Dynamic Proteome Profiling Research Articles

Comparative Analysis of Schistosoma japonicum from Pairing-to-Sexual Maturation based on iTRAQ Proteomics

Objective: Schistosomiasis, which is caused by the schistosome worm, poses significant health challenges. Understanding the sexual development and maturation of schistosomes would provide valuable insight for preventing the transmission of schistosomiasis and pathologic damage to the host. Methods: Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to monitor the dynamic proteomic profiles in Schistosoma japonicum during development from pairing-to-maturation. RNA interference (RNAi) experiments were used to elucidate sex-biased gene function. Results: Of the 2927 identified proteins, 58.6% showed differential expression after comparing sexes and developmental stages. Both male and female worms displayed a similar number of gender-differentially expressed proteins after pairing. However, these proteins exhibited significant gender-specific functions, with reproduction central in females, while males were enriched in metabolic processes. Females exhibited 73% of their time-variant protein expression during 22-26 days post-infection (dpi), while males had 62% during 18-22 dpi, indicating earlier maturation in males. Functional analysis revealed different peptidases expressed during male and female development and maturation. Sex-biased SjU2AF exhaustion led to worm development delay, abnormal reproductive organ development, and death. Conclusions: Comparative proteomics enhances our understanding of mechanisms underlying schistosome maturation and reveals a new potential target for chemotherapy and vaccines.

Metal organic layers enabled cell surface engineering coupling biomembrane fusion for dynamic membrane proteome profiling.

Systematically dissecting the highly dynamic and tightly communicating membrane proteome of living cells is essential for the system-level understanding of fundamental cellular processes and intricate relationship between membrane-bound organelles constructed through membrane traffic. While extensive efforts have been made to enrich membrane proteins, their comprehensive analysis with high selectivity and deep coverage remains a challenge, especially at the living cell state. To address this problem, we developed the cell surface engineering coupling biomembrane fusion method to map the whole membrane proteome from the plasma membrane to various organelle membranes taking advantage of the exquisite interaction between two-dimensional metal-organic layers and phospholipid bilayers on the membrane. This approach, which bypassed conventional biochemical fractionation and ultracentrifugation, facilitated the enrichment of membrane proteins in their native phospholipid bilayer environment, helping to map the membrane proteome with a specificity of 77% and realizing the deep coverage of the HeLa membrane proteome (5087 membrane proteins). Furthermore, membrane N-phosphoproteome was profiled by integrating the N-phosphoproteome analysis strategy, and the dynamic membrane proteome during apoptosis was deciphered in combination with quantitative proteomics. The features of membrane protein N-phosphorylation modifications and many differential proteins during apoptosis associated with mitochondrial dynamics and ER homeostasis were found. The method provided a simple and robust strategy for efficient analysis of membrane proteome, offered a reliable platform for research on membrane-related cell dynamic events and expanded the application of metal-organic layers.

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Understanding the effects of nutrition and post-exercise nutrition on skeletal muscle protein turnover: Insights from stable isotope studies

Dynamic Proteome Profiling of Protein Fractional and Molar Synthesis Rates in Human Muscle in vivo

This study aimed to develop a method for measuring the abundance and synthesis rate of human muscle proteins in molar rather than fractional (%/d) terms. Losses in proteostasis are implicated in ageing and chronic diseases but research is hindered by a lack of methods for studying protein dynamics in humans. Protein‐specific fractional synthesis rate (FSR) can be measured by stable isotope labelling and peptide mass spectrometry (MS). However, FSR is a measurement of an unknown whole, i.e. protein abundance. If protein abundance changes or is different between conditions, the interpretation of FSR data may be confounded.Three physically active males (21 ± 1 years; height 178 ± 1 cm; weight 75 ± 5 kg) gave their informed consent to the ethically approved procedures. Newly synthesised proteins were labelled by oral consumption of deuterium oxide (50 ml of 99.8 atom %) taken four times per day during days 1–5, and two times per day during days 6–12. Samples of blood and vastus lateralis were collected after an overnight fast on days 0, 4, 8 and 12. Precursor enrichment and incorporation of deuterium oxide into muscle proteins were determined by liquid chromatography tandem MS. Protein abundances were normalised to 30 fmol yeast alcohol dehydrogenase added during sample preparation. FSR (%/d) was calculated from time‐dependent changes in peptide mass isotopomer abundances. FSR and abundance data (fmol/ μg protein) were combined to calculate molar synthesis rates (MSR; fmol/ μg protein/ d).Abundance data were collected for 1,368 proteins that had at least 1 unique peptide. FSR data were calculated from 3,944 peptides representing 940 proteins (average 3 peptides per protein, range 1 – 53 peptides per protein). In total, 769 proteins had abundance and FSR data in two or more participants. The median (M), lower‐(Q1) and upper‐quartile (Q3) values for protein FSR (%/d) were M = 4.39, Q1 = 0.96, Q3 = 7.84. Alpha skeletal muscle actin had the lowest FSR (0.28 ± 0.1 %/d) whereas the highest recorded FSR was 54.43 ± 8.9 %/d (nuclear protein localization protein 4 homolog). tRNA ligases were common amongst top‐ranked proteins by FSR but were ranked amongst the lowest in abundance e.g. Leucine‐‐tRNA ligase was the lowest abundance (5 amol/ μg protein) protein detected. The most abundant protein was myoglobin (1.52 pmol/ μg protein). Average MSR (fmol/ μg protein/ d) was M = 0.05 (Q1 = 0.017, Q3 = 0.13). Metabolic enzymes were amongst the top‐ranked proteins by MSR (range 1–10 fmol/ μg protein/ d) alongside cullin‐associated NEDD8‐dissociated protein 2 (0.9 ± 0.2 fmol/ μg protein/ d) and antioxidant enzymes, including mitochondrial superoxide dismutase [Mn], peroxiredoxin 3, peroxiredoxin 2 and glutathione S‐transferase‐P (each ~0.5 fmol/ μg protein/ d).MSR proteomic data offers new insight to the allocation of cellular resources. Future applications investigating muscle adaptation or differences between health and disease may uncover unexpected insight regarding which proteins are most synthesised and highlight new avenues of research in human muscle physiology.

Dynamic proteome profiling of human pluripotent stem cell-derived pancreatic progenitors.

A comprehensive characterization of the molecular processes controlling cell fate decisions is essential to derive stable progenitors and terminally differentiated cells that are functional from human pluripotent stem cells (hPSCs). Here, we report the use of quantitative proteomics to describe early proteome adaptations during hPSC differentiation toward pancreatic progenitors. We report that the use of unbiased quantitative proteomics allows the simultaneous profiling of numerous proteins at multiple time points, and is a valuable tool to guide the discovery of signaling events and molecular signatures underlying cellular differentiation. We also monitored the activity level of pathways whose roles are pivotal in the early pancreas differentiation, including the Hippo signaling pathway. The quantitative proteomics data set provides insights into the dynamics of the global proteome during the transition of hPSCs from a pluripotent state toward pancreatic differentiation.

Reliability of Protein Abundance and Synthesis Measurements in Human Skeletal Muscle

The repeatability of dynamic proteome profiling (DPP), which is a novel technique for measuring the relative abundance (ABD) and fractional synthesis rate (FSR) of proteins in humans, is investigated. LC-MS analysis is performed on muscle samples taken from male participants (n=4) that consumed 4×50mL doses of deuterium oxide (2 H2 O) per day for 14 days. ABD is measured by label-free quantitation and FSR is calculated from time-dependent changes in peptide mass isotopomer abundances. One-hundred one proteins have at least one unique peptide and are used in the assessment of protein ABD. Fifty-four of these proteins meet more stringent criteria and are used in the assessment of FSR data. The median (M), lower-, (Q1 ) and upper-quartile (Q3 ) values for protein FSR (%/d) are M=1.63, Q1 =1.07, and Q3 =3.24, respectively. The technical CV of ABD data has a median value of 3.6% (Q1 1.7% to Q3 6.7%), whereas the median CV of FSR data is 10.1% (Q1 3.5% to Q3 16.5%). These values compare favorably against other assessments of technical repeatability of proteomics data, which often set a CV of 20% as the upper bound of acceptability.

Dynamic N-glycoproteome analysis of maize seedling leaves during de-etiolation using Concanavalin A lectin affinity chromatography and a nano-LC-MS/MS-based iTRAQ approach.

The identification of N -glycosylated proteins with information about changes in the level of N -glycosylation during de-etiolation provides a database that will aid further research on plant N -glycosylation and de-etiolation. N-glycosylation is one of the most prominent and abundant protein post-translational modifications in all eukaryotes and in plants it plays important roles in development, stress tolerance and immune responses. Because light-induced de-etiolation is one of the most dramatic developmental processes known in plants, seedlings undergoing de-etiolation are an excellent model for investigating dynamic proteomic profiles. Here, we present a comprehensive, quantitative N-glycoproteomic profile of maize seedlings undergoing 12h of de-etiolation obtained using Concanavalin A (Con A) lectin affinity chromatography enrichment coupled with a nano-LC-MS/MS-based iTRAQ approach. In total, 1084 unique N-glycopeptides carrying 909 N-glycosylation sites and corresponding to 609 proteins were identified and quantified, including 186 N-glycosylation sites from 162 proteins that were significantly regulated over the course of the 12h de-etiolation period. Based on hierarchical clustering analysis, the significantly regulated N-glycopeptides were divided into seven clusters that showed different N-glycosylation patterns during de-etiolation. We found no obvious difference in the enriched MapMan bincode categories for each cluster, and these clustered significantly regulated N-glycoproteins (SRNPs) are enriched in miscellaneous, protein, cell wall and signaling, indicating that although the N-glycosylation regulation patterns of these SRNPs might differ, they are involved in similar biological processes. Overall, this study represents the first large-scale quantitative N-glycoproteome of the model C4 plant, maize, which is one of the most important cereal and biofuel crops. Our results greatly expand the maize N-glycoproteomic database and also shed light on the potential roles of N-glycosylation modification during the greening of maize leaves.

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high‐fat diet and resistance exercise

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise. Precursor enrichment and the rate of incorporation of deuterium oxide into newly synthesized muscle proteins were determined by mass spectrometry. Ninety proteins were included in the DPP, with 28 proteins exhibiting significant responses to REX. The most common pattern of response was an increase in turnover, followed by an increase in abundance with no detectable increase in protein synthesis. Here, we provide novel evidence that demonstrates that the contribution of synthesis and breakdown to changes in protein abundance induced by REX differ on a protein-by-protein basis. We also highlight the importance of the degradation of individual muscle proteins after exercise in human skeletal muscle.-Camera, D. M., Burniston, J. G., Pogson, M. A., Smiles, W. J., Hawley, J. A. Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

Selection of AECOPD-specific immunomodulatory biomarkers by integrating genomics and proteomics with clinical informatics.

Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) as a serious event has high mortality and medical costs. Systemic inflammation and immune response are the major factors influencing the outcome and quality of patient with AECOPD. On basis of identification and validation of AECOPD-specific inflammatory biomarkers, the present study aimed to identify AECOPD-specific immunomodulatory mediators by evaluating dynamic genomic and proteomic profiles of peripheral blood mononuclear cells (PBMCs) and plasma in patients with AECOPD on day 1, 3, and 10 after the hospital admission, to compare with healthy controls or patients with stable COPD. We found that genes and proteins of C1QC and C1RL were co-differentially up-expressed in patients with COPD or AECOPD, while haptoglobin (HP), ORM1, SERPING1, and C3 were identified as a panel of AECOPD-specific immunomodulatory mediators. We also found that inflammatory stimuli could up-regulate osteopontin (OPN)-associated HP expression through the PI3K signal pathway in A549 cells. Block of autocrine production of OPN by gene inhibition could reduce HP production from inflammation-induced lung epithelial cells. The complex network of AECOPD- or COPD-specific immunomodulatory mediators will benefit the development of precision or personalized medicine strategies for prevention and treatment of AECOPD.

Dynamic Proteome Profiling of the Human Muscle Response to Resistance Exercise Training

Resistance exercise is a potent stimulus for muscle adaptation that is underpinned by selective changes in protein abundance. Our objective was to use Dynamic Proteome Profiling to calculate the contributions of synthesis and degradation to changes in abundance on a protein by protein basis. After 3 days of dietary control (65% Carbohydrate, 20% Fat, 15% Protein), two groups (n=8, in each) of age‐matched (37 ± 5 y old), overweight (BMI: 26 ± 3), untrained (38 ± 6VO2max ml/kg/min) males consumed a high‐fat diet (20% Carbohydrate, 65% Fat, 15% Protein) and ingested deuterium oxide (200 ml/d) during a 9‐day intervention period. Venous blood samples were collected daily and samples of vastus lateralis were collected prior to (i.e. day 0 control) and on days 3, 6 and 9 of the intervention. Participants were assigned either to a non‐exercise group or an exercise group that performed resistance training (leg press, leg extension and dumbbell squats; each 4 × 10 repetitions at 80%1RM) on days 1, 4 and 7. Blood and muscle samples were analysed by mass spectrometry to determine precursor enrichment and the rate of incorporation of deuterium in to newly synthesised proteins. The relative abundance of each protein was analysed by label‐free profiling and the contribution of protein degradation was then calculated. Precursor (body water) enrichment increased at a rate of 0.14 ± 0.05 %/d and reached 2.14 ± 0.08 % after 9 days. Muscles were analysed as myofibrillar or sarcoplasmic fractions. The average rate of synthesis of mixed sarcoplasmic proteins was similar in control (2.05 ± %/d) and (2.19 ± %/d) exercised muscle. In the myofibrillar fraction, the average rate of synthesis was 45 % greater (P<0.05) in exercised (1.58 ± %/d) compared to control (1.09 ± %/d) muscles. In total, 90 proteins were included in the Dynamic Proteome Profiling. There were no significant between‐group differences in the muscle proteome prior to exercise training and no changes in protein abundance occurred in the muscle of participants in the non‐exercise group. In contrast, 24 proteins exhibited statistically significant differences in either synthesis rate or abundance in response to resistance training. The most common pattern of response was an increase in turnover. For example, the synthesis rate of myosin heavy chain IIa was 2‐fold greater in exercised muscle but this was not associated with a statistically significant increase in protein abundance. Conversely, 3 glycolytic enzymes (beta‐enolase, phosphoglycerate mutase 2 and triosephosphate isomerase) became significantly less abundant in exercise‐trained muscle despite exhibiting significantly elevated rates of synthesis. This suggests protein degradation was increased to a greater extent than the measured increase in synthesis. Our findings provide empirical evidence that the contributions that synthesis and degradation make to changes in protein abundance induced by resistance exercise differs on a protein by protein basis.Support or Funding InformationThis project was funded, in part by a CRN Grant 2013000443 to JAH

Proteomic and phosphoproteomic analysis reveals the response and defense mechanism in leaves of diploid wheat T. monococcum under salt stress and recovery

Salinity is a major abiotic stress factor affecting crops production and productivity. Triticum monococcum is closely related to Triticum urartu (AUAU), which is used as a model plant of wheat A genome study. Here, salt stress induced dynamic proteome and phosphoproteome profiling was focused. The T. monococcum seedlings were initially treated with different concentrations of NaCl ranging from 80 to 320mM for 48h followed by a recovery process for 48h prior to proteomic and phosphoproteomic analysis. As a result, a total of 81 spots corresponding to salt stress and recovery were identified by MALDI-TOF/TOF-MS from 2-DE gels. These proteins were mainly involved in regulatory, stress defense, protein folding/assembly/degradation, photosynthesis, carbohydrate metabolism, energy production and transportation, protein metabolism, and cell structure. Pro-Q Diamond staining was used to detect the phosphoproteins. Finally, 20 spots with different phosphorylation levels during salt treatment or recovery compared with controls were identified. A set of potential salt stress response and defense biomarkers was identified, such as cp31BHv, betaine-aldehyde dehydrogenase, leucine aminopeptidase 2, Cu/Zn superoxide dismutase, and 2-Cys peroxiredoxin BAS1, which could lead to a better understanding of the molecular basis of salt response and defense in food crops. Biological significanceSoil salinity reduces the yield of the major crops, which is one of the severest problems in irrigated agriculture worldwide. However, how crops response and defense during different levels of salt treatment and recovery processes is still unclear, especially at the post-translational modification level. T. monococcum is a useful model for common wheat. Thus, proteomic and phosphoproteomic analyses of T. monococcum leaves were performed in our study, which provided novel insights into the underlying salt response and defense mechanisms in wheat and other crops.

Protein expression profiling in head fragments during planarian regeneration after amputation.

Following amputation, a planarian tail fragment can regrow into a complete organism including a well-organized brain within about 2-3 weeks, thus restoring the structure and function to presurgical levels. Despite the enormous potential of these animals for regenerative medicine, our understanding of the exact mechanism of planarian regeneration is incomplete. To better understand the molecular nature of planarian head regeneration, we applied two-dimensional electrophoresis (2-DE)/matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)/time-of-flight mass spectrometry (TOF MS) technique to analyze the dynamic proteomic expression profiles over the course of 6 to 168 h post-decapitation. This approach identified a total of 141 differentially expressed proteins, 47 of which exhibited exceptionally high fold changes (≥3-fold change). Of these, Rx protein, an important regulator of head and brain development, was considered to be closely related to planarian head regeneration because of its exceptional high expression almost throughout the time course of regeneration process. Functional annotation analysis classified the 141 proteins into eight categories: (1) signaling, (2) Ca(2+) binding and translocation, (3) transcription and translation, (4) cytoskeleton, (5) metabolism, (6) cell protection, (7) tissue differentiation, and (8) cell cycle. Signaling pathway analysis indicated that Wnt1/Ca(2+) signaling pathway was activated during head regeneration. Integrating the analyses of proteome expression profiling, functional annotation, and signaling pathway, amputation-induced head reformation requires some mechanisms to promote cell proliferation and differentiation, including differential regulation of proapoptotic and antiapoptotic proteins, and the regulation of proliferation and differentiation-related proteins. Importantly, Wnt1/Ca(2+) signaling pathway upregulates Rx expression, finally facilitating the differentiation of neoblasts into various cell types. Taken together, our study demonstrated that proteomic analysis approach used by us is a powerful tool in understanding molecular process related to head regeneration of planarian.

Comparative analysis of the dynamic proteomic profiles in berry skin between red and white grapes (Vitis vinifera L.) during fruit coloration

The aim of this study was to deepen our understanding of the proteomic profiles related to skin coloration at the whole proteome level through the analysis of multiple protein spots. A comparative proteomics analysis was conducted on the berry skins of red and white progeny derived from a single cross, ‘Jingxiu’ (red)בMuscat of Alexandria’ (white), to minimize genetic background differences. Among the 1000–1800 spots assessed, the levels of 57, 70 and 26 proteins were lower in the skins of white progeny, compared to the skins of red progeny at about two weeks before veraison, at veraison and at maturity, respectively. Alternatively, the levels of 54, 50 and 23 proteins were higher at about two weeks before veraison, at veraison and at maturity, respectively. The levels of proteins involved in anthocyanin biosynthesis, especially UFGT and AOMT, were lower in the skins of white progeny at veraison and at maturity, and the levels of most proteins involved in amino acid metabolism were found to be lower before veraison. Almost all differentially accumulated proteins involved in energy production were found before and at veraison, while there was no difference detected in the levels of glycolysis, tricarboxylic acid cycle and photosynthesis between the skins of red and white progeny. The differential accumulation of enzyme proteins involved in anthocyanin synthesis had a decisive influence on the different skin colorization of red and white progeny. Many proteins related to metabolism and energy production were also differentially accumulated. In addition, many differences in the skin proteomes of white and red progeny were apparent before veraison, i.e. before skins developed coloration.

Comparative Analysis of Dynamic Proteomic Profiles between in Vivo and in Vitro Produced Mouse Embryos during Postimplantation Period

Assisted reproductive technology (ART) increasingly is associated with long-term side-effects on postnatal development and behaviors. High-throughput gene expression analysis has been extensively used to explore mechanisms responsible for these disorders. Our study, for the first time, provides a comparative proteomic analysis between embryos after in vivo fertilization and development (IVO, control) and in vitro fertilization and culture (IVP). By comparing the dynamic proteome during the postimplantation period, we identified 300 and 262 differentially expressed proteins (DEPs) between IVO and IVP embryos at embryonic day 7.5 (E7.5) and E10.5, respectively. Bioinformatic analysis showed many DEPs functionally associated with post-transcriptional, translational, and post-translational regulation, and these observations were consistent with correlation analysis between mRNA and protein abundance. In addition to altered gene expression due to IVP procedures, our findings suggest that aberrant processes at these various levels also contributed to proteomic alterations. In addition, numerous DEPs were involved in energy and amino acid metabolism, as well as neural and sensory development. These DEPs are potential candidates for further exploring the mechanism(s) of ART-induced intrauterine growth restriction and neurodevelopmental disorders. Moreover, significant enrichment of DEPs in pathways of neurodegenerative diseases implies the potentially increased susceptibility of ART offspring to these conditions as adults.

Relationships between Hematopoiesis and Hepatogenesis in the Midtrimester Fetal Liver Characterized by Dynamic Transcriptomic and Proteomic Profiles

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation.