Plant expression of NifD protein variants resistant to mitochondrial degradation.

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

iTRAQ-Based Proteomic Analysis Reveals Recovery of Impaired Mitochondrial Function in Ischemic Myocardium by Shenmai Formula.

Shenmai formula (SM) has been a traditional medicinal remedy for treating cardiovascular diseases in China for 800 years; however, its mechanism of action remains unclear. To explore the mechanism underlying cardioprotective effects of SM, iTRAQ-based proteomic approach was applied to analyze protein of myocardium in rats with myocardial ischemic injury. Upon treatment with SM and its two major components Red ginseng (RG) and Radix Ophiopogonis (OP), 101 differentially expressed proteins were filtered from a total of 712 detected and annotated proteins. They can be classified according to their locations and functions, while most of them are located in intracellular organelle, participating in cellular metabolic process. The functions of them are mostly associated with mitochondrial oxidative phosphorylation/respiration. The differentially expressed proteins were validated by liquid chromatography-tandem mass spectrometry and Western blotting (ATP5D, NDUFB10, TNNC1). Further in vitro experiments found that SM could attenuate hypoxia induced impairment of mitochondrial membrane potential and cellular ATP concentration in neonatal rat ventricular myocytes. Interestingly, the result of quantitative mitochondrial biogenesis assays revealed that SM had dominant positive effects on the maximum respiration, ATP-coupled respiration, and spare capacity of mitochondria in response to hypoxia. Hence, our findings suggest that SM promotes mitochondrial function to protect cardiomyocytes against hypoxia, which provides a possible illustration for conventional botanical therapy on a molecular level.

Species-specific identification of collagen components in Colla corii asini using a nano-liquid chromatography tandem mass spectrometry proteomics approach.

Colla corii asini (CCA) is a protein-based traditional Chinese medicine made from donkey skins. Because it has the ability to nourish blood, its demand is increasing rapidly. The shortage of donkey skins increases the risk of the adulteration of CCA products with other animal skins. To ensure the drug efficacy and safety of CCA products, a proteomics technique was applied to reveal proteins in the skins of donkey, horse, cattle, and pig. Species-specific peptides for each animal species were predicted using bioinformatics, and their presence in the skins and gelatin samples was examined by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). One unique marker peptide for each animal species was selected to develop an LC-MS/MS multiple reaction monitoring method. The capability of this method to identify donkey, horse, cattle, and pig materials was demonstrated by analyzing in-house-made donkey gelatins containing different amounts of other animal skins and commercial CCA products. The adulteration of non-donkey species could be sensitively detected at a low level of 0.5%. Hybrid animals, such as mules and hinnies, were also differentiated from donkeys. We provide a practical tool for the quality control of CCA products. The strategy can also be used to study other important traditional Chinese medicines which contain animal proteins.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Applications in Quantitative Proteomics.

Recent advances in inductively coupled plasma mass spectrometry (ICP-MS) hyphenated to different separation techniques have promoted it as a valuable tool in protein/peptide quantification. These emerging ICP-MS applications allow absolute quantification by measuring specific elemental responses. One approach quantifies elements already present in the structure of the target peptide (e.g. phosphorus and sulphur) as natural tags. Quantification of these natural tags allows the elucidation of the degree of protein phosphorylation in addition to absolute protein quantification. A separate approach is based on utilising bi-functional labelling substances (those containing ICP-MS detectable elements), that form a covalent chemical bond with the protein thus creating analogs which are detectable by ICP-MS. Based on the previously established stoichiometries of the labelling reagents, quantification can be achieved. This technique is very useful for the design of precise multiplexed quantitation schemes to address the challenges of biomarker screening and discovery. This review discusses the capabilities and different strategies to implement ICP-MS in the field of quantitative proteomics.

Development of an Ion Mobility Spectrometry-Orbitrap Mass Spectrometer Platform.

Complex samples benefit from multidimensional measurements where higher resolution enables more complete characterization of biological and environmental systems. To address this challenge, we developed a drift tube-based ion mobility spectrometry-Orbitrap mass spectrometer (IMS-Orbitrap MS) platform. To circumvent the time scale disparity between the fast IMS separation and the much slower Orbitrap MS acquisition, we utilized a dual gate and pseudorandom sequences to multiplex the injection of ions and allow operation in signal averaging (SA), single multiplexing (SM), and double multiplexing (DM) IMS modes to optimize the signal-to-noise ratio of the measurements. For the SM measurements, a previously developed algorithm was used to reconstruct the IMS data. A new algorithm was developed for the DM analyses involving a two-step process that first recovers the SM data and then decodes the SM data. The algorithm also performs multiple refining procedures to minimize demultiplexing artifacts. The new IMS-Orbitrap MS platform was demonstrated by the analysis of proteomic and petroleum samples, where the integration of IMS and high mass resolution proved essential for accurate assignment of molecular formulas.

The Plant Ovule Secretome: A Different View toward Pollen-Pistil Interactions.

During plant sexual reproduction, continuous exchange of signals between the pollen and the pistil (stigma, style, and ovary) plays important roles in pollen recognition and selection, establishing breeding barriers and, ultimately, leading to optimal seed set. After navigating through the stigma and the style, pollen tubes (PTs) reach their final destination, the ovule. This ultimate step is also regulated by numerous signals emanating from the embryo sac (ES) of the ovule. These signals encompass a wide variety of molecules, but species-specificity of the pollen-ovule interaction relies mainly on secreted proteins and their receptors. Isolation of candidate genes involved in pollen-pistil interactions has mainly relied on transcriptomic approaches, overlooking potential post-transcriptional regulation. To address this issue, ovule exudates were collected from the wild potato species Solanum chacoense using a tissue-free gravity-extraction method (tf-GEM). Combined RNA-seq and mass spectrometry-based proteomics led to the identification of 305 secreted proteins, of which 58% were ovule-specific. Comparative analyses using mature ovules (attracting PTs) and immature ovules (not attracting PTs) revealed that the last maturation step of ES development affected almost half of the ovule secretome. Of 128 upregulated proteins in anthesis stage, 106 were not regulated at the mRNA level, emphasizing the importance of post-transcriptional regulation in reproductive development.

The colostrum proteome, ruminant nutrition and immunity: a review.

In this review authors address colostrum proteins implications in different domestic ruminant species. The colostrogenesis process and how different factors, such as litter size or nutrition during gestation can alter the different components concentrations in colostrum are also reviewed. The different colostrum fractions will be described, focusing on high and low abundant proteins. This review describes the major function of such proteins and their role on the passive immune transfer and nutrition in the newborn animal. It will be also performed a comprehensive review on different techniques and commercial kits available for high abundant protein depletion in colostrum. We will finally focus on how proteomics has been used to address this issue and how it can contribute to the major questions about colostrum associated immunology.

Development of continuous microwave-assisted protein digestion with immobilized enzyme.

In this study, an easy and efficiency protein digestion method called continuous microwave-assisted protein digestion (cMAED) with immobilized enzyme was developed and applied for proteome analysis by LC-MS(n). Continuous microwave power outputting was specially designed and applied. Trypsin and bromelain were immobilized onto magnetic micropheres. To evaluate the method of cMAED, bovine serum albumin (BSA) and protein extracted from ginkgo nuts were used as model and real protein sample to verify the digestion efficiency of cMAED. Several conditions including continuous microwave power, the ratio of immobilized trypsin/BSA were optimized according to the analysis of peptide fragments by Tricine SDS-PAGE and LC-MS(n). Subsequently, the ginkgo protein was digested with the protocols of cMAED, MAED and conventional heating enzymatic digestion (HED) respectively and the LC-MS(n) profiles of the hydrolysate was compared. Results showed that cMAED combined with immobilized enzyme was a fast and efficient digestion method for protein digestion and microwave power tentatively affected the peptide producing. The cMAED method will be expanded for large-scale preparation of bioactive peptides and peptide analysis in biological and clinical research.

Novel pathophysiological markers are revealed by iTRAQ-based quantitative clinical proteomics approach in vascular dementia.

Vascular dementia (VaD) is a leading cause of dementia in the elderly together with Alzheimer's disease with limited treatment options. Poor understanding of the pathophysiology underlying VaD is hindering the development of new therapies. Hence, to unravel its underlying molecular pathology, an iTRAQ-2D-LC-MS/MS strategy was used for quantitative analysis of pooled lysates from Brodmann area 21 of pathologically confirmed cases of VaD and matched non-neurological controls. A total of 144 differentially expressed proteins out of 2281 confidently identified proteins (false discovery rate=0.3%) were shortlisted for bioinformatics analysis. Western blot analysis of selected proteins using samples from individual patients (n=10 per group) showed statistically significant increases in the abundance of SOD1 and NCAM and reduced ATP5A in VaD. This suggested a state of hypometabolism and vascular insufficiency along with an inflammatory condition during VaD. Elevation of SOD1 and increasing trend for iron-storage proteins (FTL, FTH1) may be indicative of an oxidative imbalance that is accompanied by an aberrant iron metabolism. The synaptic proteins did not exhibit a generalized decrease in abundance (e.g. syntaxin) in the VaD subjects. This reported proteome offers a reference data set for future basic or translational studies on VaD. BIOLOGICAL SIGNIFICANCE: Our study is the first quantitative clinical proteomic study where iTRAQ-2D-LC-MS/MS strategy has been used to identify the differential proteome in the VaD cortex by comparing VaD and matched control subjects. We generate testable hypothesis about the involvement of various proteins in the vascular and parenchymal events during the evolution of VaD that finally leads to malfunction and demise of brain cells. This study also establishes quantitative proteomics as a complementary approach and viable alternative to existing neurochemical, electron microscopic and neuroimaging techniques that are traditionally being used to understand the molecular pathology of VaD. Our study could inspire fellow researchers to initiate similar retrospective studies targeting various ethnicities, age-groups or sub-types of VaD using brain samples available from brain banks across the world. Meta-analysis of these studies in the future may be able to shortlist candidate proteins or pathways for rationale exploration of therapeutic targets or biomarkers for VaD.

Quantitative proteomic analysis reveals the neuroprotective effects of huperzine A for amyloid beta treated neuroblastoma N2a cells.

Alzheimer's disease is a worldwide metabolic disease and an economically costly disease to society, so more medicines need to be developed to treat this disease. Huperzine A, a novel lycopodium alkaloid isolated from traditional Chinese medicine Huperzia serrata (Qian Ceng Ta), has been shown to possess multiple neuroprotective effects for Alzheimer's disease, but the precise pharmacological mechanism of huperzine A is unclear and needs to be further investigated. In this study, proteins from untreated N2a cells (Con group), cells preincubated with huperzine A followed by Aß (1-42) oligomers treatment (HupA group) and cells treated with Aß (1-42) oligomers (Aß group) with five biological replicates in each cohort, were processed in a centrifugal proteomic reactor and quantified by label-free quantitation. A total of 2860 proteins were quantified with high confidence, and 198 proteins were significantly changed (with p-value < 0.05) between HupA and Aß cohorts. The pathway and direct protein-protein interaction network analysis showed that huperzine A protects N2a cells against Aß oligomer-induced cell death by downregulation of cellular tumor antigen p53 (Trp53) expression.

The effect of selenium enrichment on baker's yeast proteome.

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p≤0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.

Analysis of low molecular weight compounds by MALDI-FTICR-MS.

This review focuses on recent applications of matrix-assisted laser desorption ionization-Fourier-transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) in qualitative and quantitative analysis of low molecular weight compounds. The scope of the work includes amino acids, small peptides, mono and oligosaccharides, lipids, metabolic compounds, small molecule phytochemicals from medicinal herbs and even the volatile organic compounds from tobacco. We discuss both direct analysis and analysis following derivatization. In addition we review sample preparation strategies to reduce interferences in the low m/z range and to improve sensitivities by derivatization with charge tags. We also present coupling of head space techniques with MALDI-FTICR-MS. Furthermore, omics analyses based on MALDI-FTICR-MS were also discussed, including proteomics, metabolomics and lipidomics, as well as the relative MS imaging for bio-active low molecular weight compounds. Finally, we discussed the investigations on dissociation/rearrangement processes of low molecular weight compounds by MALDI-FTICR-MS.

Considerations for target validation and industrial approaches.

Target validation in health and disease integrates the modulation of a certain molecular target with an expected biological/biochemical/physiological or pathophysiological response or effect. The current state-of-the-art in target validation requires the interface of multiple complementary approaches and technologies to define the mechanistic connectivity between a molecular target and underlying micro- and macrobiotic processes. Target validation also represents the basis for "drug target validation" with focus on therapeutic applications. The concepts of "target validation" and "drug-based therapeutic intervention" continue to coevolve as new classes of therapeutic agents and delivery systems emerge and enable us to target or modulate previously inaccessible molecular entities.

Derivatisation of arginine residues with malondialdehyde for the analysis of peptides and protein digests by LC-ESI-MS/MS.

In this study a selective tagging strategy for the derivatisation of arginine residues in peptides is presented. It is based on the reaction of the guanidine group of the arginine side-chain with malondialdehyde (MDA) under strongly acidic conditions, in which a stable pyrimidine ring is formed. The reaction conditions have been optimised so that quantitative modification can be achieved for a variety of peptides. The label has a strong influence on the polarity and basicity of the arginine side-chain and thus on the chromatographic and mass spectrometric properties of arginine-containing peptides. For example, retention, particularly of small and polar peptides as well as arginine-rich peptides, is significantly increased by derivatisation, and therefore sensitivity is also enhanced in liquid chromatography-mass spectrometry (LC-MS). The arginine side-chain also has a strong impact on the fragmentation behaviour of peptides in tandem mass spectrometry. This has been investigated for standard peptides for which, in some cases, significantly more fragment ions were formed after derivatisation. Finally, the method was tested for tryptic digests of standard proteins to demonstrate how the tagging strategy can give improved or complementary information for protein identification.

Chemical proteomics and its application to drug discovery.

The completion of the human genome sequencing project has provided a flood of new information that is likely to change the way scientists approach the study of complex biological systems. A major challenge lies in translating this information into new and better ways to treat human disease. The multidisciplinary science of chemical proteomics can be used to distill this flood of new information. This approach makes use of synthetic small molecules that can be used to covalently modify a set of related enzymes and subsequently allow their purification and/or identification as valid drug targets. Furthermore, such methods enable rapid biochemical analysis and small-molecule screening of targets thereby accelerating the often difficult process of target validation and drug discovery.