Dimethyl Labeling Research Articles

The mechanism of iron-compensation for manganese deficiency of Streptococcus pneumoniae

Given their involvement in catalysis, infection, and biofilm formation, Fe and Mn are essential for bacterial survival and virulence. In this study, we found that Streptococcus pneumoniae (S. pneumoniae) could grow in the Mn-deficient medium (MDCM). Furthermore, findings showed that the Fe concentration in the bacterium increased when the Mn concentration decreased. In addition, it was noted that supplementing MDCM with Fe resulted in the recovery of bacterial growth. Quantitative proteomics using stable-isotope dimethyl labeling was performed to investigate the adaptive growth mechanism of S. pneumoniae under Mn-deficient conditions. It was found that the expression levels of 25 proteins were downregulated, whereas those of 54 proteins were upregulated in S. pneumoniae grown in MDCM. It was also noted that several of the downregulated proteins were involved in cell energy metabolism, amino acid synthesis, and reduction of oxidation products. More importantly, several ATP-binding cassette transporters related to Fe uptake, such as PiuA, PiaA, PitA, and SPD_1609, were overexpressed for increased Fe uptake from the MDCM. The results suggest that Mn deficiency disturbs multiple metabolic processes in S. pneumoniae. Furthermore, it causes a compensatory effect of Fe for Mn, which is beneficial for the survival of the bacterium in extreme environments. SignificanceThe relationship between manganese and iron metabolism in S. pneumoniae has not been clearly revealed. In this paper, we suggest that Mn limitation disturbs multiple metabolic processes and evidently decreases the ATP level in the bacterium. In order to survive in this extreme environment, bacteria upregulated three type of Fe ion transporters PiuABC (heme), PiaABC (ferrichrome) and PitABC (Fe3+) to uptake enough Fe ions to response to Mn deficiency. Therefore, this study reveals a bacterial mechanism of Fe compensation for Mn, and provides new insight for investigating the relativeness of Fe and Mn metabolism of bacteria.

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.

Proteomic differences between focal and diffuse traumatic brain injury in human brain tissue

The early molecular response to severe traumatic brain injury (TBI) was evaluated using biopsies of structurally normal-appearing cortex, obtained at location for intracranial pressure (ICP) monitoring, from 16 severe TBI patients. Mass spectrometry (MS; label free and stable isotope dimethyl labeling) quantitation proteomics showed a strikingly different molecular pattern in TBI in comparison to cortical biopsies from 11 idiopathic normal pressure hydrocephalus patients. Diffuse TBI showed increased expression of peptides related to neurodegeneration (Tau and Fascin, p < 0.05), reduced expression related to antioxidant defense (Glutathione S-transferase Mu 3, Peroxiredoxin-6, Thioredoxin-dependent peroxide reductase; p < 0.05) and increased expression of potential biomarkers (e.g. Neurogranin, Fatty acid-binding protein, heart p < 0.05) compared to focal TBI. Proteomics of human brain biopsies displayed considerable molecular heterogeneity among the different TBI subtypes with consequences for the pathophysiology and development of targeted treatments for TBI.

Unbiased Proteomic Study of the Axons of Cultured Rat Cortical Neurons.

The axon is a long projection connecting a neuron to its targets. Here, the axons of cultured rat cortical neurons were isolated with micropatterned chips that enable the separation of axons from their cell bodies. Proteins extracted from isolated axons and whole neurons were subjected to analyses using two-dimensional liquid chromatography-tandem mass spectrometry (2D-LC-MS/MS) analyses without and with stable isotope dimethyl labeling, resulting in the identification of >2500 axonal proteins and 103 axon-enriched proteins. A strong correlation exists between the abundances of axonal proteins and their counterparts in whole neurons. The proteomic results confirm the axonal protein constituents of the subcellular structures documented in earlier electron microscopic studies. Cortical axons have proteins that are components of machineries for protein degradation and the synthesis of soluble, membrane, and secretory proteins, although axons lack conventional Golgi apparatus. Despite the fact that axons lack nucleus, nuclear proteins were identified, and 67 of them were found enriched in axons. Some of the results obtained by the MS-based studies were validated by quantitative Western blotting and immunofluorescence staining analyses. The results represent the first comprehensive description of the axonal protein landscape. The MS proteomics data are available via ProteomeXchange with identifier PXD005527.

Validation of In Vivo Protein Surface Accessibility Method

Measuring changes in surface accessibility is a way to probe conformational changes of proteins. Traditionally, in vitro methods of resolving protein structures have relied on the isolation and purification of proteins, but this artificial protein environment may not reflect its functional native conformation. Therefore, this project seeks to develop a novel approach to identify and quantify changes in protein surface accessibility caused by protein misfolding or altered biomolecule interactions in vivo on a proteome‐wide scale. We want to find out if this approach, chemically labeling the ɛ‐amino group of lysine residues, will yield the same results in vitro as it did in vivo, thus validating the accuracy of results generated with this technique. This dimethylation method is expected to yield biologically relevant information as it allows for the screening of proteomes for structural perturbations as well as identification of novel structural information for proteins not yet crystallized.Using GAPDH as a control, we showed that the GAPDH tetramer stays intact upon dimethylation using native gel electrophoresis. Using data from crystal structures of GAPDH, the GAPDH tetramer contains one predicted inaccessible lysine residue and we were able to detect this inaccessible lysine residue with our in vitro dimethylation experiment. In addition, under time‐limited conditions, the dimethylation reaction does not go to completion and appears to preferentially occur at certain lysine sites. The extent of dimethylation does not scale with relative surface accessibility and rather requires a minimal accessibility to the ɛ‐amino group of lysine residues.The dimethylation labeling method is reproducible in vitro as well as in vivo. The GAPDH experiments show that the method chemically labels proteins that are still in their native conformation. Limiting conditions of the dimethylation reaction provides additional structural information of the protein, which allow us to validate predicted structural models and/or data. This technique can be utilized to screen the proteome for structural alterations, which is especially important in diseases or drug interactions.Support or Funding InformationNIH NCI Enabling Technologies Grant (IMAT) R33CA212973‐01 to John R. Yates IIIThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Secretome Analysis of Hypoxia-Induced 3T3-L1 Adipocytes Uncovers Novel Proteins Potentially Involved in Obesity.

In the obese state, as adipose tissue expands, adipocytes become hypoxic and dysfunctional, leading to changes in the pattern of adipocyte-secreted proteins. To better understand the role of hypoxia in the mechanisms linked to obesity, we comparatively analyzed the secretome of murine differentiated 3T3-L1 adipocytes exposed to normoxia or hypoxia for 24 h. Proteins secreted into the culture media were precipitated by trichloroacetic acid and then digested with trypsin. The peptides were labeled with dimethyl labeling and analyzed by reversed phase nanoscale liquid chromatography coupled to a quadrupole Orbitrap mass spectrometer. From a total of 1508 identified proteins, 109 were differentially regulated, of which 108 were genuinely secreted. Factors significantly downregulated in hypoxic conditions included adiponectin, a known adipokine implicated in metabolic processes, as well as thrombospondin-1 and -2, and matrix metalloproteinase-11, all multifunctional proteins involved in extracellular matrix (ECM) homeostasis. Findings were validated by Western blot analysis. Expression studies of the relative genes were performed in parallel experiments in vitro, in differentiated 3T3-L1 adipocytes, and in vivo, in fat tissues from obese versus lean mice. Our observations are compatible with the concept that hypoxia may be an early trigger for both adipose cell dysfunction and ECM remodeling.

A cross-sequence labeling equipment for high-throughput proteome based on pseudo-isobaric dimethyl labeling

Facing the needs of multidimensional and multi-sample proteome quantification in the field of biology and precision medicine, high-throughput quantitative labeling and analysis have become the trend in recent years. A novel cross-sequence labeling equipment for high-throughput proteome based on pseudo-isobaric dimethyl labeling (pIDL-StageTip) was established. The equipment needs only simple devices and centrifugal force. The throughput of quantitative labeling is effectively increased, and the controllability of the two-step labeling reaction time at the terminals of the peptides and the ease of operation are ensured. By optimizing the labeling conditions of NaBD3CN and NaBH3CN under acidic conditions respectively, labeling efficiency of the standard proteolysis achieved 100% and labeling selectivity was more than 95%. Even in the complex human proteome, the labeling efficiency was greater than 99% and the labeling selectivity was 100%. The quantitative method based on the equipment showed high quantitative accuracy and precision. The equipment provides a reliable solution for quantitative proteome labeling with high operability, accuracy, and throughput.

Proteomic changes in ‘Ambrosia’ apple fruit during cold storage and in response to delayed cooling treatment

‘Ambrosia’ apple is one of the emerging apple cultivars that have been gaining popularity in North America due to its superior eating quality. An effective postharvest storage regime has been established for optimal storage and quality maintenance. Unfortunately, a postharvest physiological disorder has often been reported, which shows symptoms similar to low temperature induced soft scald in ‘Honeycrisp’. Therefore, a delayed cooling strategy prior to storage has been developed and implemented as a successful and economical method to prevent this disorder. In order to reveal the molecular mechanism of soft scald development and delayed cooling in combating this low temperature induced disorder, a quantitative proteomic investigation employing stable isotope dimethylation labeling by peptides was conducted on ‘Ambrosia’ apples from three commercial orchards in BC, Canada. Quantitative changes in protein abundance in association with disorder development and in response to delayed cooling treatment after one and three month storage were found. Among the quantified proteins, 495 and 575 proteins were commonly presented in three biological replicates after one and three month storage; respectively. Among them, the abundance of 78 and 88 proteins were found to be significantly changed in response to delayed cooling as compared with normal cold storage for one and three months, respectively. These identified proteins were functionally annotated using MAPMAN software, which identified glycolysis, lipid metabolism, amino acids (including GABA shunt), hormone response, stress and signaling, redox and glutathione metabolism as the major pathways influenced by the delayed cooling treatment. A principal component analysis (PCA) revealed groups of proteins that play a major role in response to disorder development and a negative response to delayed cooling. In addition, an ABC transporter protein F family is correlated to the delayed cooling treatment. This study demonstrates the potential mechanism of the biological effect of delayed cooling on apple fruit at the proteomic level. It also provides in-depth insight on molecular mechanisms of the delayed cooling treatment in apples.

Characterization of the functions and proteomes associated with membrane rafts in chicken sperm.

Cellular membranes are heterogeneous, and this has a great impact on cellular function. Despite the central role of membrane functions in multiple cellular processes in sperm, their molecular mechanisms are poorly understood. Membrane rafts are specific membrane domains enriched in cholesterol, ganglioside GM1, and functional proteins, and they are involved in the regulation of a variety of cellular functions. Studies of the functional characterization of membrane rafts in mammalian sperm have demonstrated roles in sperm-egg binding and the acrosomal reaction. Recently, our biochemical and cell biological studies showed that membrane rafts are present and might play functional roles in chicken sperm. In this study, we isolated membrane rafts from chicken sperm as a detergent-resistant membranes (DRM) floating on a density gradient in the presence of 1% Triton X-100, and characterized the function and proteomes associated with these domains. Biochemical comparison of the DRM between fresh and cryopreserved sperm demonstrated that cryopreservation induces cholesterol loss specifically from membrane rafts, indicating the functional connection with reduced post-thaw fertility in chicken sperm. Furthermore, using an avidin-biotin system, we found that sperm DRM is highly enriched in a 60 KDa single protein able to bind to the inner perivitelline layer. To identify possible roles of membrane rafts, quantitative proteomics, combined with a stable isotope dimethyl labeling approach, identified 82 proteins exclusively or relatively more associated with membrane rafts. Our results demonstrate the functional distinctions between membrane domains and provide compelling evidence that membrane rafts are involved in various cellular pathways inherent to chicken sperm.

Integrated platform with combination of on-line protein digestion, isotope dimethyl labeling and multidimensional peptide separation for high-throughput proteome quantification

In recent years, various enzymatic microreactors and on-line enzyme digestion strategies have been widely applied in high throughput proteome analysis. However, the incomplete and irreproducible digestion would introduce some unexpected variations in comparative proteome quantification when the samples are digested and then chemically isotope labeled in different aliquots. To address these problems, we developed an integrated platform for high throughput proteome quantification with combination of on-line low miss-cleavage protein digestion by an ultra-performance immobilized enzymatic reactor, on-line dimethyl labeling onto a C18 precolumn, peptide separation by two-dimensional nano liquid chromatography and MS detection. Compared to traditional off-line method, such a platform exhibits obvious advantages such as high sensitivity, throughput, accuracy, precision and ease of automation. All these results demonstrated that such a platform might become a promising technique for the quantitative proteome analysis.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker

AbstractA genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low‐abundance prey proteins after intracellular photocrosslinking and prey–bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease–chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

Quantitative and Comparative Profiling of Protease Substrates through a Genetically Encoded Multifunctional Photocrosslinker.

A genetically encoded, multifunctional photocrosslinker was developed for quantitative and comparative proteomics. By bearing a bioorthogonal handle and a releasable linker in addition to its photoaffinity warhead, this probe enables the enrichment of transient and low-abundance prey proteins after intracellular photocrosslinking and prey-bait separation, which can be subject to stable isotope dimethyl labeling and mass spectrometry analysis. This quantitative strategy (termed isoCAPP) allowed a comparative proteomic approach to be adopted to identify the proteolytic substrates of an E. coli protease-chaperone dual machinery DegP. Two newly identified substrates were subsequently confirmed by proteolysis experiments.

MdFDIA: A Mass Defect Based Four-Plex Data-Independent Acquisition Strategy for Proteome Quantification.

Data-independent acquisition (DIA) has recently emerged as a powerful quantitative approach for large-scale proteome quantification, providing a sensitive and reproducible alternative to data-dependent acquisition (DDA). However, lack of compatible multiplexed quantification methods is a bottleneck of DIA. To alleviate this challenge, we present a mass defect based four-plex data-independent acquisition strategy, termed "MdFDIA", for parallel analysis of four different protein samples in a DIA experiment without the additional complexity of tandem mass spectrometry (MS2) spectra. MdFDIA is a hybrid approach that combines stable isotope labeling with amino acids in cell culture (SILAC) and dimethyl labeling. Briefly, the isotopes 13C615N2-lysine (+8.0142 Da, light) and D8-lysine (+8.0512 Da, heavy) were metabolically embedded in different proteome samples during cell culture. Then, two 13C615N2-lysine and D8-lysine labeled protein samples were digested with Lys-C, followed by in vitro labeling with light (213CD2H, +34.06312 Da) and heavy (2CD3, +34.06896 Da) dimethyl groups, respectively, producing four different pseudoisobaric labeled protein samples. The labeled samples were then equally mixed and analyzed by DIA. The subtle mass differences between the four labeled forms in MS2 scans can be resolved on an Orbitrap Fusion Lumos instrument to facilitate quantification without abundance information encoded in MS2 spectra. Additionally, a systematic investigation was carried out and revealed that MdFDIA enabled a significant decrease of the adverse impact on the accuracy of the quantitative assays arising from the chromatographic isotope effect, especially the deuterium effect, which typically occurs in a DDA experiment. Additionally, MdFDIA provided a method for validating the fragment type in the DIA spectra identification result. Furthermore, MdFDIA was applied to quantitative proteome analyses of four different breast cancer cell lines, demonstrating the feasibility of this strategy for biological applications.

Tryptic Peptides Bearing C-Terminal Dimethyllysine Need to Be Considered during the Analysis of Lysine Dimethylation in Proteomic Study.

Lysine methylation plays important roles in structural and functional regulation of chromatin. Although trypsin is the most widely used protease in mass spectrometry-based proteomic analysis for lysine methylation substrates, the proteolytic activity of trypsin on dimethylated lysine residues remains an arguable issue. In this study, we tested the ability of trypsin to cleave dimethylated lysine residues in synthetic peptides, purified albumin, and whole cell lysate, and found that the C-terminal of dimethylated lysine residue could be cleaved in a protein sequence-dependent manner. Kinetic studies revealed that the optimal digestion time and enzyme-to-substrate ratio for the cleavage of dimethylated lysine by trypsin was around 16 h and 1:50, respectively. We further showed the tryptic C-terminal lysine-dimethylated (C-Kme2) peptides could contribute to a significant portion of substrate identification in the proteomic study, which utilizes the chemical dimethylation labeling approach. More than 120 tryptic C-Kme2 peptides (7% of total peptides identified) were identified in chemically lysine-dimethyl-labeled HeLa whole cell lysate by a single-shot nanoflow high performance liquid chromatography with tandem mass spectrometry (nano-HPLC-MS/MS) analysis. Moreover, in an assay for substrate identification of protease Glu-C using stable isotope dimethyl labeling approach, our data showed the tryptic C-Kme2 peptides accounted for more than 13% of total tryptic peptides. Additionally, our in vivo methylome profiling data revealed some C-Kme2 peptides, which is of importance to identification and quantification of biologically relevant protein and lysine-methylated site. Therefore, we reason that the tryptic peptides bearing C-terminal dimethylated lysine need to be considered in the mass spectrometric analysis of lysine dimethylation.

Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents.

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and β-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and β-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.

Salinity-Induced Palmella Formation Mechanism in Halotolerant Algae Dunaliella salina Revealed by Quantitative Proteomics and Phosphoproteomics.

Palmella stage is critical for some unicellular algae to survive in extreme environments. The halotolerant algae Dunaliella salina is a good single-cell model for studying plant adaptation to high salinity. To investigate the molecular adaptation mechanism in salinity shock-induced palmella formation, we performed a comprehensive physiological, proteomics and phosphoproteomics study upon palmella formation of D. salina using dimethyl labeling and Ti4+-immobilized metal ion affinity chromatography (IMAC) proteomic approaches. We found that 151 salinity-responsive proteins and 35 salinity-responsive phosphoproteins were involved in multiple signaling and metabolic pathways upon palmella formation. Taken together with photosynthetic parameters and enzyme activity analyses, the patterns of protein accumulation and phosphorylation level exhibited the mechanisms upon palmella formation, including dynamics of cytoskeleton and cell membrane curvature, accumulation and transport of exopolysaccharides, photosynthesis and energy supplying (i.e., photosystem II stability and activity, cyclic electron transport, and C4 pathway), nuclear/chloroplastic gene expression regulation and protein processing, reactive oxygen species homeostasis, and salt signaling transduction. The salinity-responsive protein–protein interaction (PPI) networks implied that signaling and protein synthesis and fate are crucial for modulation of these processes. Importantly, the 3D structure of phosphoprotein clearly indicated that the phosphorylation sites of eight proteins were localized in the region of function domain.

Complementary proteomic approaches reveal mitochondrial dysfunction, immune and inflammatory dysregulation in a mouse model of Gulf War Illness

PurposeLong‐term consequences of combined pyridostigmine bromide (PB) and permethrin (PER) exposure in C57BL6/J mice using a well‐characterized mouse model of exposure to these Gulf War (GW) agents were explored at the protein level.Experimental designWe used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane‐bound protein fractions from brain samples using two orthogonal isotopic labeling LC‐MS/MS proteomic approaches—stable isotope dimethyl labeling and iTRAQ.ResultsThe use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both distinct and overlapping datasets. This combined analysis identified changes in several mitochondrial, as well as immune and inflammatory pathways after GW agent exposure.Conclusions and clinical relevanceThe work discussed here provides insight into GW agent exposure dependent mechanisms that adversely affect mitochondrial function and immune and inflammatory regulation. Collectively, our work identified key pathways which were chronically impacted in the mouse CNS following acute GW agent exposure, this may lead to the identification of potential targets for therapeutic intervention in the future.Long‐term consequences of combined PB and PER exposure in C57BL6/J mice using a well‐characterized mouse model of exposure to these GW agents were explored at the protein level. Expanding on earlier work, we used orthogonal proteomic approaches to identify pathways that are chronically impacted in the mouse CNS due to semiacute GW agent exposure early in life. These analyses were performed on soluble and membrane‐bound protein fractions from brain samples using two orthogonal isotopic labeling LC‐MS/MS proteomic approaches—stable isotope dimethyl labeling and iTRAQ. The use of these approaches allowed for greater coverage of proteins than was possible by either one alone and revealed both distinct and overlapping datasets. This combined analysis identified changes in several mitochondrial, as well as immune and inflammatory pathways after GW agent exposure. The work discussed here provides insight into GW agent exposure dependent mechanisms that adversely affect mitochondrial function and immune and inflammatory regulation at 5 months postexposure to PB + PER.

Shotgun proteomic analysis to study the decrease of xenograft tumor growth after rosemary extract treatment

The antiproliferative activity of Rosemary (Rosmarinus officinalis) has been widely studied in different in vitro and in vivo models, which demonstrate that rosemary extracts inhibit the cellular proliferation due to its ability to interact with a wide spectrum of molecular targets. However, a comprehensive proteomics study in vivo has not been carried out yet. In the present work, the effects of rosemary extract on xenograft tumor growth has been studied and, for the first time, a shotgun proteomic analysis based on nano-LC–MS/MS together with stable isotope dimethyl labeling (DML) has been applied to investigate the global protein changes in vivo. Our results show that the daily administration of a polyphenol-enriched rosemary extract reduces the progression of colorectal cancer in vivo with the subsequent deregulation of 74 proteins. The bioinformatic analysis of these proteins indicates that the rosemary extract mainly alters the RNA Post-Transcriptional Modification, the Protein Synthesis and the Amino Acid Metabolism functions and suggests the inactivation of the oncogene MYC. These results demonstrate the high utility of the proposed analytical methodology to determine, simultaneously, the expression levels of a large number of protein biomarkers and to generate new hypothesis about the molecular mechanisms of this extract in vivo.

Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies.

Site-specific solvent accessibility of the primary amines (mainly lysine or the N-termini) on proteins is of great interest in many research areas because amines are an important functional group for protein conjugation. In this study, we coupled dimethyl labeling via reductive amination with liquid chromatography-mass spectrometry (LC-MS) to fully characterize the solvent accessibility of lysine residues and the N-termini on human immunoglobulin G (IgG). Circular dichroism (CD) and fluorescence spectroscopy revealed that dimethyl labeling did not alter the conformation of the native IgG molecule. Based on intact protein measurements, up to 28 (light chain) and 66 (heavy chain) dimethyl tags, covering all lysine residues and the N-termini, were sequentially incorporated into IgG molecules in 1000 s. All labeled sites were identified and quantified by a bottom-up proteomics approach. Some highly exposed hot-spots (for example, the N-termini of both the heavy and the light chains) and some buried sites (for example, K415 in the heavy chain and K39 in the light chain) were unambiguously revealed. This method was also used to characterize aggregation-induced structural changes in IgGs by increasing the temperature. Substantial changes in the labeling percentage of many lysine sites were observed, indicating a non-native aggregation triggered by thermal stress. Due to high labeling yields and the van der Waals surface of the labeling reagents being comparable to that of water, dimethyl labeling is a highly promising technique for probing the amine's surface topography of proteins. It can also be used as a complementary approach to other methods for resolving the higher-order structure of proteins by LC-MS.

Wheat (Triticum aestivum L.) grain proteome response to elevated [CO2] varies between genotypes

The impact of rising carbon dioxide concentration ([CO2]) in the atmosphere on wheat grain protein concentration and proteome was investigated in this study. Wheat genotypes (H45, SB003, SB062 and Yitpi) were grown in the Australian Grains Free-Air CO2 Enrichment (AGFACE) facility, Horsham, Victoria, Australia under ambient [CO2] (a[CO2], 391 μmol mol−1) and elevated [CO2] (e[CO2], 550 ± 20 μmol mol−1). Grain yield and grain protein concentration were measured. Global grain proteome comparison was carried out using stable isotope dimethyl labelling followed by liquid chromatography - mass spectrometry (LC-MS/MS). Grain yield was significantly increased at e[CO2], whereas protein concentration was significantly decreased and responses varied between genotypes. Proteome-wide analysis revealed that protein composition was also altered under e[CO2]. Grain protein concentration and composition of SB003 was very responsive to e[CO2]. Mainly storage proteins were decreased at e[CO2] and the responses varied between genotypes. These findings suggest that e[CO2] may have a major impact on grain protein quality and thus bread quality and human and animal nutrition. Further, these findings suggest that [CO2] insensitive cultivars can be identified for grain quality improvement under changing climate.