Quantitation Of Post-translational Modifications Research Articles

Differential Abundance of Protein Acylation in Mycobacterium tuberculosis Under Exposure to Nitrosative Stress.

Human macrophages generate antimicrobial reactive nitrogen species in response to infection by Mycobacterium tuberculosis (Mtb). Exposure to these redox-reactive compounds induces stress response in Mtb, which can affect posttranslational modifications (PTM). Here, we present the global analysis of the PTM acylation of Mtb proteins in response to a sublethal dose of nitrosative stress in the form of nitric oxide (NO) using label free quantification. A total of 6437 acylation events were identified on 1496 Mtb proteins, and O-acylation accounted for 92.2% of the events identified, while 7.8% were N-acylation events. About 22% of the sites identified were found to be acylated by more than one acyl-group. Furthermore, the abundance of each acyl-group decreased as their molecular weight increased. Quantitative PTM analysis revealed differential abundance of acylation in proteins involved in stress response, iron ion homeostasis, growth, energy metabolism, and antimicrobial resistance (AMR) induced by nitrosative stress over time. The results reveal a potential role of Mtb protein acylation in the bacterial stress responses and AMR. To our knowledge, this is the first report on global O-acylation profile of Mtb in response to NO. This will significantly improve our understanding of the changes in Mtb acylation under nitrosative stress, highly relevant for global health.

Comprehensive multi-attribute method workflow for biotherapeutic characterization and current good manufacturing practices testing.

The multi-attribute method (MAM) is a liquid chromatography-mass spectrometry (LC-MS)-based method that is used to directly characterize and monitor numerous product quality attributes (PQAs) at the amino acid level of a biopharmaceutical product. MAM enables identity testing based on primary sequence verification, detection and quantitation of post-translational modifications and impurities. This ability to simultaneously and directly determine PQAs of therapeutic proteins makes MAM a more informative, streamlined and productive workflow than conventional chromatographic and electrophoretic assays. MAM relies on proteolytic digestion of the sample followed by reversed-phase chromatographic separation and high-resolution LC-MS analysis in two phases. First, a discovery study to determine quality attributes for monitoring is followed by the creation of a targeted library based on high-resolution retention time plus accurate mass analysis. The second aspect of MAM is the monitoring phase based on the target peptide library and new peak detection using differential analysis of the data to determine the presence, absence or change of any species that might affect the activity or stability of the biotherapeutic. The sample preparation process takes between 90 and 120 min, whereas the time spent on instrumental and data analyses might vary from one to several days for different sample sizes, depending on the complexity of the molecule, the number of attributes to be monitored and the information to be detailed in the final report. MAM is developed to be used throughout the product life cycle, from process development through upstream and downstream processes to quality control release or under current good manufacturing practices regulations enforced by regulatory agencies.

QPTMplants: an integrative database of quantitative post-translational modifications in plants.

As a crucial molecular mechanism, post-translational modifications (PTMs) play critical roles in a wide range of biological processes in plants. Recent advances in mass spectrometry-based proteomic technologies have greatly accelerated the profiling and quantification of plant PTM events. Although several databases have been constructed to store plant PTM data, a resource including more plant species and more PTM types with quantitative dynamics still remains to be developed. In this paper, we present an integrative database of quantitative PTMs in plants named qPTMplants (http://qptmplants.omicsbio.info), which hosts 1 242 365 experimentally identified PTM events for 429 821 nonredundant sites on 123 551 proteins under 583 conditions for 23 PTM types in 43 plant species from 293 published studies, with 620 509 quantification events for 136 700 PTM sites on 55 361 proteins under 354 conditions. Moreover, the experimental details, such as conditions, samples, instruments and methods, were manually curated, while a variety of annotations, including the sequence and structural characteristics, were integrated into qPTMplants. Then, various search and browse functions were implemented to access the qPTMplants data in a user-friendly manner. Overall, we anticipate that the qPTMplants database will be a valuable resource for further research on PTMs in plants.

Fast and efficient digestion of adeno associated virus (AAV) capsid proteins for liquid chromatography mass spectrometry (LC-MS) based peptide mapping and post translational modification analysis (PTMs)

Adeno-associated virus (AAV) represent a widely used delivery mechanism for gene therapy treatments currently being developed. The size and complexity of these molecules requires the development of sensitive analytical methods for detailed product characterization. Among the quality attributes that need to be monitored, characterization of the AAV capsid protein amino acid sequences and any associated post translational modifications (PTM) present, should be performed. As commonly used for recombinant protein analysis, LC-MS based peptide mapping can provide sequence coverage and PTM information to improve product understanding and the development and deployment of the associated manufacturing processes. In the current study, we report a fast and efficient method to digest AAV5 capsid proteins in only 30 min prior to peptide mapping analysis. The performance of different proteases in digesting AAV5 was compared and the benefits of using nanoflow liquid chromatography for separation prior to high resolution mass spectrometry to obtain 100% sequence coverage are highlighted. Characterization and quantitation of PTMs on AAV5 capsid proteins when using pepsin as a single protease is reported, thereby demonstrating the potential of this method to aid with complete characterization of AAV serotypes in gene therapy development laboratories.

Isotopically Dimethyl Labeling-Based Quantitative Proteomic Analysis of Phosphoproteomes of Soybean Cultivars.

Isotopically dimethyl labeling was applied in a quantitative post-translational modification (PTM) proteomic study of phosphoproteomic changes in the drought responses of two contrasting soybean cultivars. A total of 9457 phosphopeptides were identified subsequently, corresponding to 4571 phosphoprotein groups and 3889 leading phosphoproteins, which contained nine kinase families consisting of 279 kinases. These phosphoproteins contained a total of 8087 phosphosites, 6106 of which were newly identified and constituted 54% of the current soybean phosphosite repository. These phosphosites were converted into the highly conserved kinase docking sites by bioinformatics analysis, which predicted six kinase families that matched with those newly found nine kinase families. The overly post-translationally modified proteins (OPP) occupies 2.1% of these leading phosphoproteins. Most of these OPPs are photoreceptors, mRNA-, histone-, and phospholipid-binding proteins, as well as protein kinase/phosphatases. The subgroup population distribution of phosphoproteins over the number of phosphosites of phosphoproteins follows the exponential decay law, Y = 4.13e−0.098X − 0.04. Out of 218 significantly regulated unique phosphopeptide groups, 188 phosphoproteins were regulated by the drought-tolerant cultivar under the water loss condition. These significantly regulated phosphoproteins (SRP) are mainly enriched in the biological functions of water transport and deprivation, methionine metabolic processes, photosynthesis/light reaction, and response to cadmium ion, osmotic stress, and ABA response. Seventeen and 15 SRPs are protein kinases/phosphatases and transcription factors, respectively. Bioinformatics analysis again revealed that three members of the calcium dependent protein kinase family (CAMK family), GmSRK2I, GmCIPK25, and GmAKINβ1 kinases, constitute a phosphor-relay-mediated signal transduction network, regulating ion channel activities and many nuclear events in this drought-tolerant cultivar, which presumably contributes to the development of the soybean drought tolerance under water deprivation process.

Functional analysis tools for post-translational modification: a post-translational modification database for analysis of proteins and metabolic pathways.

Post-translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high-resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post-Translational Modifications (FAT-PTM) database (https://bioinformatics.cse.unr.edu/fat-ptm/), which currently supports eight different types of PTM and over 49000 PTM sites identified in large-scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT-PTM database currently supports tools to visualize protein-centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein-centric metabolic pathways and groups of proteins that are co-modified by multiple PTMs. Overall, the FAT-PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.

Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis

Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied both CsCl density gradient (CDG) centrifugation-based protein fractionation and a dimethyl-labeling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl chemical labeling with chromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-based computational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, named Stable isotope-based Quantitation-Dimethyl labeling (SQUA-D), and finally the confirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7456 unambiguous acetylation sites were found from 2638 different acetylproteins, and 5250 acetylation sites, including 5233 sites on lysine side chain and 17 sites on protein N termini, were identified repetitively. Out of these repetitively discovered acetylation sites, 4228 sites on lysine side chain (i.e. 80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signaling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and 31 hormone-suppressed acetylpeptide groups or called unique PTM peptide arrays (UPAs) that share the identical unique PTM site pattern (UPSP). This CDG centrifugation protein fractionation in combination with dimethyl labeling-based quantitative PTM proteomics, and SQUA-D may be applied in the quantitation of any PTM proteins in any model eukaryotes and agricultural crops as well as tissue samples of animals and human beings.

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies.

Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)—a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits—conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit–N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

Quantitation, networking, and function of protein phosphorylation in plant cell

Protein phosphorylation is one of the most important post-translational modifications (PTMs) as it participates in regulating various cellular processes and biological functions. It is therefore crucial to identify phosphorylated proteins to construct a phosphor-relay network, and eventually to understand the underlying molecular regulatory mechanism in response to both internal and external stimuli. The changes in phosphorylation status at these novel phosphosites can be accurately measured using a 15N-stable isotopic labeling in Arabidopsis (SILIA) quantitative proteomic approach in a high-throughput manner. One of the unique characteristics of the SILIA quantitative phosphoproteomic approach is the preservation of native PTM status on protein during the entire peptide preparation procedure. Evolved from SILIA is another quantitative PTM proteomic approach, AQUIP (absolute quantitation of isoforms of post-translationally modified proteins), which was developed by combining the advantages of targeted proteomics with SILIA. Bioinformatics-based phosphorylation site prediction coupled with an MS-based in vitro kinase assay is an additional way to extend the capability of phosphosite identification from the total cellular protein. The combined use of SILIA and AQUIP provides a novel strategy for molecular systems biological study and for investigation of in vivo biological functions of these phosphoprotein isoforms and combinatorial codes of PTMs.

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.

Existing bioinformatics tools for the quantitation of post-translational modifications

Mass spectrometry (MS)-based proteomics, by itself, is a vast and complex area encompassing various mass spectrometers, different spectra, and search result representations. When the aim is quantitation performed in different scanning modes at different MS levels, matters become additionally complex. Quantitation of post-translational modifications (PTM) represents the greatest challenge among these endeavors. Many different approaches to quantitation have been described and some of these can be directly applied to the quantitation of PTMs. The amount of data produced via MS, however, makes manual data interpretation impractical. Therefore, specialized software tools meet this challenge. Any software currently able to quantitate differentially labeled samples may theoretically be adapted to quantitate differential PTM expression among samples as well. Due to the heterogeneity of mass spectrometry-based proteomics; this review will focus on quantitation of PTM using liquid chromatography followed by one or more stages of mass spectrometry. Currently available free software, which either allow analysis of PTM or are easily adaptable for this purpose, is briefly reviewed in this paper. Selected studies, especially those related to phosphoproteomics, shall be used to highlight the current ability to quantitate PTMs.

Systematic Mapping of Posttranslational Modifications in Human Estrogen Receptor-α with Emphasis on Novel Phosphorylation Sites

A systematic study of posttranslational modifications of the estrogen receptor isolated from the MCF-7 human breast cancer cell line is reported. Proteolysis with multiple enzymes, mass spectrometry, and tandem mass spectrometry achieved very high sequence coverage for the full-length 66-kDa endogenous protein from estradiol-treated cell cultures. Nine phosphorylated serine residues were identified, three of which were previously unreported and none of which were previously observed by mass spectrometry by any other laboratory. Two additional modified serine residues were identified in recombinant protein, one previously reported but not observed here in endogenous protein and the other previously unknown. Although major emphasis was placed on identifying new phosphorylation sites, N-terminal loss of methionine accompanied by amino acetylation and a lysine side chain acetylation (or possibly trimethylation) were also detected. The use of both HPLC-ESI and MALDI interfaced to different mass analyzers gave higher sequence coverage and identified more sites than could be achieved by either method alone. The estrogen receptor is critical in the development and progression of breast cancer. One previously unreported phosphorylation site identified here was shown to be strongly dependent on estradiol, confirming its potential significance to breast cancer. Greater knowledge of this array of posttranslational modifications of estrogen receptor, particularly phosphorylation, will increase our understanding of the processes that lead to estradiol-induced activation of this protein and may aid the development of therapeutic strategies for management of hormone-dependent breast cancer.