Global substrate specificity profiling of post‐translational modifying enzymes

Enzymes that modify the proteome, referred to as post‐translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide‐based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP‐MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems‐level understanding of PTM enzyme regulation and function.     

Comprehensive two-dimensional liquid chromatography mass spectrometric profiling of the rat hippocampal proteome

In this study, we performed the first high‐throughput and comprehensive proteomic profiling of the rat hippocampal proteome. Using a combination of 2‐D LC‐MS and data analysis with the Rosetta Elucidator^® system, we identified 1340 unique proteins. Functional classification showed that most of these were associated with synaptic function and comprised a high proportion of phosphorylated proteins and analytically challenging classes of membrane proteins such as ion channel receptor subunits.     

Protein labeling by iTRAQ: a new tool for quantitative mass spectrometry in proteome research

A novel, MS-based approach for the relative quantification of proteins, relying on the derivatization of primary amino groups in intact proteins using isobaric tag for relative and absolute quantitation (iTRAQ) is presented. Due to the isobaric mass design of the iTRAQ reagents, differentially labeled proteins do not differ in mass; accordingly, their corresponding proteolytic peptides appear as single peaks in MS scans. Because quantitative information is provided by isotope-encoded reporter ions that can only be observed in MS/MS spectra, we analyzed the fragmentation behavior of ESI and MALDI ions of peptides generated from iTRAQ-labeled proteins using a TOF/TOF and/or a QTOF instrument. We observed efficient liberation of reporter ions for singly protonated peptides at low-energy collision conditions. In contrast, increased collision energies were required to liberate the iTRAQ label from lysine side chains of doubly charged peptides and, thus, to observe reporter ions suitable for relative quantification of proteins with high accuracy. We then developed a quantitative strategy that comprises labeling of intact proteins by iTRAQ followed by gel electrophoresis and peptide MS/MS analyses. As proof of principle, mixtures of five different proteins in various concentration ratios were quantified, demonstrating the general applicability of the approach presented here to quantitative MS-based proteomics.     

Solvent-induced proteome profiling for proteomic quantitation and target discovery of small molecular drugs

Target identification by modification-free proteomic approaches can potentially reveal the pharmacological mechanism of small molecular compounds. By combining the recent solvent-induced protein precipitation (SIP) method with TMT-labeling quantitative proteomics, we propose solvent-induced proteome profiling (SIPP) approach to identify small molecule–protein interactions. The SIPP approach enables to depict denaturation curves of the target protein by varying concentrations of organic solvents to induce unfolding and precipitation of the cellular proteome. By using this approach, we have successfully identified the known targets of market drugs and natural products and extended the proteome information of SIP for target identification.     

Proteome profiling of Populus euphratica Oliv. upon heat stress

BACKGROUND AND AIMS: Populus euphratica is a light-demanding species ecologically characterized as a pioneer. It grows in shelter belts along riversides, being part of the natural desert forest ecosystems in China and Middle Eastern countries. It is able to survive extreme temperatures, drought and salt stress, marking itself out as an important plant species to study the mechanisms responsible for survival of woody plants under heat stress. METHODS: Heat effects were evaluated through electrolyte leakage on leaf discs, and LT(50) was determined to occur above 50 degrees C. Protein accumulation profiles of leaves from young plants submitted to 42/37 degrees C for 3 d in a phytotron were determined through 2D-PAGE, and a total of 45 % of up- and downregulated proteins were detected. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)/TOF analysis, combined with searches in different databases, enabled the identification of 82 % of the selected spots. KEY RESULTS: Short-term upregulated proteins are related to membrane destabilization and cytoskeleton restructuring, sulfur assimilation, thiamine and hydrophobic amino acid biosynthesis, and protein stability. Long-term upregulated proteins are involved in redox homeostasis and photosynthesis. Late downregulated proteins are involved mainly in carbon metabolism. CONCLUSIONS: Moderate heat response involves proteins related to lipid biogenesis, cytoskeleton structure, sulfate assimilation, thiamine and hydrophobic amino acid biosynthesis, and nuclear transport. Photostasis is achieved through carbon metabolism adjustment, a decrease of photosystem II (PSII) abundance and an increase of PSI contribution to photosynthetic linear electron flow. Thioredoxin h may have a special role in this process in P. euphratica upon moderate heat exposure.     

Putting proteomics on target: activity-based profiling of ubiquitin and ubiquitin-like processing enzymes

Modification of proteins with ubiquitin (Ub) and Ub-like modifiers (Ubls) plays a fundamental role in cell biology. As a consequence, proteomics-based efforts were developed to characterize proteins that are modified by Ub or Ubls. A more focused functional proteomics strategy relies on active-site probes based on the Ub/Ubl scaffold, which specifically targets Ub/Ubl-processing enzymes. Activity-based profiling with such tools led to the identification of novel gene products with Ub/Ubl-processing activity and uncovered novel control mechanisms regulating their activity. This review discusses recent advances in chemistry-based functional proteomics applications, and how this information can provide a framework for drug development against Ub/Ubl-processing enzymes.     

Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry

There is significant interest in characterization of the human plasma proteome due to its potential for providing biomarkers applicable to clinical diagnosis and treatment and for gaining a better understanding of human diseases. We describe here a strategy for comparative proteome analyses of human plasma, which is applicable to biomarker identifications for various disease states. Multidimensional liquid chromatography-mass spectrometry (LC-MS/MS) has been applied to make comparative proteome analyses of plasma samples from an individual prior to and 9 h after lipopolysaccharide (LPS) administration. Peptide peak areas and the number of peptide identifications for each protein were used to evaluate the reproducibility of LC-MS/MS and to compare relative changes in protein concentration between the samples following LPS treatment. A total of 804 distinct plasma proteins (not including immunoglobulins) were confidently identified with 32 proteins observed to be significantly increased in concentration following LPS administration, including several known inflammatory response or acute-phase mediators such as C-reactive protein, serum amyloid A and A2, LPS-binding protein, LPS-responsive and beige-like anchor protein, hepatocyte growth factor activator, and von Willebrand factor, and thus, constituting potential biomarkers for inflammatory response.     

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.     

草菇子实体不同成熟阶段的比较蛋白质组学分析

采用iTRAQ标记结合二维液相色谱串联质谱技术对草菇不同成熟阶段的差异蛋白质组进行研究。首先将提取的草菇不同成熟阶段蛋白样品进行SDS-PAGE分析,其次将经二维液相色谱串联质谱技术获取的串联质谱数据通过MASCOT软件搜库,之后对鉴定蛋白质数据进行了KEGG代谢通路分析。试验共计获得2 335个不同肽段, 鉴定到1 039个蛋白质,其中1 030个蛋白质具有定量信息。与蛋形期相比,在伸长期和成熟期阶段显著上调蛋白质85个,下调蛋白质103个。KEGG代谢通路分析结果显示,草菇不同成熟阶段中的68个差异蛋白质可定位于4种伞菌目模式真菌（灰盖鬼伞、双色蜡蘑、可可丛枝病菌和裂褶菌）的45个不同生物代谢途径,全景展示出草菇成熟阶段差异表达蛋白质定位的代谢网络。结果表明,iTRAQ标记技术结合二维液相色谱串联质谱可对不同生长发育时期的草菇蛋白样品进行有效地分离和鉴定。     

Quantitative analysis of free fatty acids in rat plasma using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with meso-tetrakis porphyrin as matrix

Quantitative analysis of free fatty acids was achieved using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a meso-tetrakis porphyrin matrix. Cesium acetate was employed as a cationizing agent. The MALDI signal was reproducible and dominated by cesiated cesium carboxylates [RCOOCs + Cs]+. The addition of two Cs ions resulted in a mass shift of 264.8 Da for each fatty acid and greatly reduced background peaks. A linear relationship between fatty acid concentration and corresponding fatty acid to internal standard peak intensity ratio was observed for three representative fatty acids analyzed across a concentration range from 4.40 to 150 microM, with correlation coefficients between 0.986 and 0.987. The application of this method was demonstrated with the analysis of free fatty acids in nonfasted and fasted rat plasmas. A total of eight free fatty acids (14:0, 16:0, 16:1, 17:0, 18:0, 18:1, 18:2, and 20:4) were detected. The relative peak height ratios of the fatty acids to the internal standard allow quantitative measurements of the free fatty acids. It was shown that the levels of free fatty acids were higher in fasted rats than in rats in a nonfasted state. This method is simple, sensitive, and fast. Thus, it provides an appealing tool for the analysis of free fatty acids or other low-molecular weight compounds during drug discovery and/or development.     

Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.     

Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes

AIMS: To set up and employ, for the biorestoration of cultural heritage (altered frescoes), an advanced and innovative biotechnology method based on the sequential use of whole viable bacterial cells and specific enzymes. METHODS AND RESULTS: The bioremediation intervention consisted of the direct application onto an artwork surface of whole bacterial cells of the Pseudomonas stutzeri A29 strain (bioaugmentation), followed by, in a final step, a purified Protease enzyme. The bioremediation was performed on a Spinello Aretino fresco that had become altered by the animal glue residues of past restoration. For the reader's interest the fresco is the 14th century Conversione di S. Efisio e battaglia (Conversion of S. Efisio and battle), size 3.5 x 7.8 m at the Pisa Camposanto Monumentale, Italy. An assessment was made of the final costs of the biological tests (whole bacterial cells, enzymes) so as to compare them with other intervention techniques. CONCLUSIONS: A successful innovative biological approach to recover valuable frescoes was set up, and the best conditions for treatment efficiency were identified. Furthermore the cost of the biological cleaning using viable bacterial cells and enzymes (P. stutzeri, Protease, Collagenase, 1 : 3 : 10, ratio respectively) was much lower than that of other conventional methods, making this biotechnology not only very interesting but also very competitive. SIGNIFICANCE AND IMPACT OF THE STUDY: New biotechnologies with an innovative, soft approach to the 'biocleaning' and 'biorestoration' of cultural heritage are in constant demand, and our results are clear evidence that such an approach has been achieved; the technique could be of significant importance towards developing other goals.     

Protein expression changes in the Sprague Dawley rat liver proteome following administration of peroxisome proliferator activated receptor alpha and gamma ligands

Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and are intimately involved in lipid metabolism and energy homeostasis. Activation of these receptors in rodents can lead to hepatomegaly and ultimately hepatic carcinogenesis although the mechanisms by which these processes occur are poorly understood. To further our understanding of these processes and to discriminate between different PPAR mediated signalling pathways, a proteomic approach has been undertaken to identify changes in protein expression patterns in Sprague Dawley rat liver following dosing with a PPARalpha agonist (Wyeth 14643), a PPARgamma agonist (Troglitazone) and a compound with mixed PPARalpha/gamma agonist activity (SB-219994). Using one-and-two-dimensional electrophoresis of tissue lysates a diverse range of protein abundance changes was observed in these tissues. Whilst a number of these proteins have PPAR response elements (PPREs) in their respective promoters, another group was detected whose expression has been documented to be sensitive to peroxisome proliferator administration. Most notably within these groups, proteins involved in lipid catabolism displayed increased expression following drug administration. A further subset of proteins, with less obvious biological implications, also showed altered expression patterns. Where available, sequences upstream of the coding regions of genes not previously known to have PPREs were searched with positional consensus matrices for the presence of PPREs in an attempt to validate these changes. Using such an approach putative PPARgamma and PPARdelta response elements were discovered upstream of the tubulin beta coding region. There was limited overlap in observed protein abundance changes between the three groups, and where this was the case (cytosolic epoxide hydrolase, peroxisomal bifunctional enzyme, hydroxymethyl glutaryl CoA, synthase, long chain acyl-CoA thioesterase), expression of these proteins had previously been shown to be under the control of PPAR activity.     

HDX‐MS reveals orthosteric and allosteric changes in apolipoprotein‐D structural dynamics upon binding of progesterone

Apolipoprotein‐D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein‐D adopts an eight‐stranded β‐barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α‐helix. Crystallography studies of recombinant apolipoprotein‐D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen‐deuterium exchange mass spectrometry (HDX‐MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX‐MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X‐ray crystallography, making HDX‐MS an invaluable orthogonal technique. Here, we report an HDX‐MS protocol for apolipoprotein‐D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand‐free apolipoprotein‐D revealed apolipoprotein‐D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein‐D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N‐terminus and C‐terminus. Together, our experiments provide insight into apolipoprotein‐D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein‐D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein‐D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein‐D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein‐D structure from X‐ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein‐D, to elucidate how proteins change flexibility when binding to small molecules.     

Metabolic profiling using combined GC-MS and LC-MS provides a systems understanding of aristolochic acid-induced nephrotoxicity in rat

We present here a combined GC-MS and LC-MS metabolic profiling approach to unraveling the pathological outcomes of aristolochic acid (AA)-induced nephrotoxicity. Urine samples were analyzed by GC-MS and LC-MS in combination with pattern recognition techniques, e.g. principal component analysis (PCA), orthogonal projection to latent structure-discriminant analysis. The work indicates that AA-induced acute renal toxicity as evidenced by histopathological examinations could be characterized by systemic disturbance of metabolic network involving free fatty acids generation, energy and amino acids metabolism, and alteration in the structure of gut microbiota. Therefore, this method is potentially applicable to the toxicological study, providing a comprehensive understanding of systems response to xenobiotic intervention.