the cases, requires a single step addition of the reagent directly to the sample. Graphical Abstract Alkylation with trialkyloxonium salts for GC-MS and LC-MS analysis.

Thermal Dissociation Assay for Time-Resolved Fluorescence Detection of Protein Post-Translational Modifications.

Post-translational modifications (PTMs) of proteins provide an important mechanism for cell signal transduction control. Impaired PTM control is a key feature in multiple different disease states, and thus the enzyme-controlling PTMs have drawn attention as highly promising drug targets. Due to the importance of PTMs, various methods to monitor PTM enzyme activity have been developed, but universal high-throughput screening (HTS), a compatible method for different PTMs, remains elusive. Here, we present a homogeneous single-label thermal dissociation assay for the detection of enzymatic PTM removal. The developed method allows the use of micromolar concentration of substrate peptide, which is expected to be beneficial when monitoring enzymes with low activity and peptide binding affinity. We prove the thermal dissociation concept functionality using peptides for dephosphorylation, deacetylation, and demethylation and demonstrate the HTS-compatible flash isothermal method for PTM enzyme activity monitoring. Using specific inhibitors, we detected literature-comparable IC50 values and Z′ factors from 0.61 to 0.72, proving the HTS compatibility of the thermal peptide-break technology.

Simultaneous Quantification of the Acetylome and Succinylome by 'One-Pot' Affinity Enrichment.

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi‐PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label‐free proteomic workflow—“one‐pot” PTM enrichment—that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label‐free, data‐independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one‐pot method are identified and quantified and peak areas are shown to be highly correlated between the one‐pot and traditional single‐PTM enrichments. The ‘one‐pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single‐PTM enrichments, the one‐pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.

Sensitive Western-Blot Analysis of Azide-Tagged Protein Post Translational Modifications Using Thermoresponsive Polymer Self-Assembly.

Western-blot (WB) is a powerful analytical technique for protein identification in complex biological samples and has been widely used in biological studies for decades. Detection specificity and sensitivity of WB largely relies on quality of the antibodies and performance of the conjugated HRP. However, the application of WB analysis for the detection of protein post-translational modifications (PTMs) is hampered by the low abundance of protein PTMs and by the limited availability of antibodies that specifically differentiate various kinds of PTMs from their protein substrates. Therefore, new recognition mechanisms and signal amplification strategies for WB analysis of protein PTMs is in high demand. In this work, we prepared a soluble polymer that detects various azide-tagged PTM proteins in WB analysis using triarylphosphine and HRP modified thermoresponsive polymer. Specific and efficient detection of azide-tagged PTM protein is achieved via the bioorthogonal reaction between azide and triarylphosphine. More importantly, the chemiluminiscent signal in the WB analysis is largely amplified by the temperature induced self-assembly of numerous thermoresponsive polymer chains carrying multiple HRPs. As a result, approximately 100 times more sensitive detection than commercial antibodies is achieved by this method using standard PTM proteins. Though, this new reagent does not directly detect native PTMs in cell, tissue or blood samples, it still has important application potential in protein PTM studies, considering the wide availability of azide-tagging techniques to a variety of PTMs.

Electrochemical detection of protein post-translational modifications: Phosphorylation and nitration of amyloid-beta (1–16)

Post-translational modifications (PTM) such as O-phosphorylation of tyrosine (Tyr) and serine (Ser) and 3-nitration of Tyr amino acid residues of proteinaceous molecules were successfully detected by electrochemistry with the amyloid-beta (1–16) peptide (Aβ16) as an example. The detection strategy was based on the effects of Tyr or Ser O-phosphorylation and Tyr 3-nitration on electrochemical activity of the peptide molecule. Square wave voltammograms of the oxidation and reduction of Aβ16 with the unmodified residues, O-phosphorylated Tyr-10 or Ser-8 (Aβ16-pTyr10 or Aβ16-pS8) and 3-nitrated Tyr-10 (Aβ16-nTyr10) were registered on carbon screen printed electrodes within the potential ranges of 0 to 1.7 V and −0.4 to −1.0 V (vs. Ag/AgCl), respectively. The Aβ16 Tyr-10 oxidation which proceeds via the hydroxyl group of benzene ring manifested as a peak at the potential maximum of about 0.6 V. O-phosphorylation of Aβ16 Tyr-10 dramatically reduced the peak at 0.6 V and produced a new oxidation wave at potentials of 1.1–1.7 V. The oxidation process registered at these elevated positive potentials has been attributed to oxidation of the phosphoryl group of benzene ring of Aβ16 Tyr-10. The role of benzene ring in the phosphoryl group oxidation was evaluated by comparing the electrochemical behavior of Aβ16-pTyr10 and Aβ16-pSer8 peptides. The addition of nitro group at the 3rd position of benzene ring of Tyr-10 caused a shift of the oxidation peak potential maximum from 0.6 to 0.75 V. Moreover, the reduction of nitro group of Aβ16-nTyr10 leading to the amino group formation was registered at −0.75 V. The results obtained for unmodified and modified Aβ16 peptides were confirmed by the data on electrochemical behavior of the corresponding free amino acids, viz. Tyr, nTyr, pTyr, and pSer. Each PTM studied was found to strongly influence the electrochemical properties of amino acids and peptides. Electrochemistry allowing to detect O-phosphorylation of Tyr and Ser and 3-nitration of Tyr residues of peptide molecules seems very promising for the study of these and, likely, many other PTM of interest in diverse in vitro systems.

Impact of Post-Translational Modifications of Crop Proteins under Abiotic Stress.

The efficiency of stress-induced adaptive responses of plants depends on intricate coordination of multiple signal transduction pathways that act coordinately or, in some cases, antagonistically. Protein post-translational modifications (PTMs) can regulate protein activity and localization as well as protein–protein interactions in numerous cellular processes, thus leading to elaborate regulation of plant responses to various external stimuli. Understanding responses of crop plants under field conditions is crucial to design novel stress-tolerant cultivars that maintain robust homeostasis even under extreme conditions. In this review, proteomic studies of PTMs in crops are summarized. Although the research on the roles of crop PTMs in regulating stress response mechanisms is still in its early stage, several novel insights have been retrieved so far. This review covers techniques for detection of PTMs in plants, representative PTMs in plants under abiotic stress, and how PTMs control functions of representative proteins. In addition, because PTMs under abiotic stresses are well described in soybeans under submergence, recent findings in PTMs of soybean proteins under flooding stress are introduced. This review provides information on advances in PTM study in relation to plant adaptations to abiotic stresses, underlining the importance of PTM study to ensure adequate agricultural production in the future.

ModProt: a database for integrating laboratory and literature data about protein post-translational modifications

Protein post-translational modifications (PTMs) play crucial roles in regulation of protein function and cell signaling, and abnormalities in protein PTMs are both causes and consequences of disease. Mass spectrometry (MS) is widely used to analyze protein PTMs. In this study, we developed an original database, ModProt (Post-Translational Modification Map of Proteome), to integrate our laboratory data and literature information regarding PTM sites. To develop the ModProt database, we constructed a web-based laboratory information management system (LIMS). This system allows us to administer the ModProt database and to view PTM site maps and corresponding protein information including amino acid sequences, official gene symbols, UniProt accessions/IDs, chromosome number/positions, and additional description. The ultimate goal of the ModProt database is to achieve PTM-based diagnosis and personalized medicine through detection of abnormal PTMs by comparing PTM site maps in healthy and disease states using the database.

Screening of protein-protein and protein-DNA interactions using microarrays: applications in biomedicine.

In this report, we focus on two different array-based technologies that enable large-scale screening of protein interactions. First, protein arrays focus on the identification of protein-protein interactions (PPIs). Second, DNA arrays have also evolved to explore the identification of protein-DNA interactions (PDIs), offering novel tools to control key biological processes. Such a tool is termed protein-binding DNA arrays (also protein-DNA arrays or protein-binding microarrays). These two array-based technologies share unrivaled screening capabilities and constitute valid approaches to address biological questions at the molecular level and, eventually, may be used in biomedical applications. Outstanding achievements of these technologies and their eventual application in biomedicine are discussed here, including the identification and characterization of biomarkers, screening of PPIs, detection of protein posttranslational modifications and biofluid profiling. Advantages and limitations of protein arrays, protein-binding arrays, and other proteomic technologies are also discussed here. Finally, we built a list of dedicated databases and on-line resources comprising updated information on human PPIs and PDIs that can serve as a toolbox for researchers in the field.

Protein Arrays: Recent Achievements and their Application to Study the Human Proteome

Protein arrays represent a step forward in proteomic technologies, as they enable to address biological and functional questions by large-scale approaches (i.e., up to several thousand proteins may be monitored in a single experiment). Here we review recent achievements of protein arrays and their classification. We also summarize current and future applications of this technology to address the study of the human proteome including protein identification, biomarker discovery, screening of protein-protein/molecule interactions, detection of protein post-translational modifications and serum profiling. Advantages and limitations of protein arrays compared to other proteomic technologies are also discussed here. The use, advances and recent applications of fluorescent microspheres as a promising alternative to planar arrays are also summarized in this report. Finally, we propose future perspectives of protein arrays from a comprehensive standpoint that includes technological, biological, economical and practical aspects.

Utilisation of Chromatographic and Electrophoretic Separation Techniques for the Detection of Protein Post-Translational Modifications

Aberrant post-translational modifications (PTMs) of proteins and the resultant disturbances of cellular signaling pathways typify many disease states. Hence the detection, localisation, and quantitation of PTMs represent important elements of disease analysis. Herein, chromatographic and electrophoretic methodology that may be exploited to first detect the protein(s) that is post-translationally modified, determine the site(s) of the PTM, quantify the stoichiometry of the modification, and then evaluate its stability is discussed. For proteins at which multiple PTMs occur, synthetic peptides that only encompass selected modification site(s) are useful for restricting analysis to specific PTMs. Suggestions for rational peptide design for studying discrete PTMs are detailed. Finally, intrinsic to these analyses of PTMs is the requirement of an evaluation of the functional outcome of the PTM.

Modification‐specific proteomics: Strategies for characterization of post‐translational modifications using enrichment techniques

More than 300 different types of protein post-translational modifications (PTMs) have been described, many of which are known to have pivotal roles in cellular physiology and disease. Nevertheless, only a handful of PTMs have been extensively investigated at the proteome level. Knowledge of protein substrates and their PTM sites is key to dissection of PTM-mediated cellular processes. The past several years have seen a tremendous progress in developing MS-based proteomics technologies for global PTM analysis, including numerous studies of yeast and other microbes. Modification-specific enrichment techniques combined with advanced MS/MS methods and computational data analysis have revealed a surprisingly large extent of PTMs in proteins, including multi-site, cooperative modifications in individual proteins. We review some of the current strategies employed for enrichment and detection of PTMs in modification-specific proteomics.