Comparative Proteomic Method Research Articles

Label-free based comparative proteomics approach revealed the changes in proteomic profiles driven by different maturities in two Chinese white truffles, Tuber panzhihuanense and Tuber latisporum

T. panzhihuanense and T. latisporum are white truffle species native to China, of which T. panzhihuanense has significant commercial potential, with high nutritional value and unique flavor. Maturity is an important factor affecting the nutrition and aroma of truffles, which determines their economic status. Here, a label-free-based comparative proteomics method was used to determine the proteomic profiles of T. panzhihuanense and T. latisporum at two different stages of maturity. The results showed that both maturity and species significantly affected the protein expression patterns. T. panzhihuanense responded stronger to maturity than T. latisporum, accompanied by a more complex interaction network between proteins. Some critical proteins were regulated by maturity and variety, including those involved in aroma formation, e.g., S-adenosyl-methionine synthetase. The enrichment of oxidation–reduction processes, glycolysis, and SNARE interactions in vesicular transport were driven by species and maturity. This study provides the first insights into the proteomic profiles of T. panzhihuanense and T. latisporum, revealing the roles of key proteins and biological processes in their maturation.

Metabolism of chiral sulfonate compound 2,3-dihydroxypropane-1-sulfonate (DHPS) by Roseobacter bacteria in marine environment.

The sulfonate compound 2,3-dihydroxypropane-1-sulfonate (DHPS) is one of the most abundant organic sulfur compounds in the biosphere. DHPS derived from dietary intake could be transformed into sulfide by intestinal microbiota and thus impacts human health. However, little is known about its sulfur transformation and subsequent impacts in marine environment. In this study, laboratory-culturing was combined with targeted metabolomic, chemical fluorescence probing, and comparative proteomic methods to examine the bioavailability of chiral DHPS (R and S isomers) for bacteria belonging to the marine Roseobacter clade. The metabolic potential of DHPS in bacteria was further assessed based on genomic analysis. Roseobacter members Ruegeria pomeroyi DSS-3, Dinoroseobacter shibae DFL 12, and Roseobacter denitrificans OCh 114 could utilize chiral DHPS for growth, producing sulfite. They all contained a similar gene cluster for DHPS metabolism but differed in the genes encoding enzymes for desulfonation. There was no significant difference in the growth rate and DHPS consumption rate for R. pomeroyi DSS-3 between R- and S-DHPS cultures, with few proteins expressed differentially were found. Proteomic data suggested that a series of hydrogenases oxidized DHPS, after which desulfonation could proceed via three distinct enzymatic pathways. Strain R. pomeroyi DSS-3 completed the desulfonation via L-cysteate sulfo-lyase, while D. shibae DFL 12 and R. denitrificans OCh 114 primarily utilized sulfolactate sulfo-lyase, and sulfopyruvate decarboxylase followed by sulfoacetaldehyde acetyltransferase, respectively, to complete desulfonation releasing the sulfonate-moiety. The sulfite could be further oxidized or incorporated into sulfate assimilation, indicated by the proteomic data. Furthermore, DHPS metabolic pathways were found primarily in marine bacterial groups, including the majority of sequenced Roseobacter genomes. Our results suggest that chiral DHPS, as a vital reduced sulfur reservoir, could be metabolized by marine bacteria, providing a resource for bacterial growth, rather than acting as a source of toxic sulfide within the marine ecosystem.

Advances of proteomics technologies for multidrug-resistant mechanisms.

Multidrug resistance (MDR) is a vital issue in cancer treatment. Drug resistance can be developed through a variety of mechanisms, including increased drug efflux, activation of detoxifying systems and DNA repair mechanisms, and escape of drug-induced apoptosis. Identifying the exact mechanism related in a particular case is a difficult task. Proteomics is the large-scale study of proteins, particularly their expression, structures and functions. In recent years, comparative proteomic methods have been performed to analyze MDR mechanisms in drug-selected model cancer cell lines. In this paper, we review the recent developments and progresses by comparative proteomic approaches to identify potential MDR mechanisms in drug-selected model cancer cell lines, which may help understand and design chemical sensitizers.

Developing Well-Annotated Species-Specific Protein Databases Using Comparative Proteogenomics.

Proteomics is a mass spectrometry-based discipline that aims to analyze proteomes and their functions. Many proteomic studies require well-developed protein databases for reference. However, most proteomes are not well-annotated, aside from model organisms. Techniques like six-frame translation, ab initio gene prediction, and EST databases can aid in maximizing the amount of proteins identified in proteomics experiments, however, each of these has its downfalls. Proteogenomics is a term used to describe the union of proteomics, genomics and transcriptomics to assist in the identification of peptides which would help build better annotated proteome databases. Here, current proteomic and proteogenomic methods will be reviewed, and an example of a comparative proteomics method using lake trout liver samples will be described.

ITRAQ-Based Proteomics Analysis and Network Integration for Kernel Tissue Development in Maize.

Grain weight is one of the most important yield components and a developmentally complex structure comprised of two major compartments (endosperm and pericarp) in maize (Zea mays L.), however, very little is known concerning the coordinated accumulation of the numerous proteins involved. Herein, we used isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomic method to analyze the characteristics of dynamic proteomics for endosperm and pericarp during grain development. Totally, 9539 proteins were identified for both components at four development stages, among which 1401 proteins were non-redundant, 232 proteins were specific in pericarp and 153 proteins were specific in endosperm. A functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the tissue development. Three and 76 proteins involved in 49 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were integrated for the specific endosperm and pericarp proteins, respectively, reflecting their complex metabolic interactions. In addition, four proteins with important functions and different expression levels were chosen for gene cloning and expression analysis. Different concordance between mRNA level and the protein abundance was observed across different proteins, stages, and tissues as in previous research. These results could provide useful message for understanding the developmental mechanisms in grain development in maize.

A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy

Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins.

Abstract B05: Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer

Abstract Pancreatic cancer is an exceptional aggressive cancer, and acquired radiation resistance in this type of cancer is common. The objective of this study was to use a comparative proteomic method to identify the proteins that are responsible for acquired radioresistance in pancreatic cancer cells. In order to eliminate the complications caused by genetic background variations, we first generated a pancreatic cancer isogenic stable cell line that differs in radioresistance. The isogenic stable cell line was generated by exposing the pancreatic cancer MIA PaCa-2 cells (an undifferentiated pancreatic cancer cell line) to a total dose of 60 Gy of ionizing radiation (IR; gamma radiation) in 30 fractions over a period of 6 weeks, a protocol that is commonly used in clinical radiotherapy. The clones that survived from the fractionated exposures showed strong radioresistance, which could be demonstrated by both cell apoptotic assays and the long-term clonogenic survival assays. We then used a mass spectrometry-based quantitative proteomic method to systematically compare the expression of proteins between the radiosensitive parental cells and the selected radioresistant cells. One protein identified to be substantially upregulated in the radioresistant cells was ecto-5’-nucleotidase (CD73), a cell surface enzyme that converts adenosine monophosphate (AMP) into adenosine and is known to play important roles in cell-cell/cell-matrix interactions and immune suppression. To examine whether the upregulated CD73 was functionally related to the acquired radioresistance of pancreatic cancer cells, we silenced or ectopically overexpressed CD73 gene in the cells and examined the effect of the expression manipulations on long-term survival and apoptosis of the cells in response to IR exposure. The results demonstrated that knockdown of CD73 in the radioresistant cells significantly decreased the long-term survival rates, and resensitized the cells to apoptosis after the cells were exposed to IR. Concomitantly, stable overexpression of CD73 in the radiosensitive parental cells significantly increased the long-term survival rates, and enhanced resistance of the cells to apoptosis upon exposure of the cells to IR. To determine if the enzyme activity of CD73 is important for its effect on radioresistance, we generated an enzyme-dead mutant CD73 and overexpressed the mutant CD73 in the parental cells. Functional assays demonstrated that the mutant CD73 affected radioresistance of pancreatic cancer cells in a similar way as wild-type CD73, suggesting that the enzyme activity is not related to its effect on radioresistance. In summary, our results suggest that upregulation of CD73 expression is an important mechanism by which pancreatic cancer cells acquire resistance to IR, and the effect of CD73 on radioresistance in pancreatic cancer cells is enzyme-independent and at least partially through affecting the sensitivity of the cells to IR-induced apoptosis. Citation Format: Anna M. Nguyen, Jianhong Zhou, Yuchun Du.{Authors}. Ecto-5’-nucleotidase (CD73) confers radioresistance in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B05.

Comparative proteome analysis of egg yolk plasma proteins during storage

Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P < 0.05) alterations in abundance at different storage times. The proteins that showed significant changes in abundance included serum albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry.

Osteoblast-released Matrix Vesicles, Regulation of Activity and Composition by Sulfated and Non-sulfated Glycosaminoglycans

Our aging population has to deal with the increasing threat of age-related diseases that impair bone healing. One promising therapeutic approach involves the coating of implants with modified glycosaminoglycans (GAGs) that mimic the native bone environment and actively facilitate skeletogenesis. In previous studies, we reported that coatings containing GAGs, such as hyaluronic acid (HA) and its synthetically sulfated derivative (sHA1) as well as the naturally low-sulfated GAG chondroitin sulfate (CS1), reduce the activity of bone-resorbing osteoclasts, but they also induce functions of the bone-forming cells, the osteoblasts. However, it remained open whether GAGs influence the osteoblasts alone or whether they also directly affect the formation, composition, activity, and distribution of osteoblast-released matrix vesicles (MV), which are supposed to be the active machinery for bone formation. Here, we studied the molecular effects of sHA1, HA, and CS1 on MV activity and on the distribution of marker proteins. Furthermore, we used comparative proteomic methods to study the relative protein compositions of isolated MVs and MV-releasing osteoblasts. The MV proteome is much more strongly regulated by GAGs than the cellular proteome. GAGs, especially sHA1, were found to severely impact vesicle-extracellular matrix interaction and matrix vesicle activity, leading to stronger extracellular matrix formation and mineralization. This study shows that the regulation of MV activity is one important mode of action of GAGs and provides information on underlying molecular mechanisms.

Potential mechanism of apoptosis induced by ultrasound in human hepatocarcinoma cells via comparative proteomic analysis.

To analyze the potential molecular mechanism of ultrasound induced apoptosis in cancer cells, comparative proteomic methods were introduced in the study. After ultrasound exposure at the intensity of 1.2 W/cm2, the human SMMC-7721 hepatocarcinoma cells were stained by trypan blue to detect the morphologic changes, and then the flow cytometry was used to examine the percentage of early apoptosis via double staining of FITC-labelled Annexin V and Propidium iodide. The proteins were separated by two-dimensional (2D) SDS polyacrylamide gel electrophoresis (PAGE). Among them, the differently expressed proteins were identified by MALDI-TOF mass spectrometry to reveal the key proteins response to ultrasound exposure. It's proved early apoptosis of cells were induced by focused ultrasound. After ultrasound exposure, the expressing characteristics of several proteins changed, in which some proteins in HSP family are associated with apoptosis initiation. It is suggested that the focused ultrasound could be applied in the assistant cancer therapy. Moreover, it is proved the comparative proteomic methods could supply information about the protein expression to analyze the metabolic processes related to bio-effects of biomedical ultrasound.

Analysis of phosphoproteome in rice pistil.

As the female reproductive part of a flower, the pistil consists of the ovary, style, and stigma, and is a critical organ for the process from pollen recognition to fertilization and seed formation. Previous studies on pollen-pistil interaction mainly focused on gene expression changes with comparative transcriptomics or proteomics method. However, studies on protein PTMs are still lacking. Here we report a phosphoproteomic study on mature pistil of rice. Using IMAC enrichment, hydrophilic interaction chromatography fraction and high-accuracy MS instrument (TripleTOF 5600), 2347 of high-confidence (Ascore ≥ 19, p ≤ 0.01), phosphorylation sites corresponding to 1588 phosphoproteins were identified. Among them, 1369 phosphorylation sites within 654 phosphoproteins were newly identified; 41 serine phosphorylation motifs, which belong to three groups: proline-directed, basophilic, and acidic motifs were identified after analysis by motif-X. Two hundred and one genes whose phosphopeptides were identified here showed tissue-specific expression in pistil based on information mining of previous microarray data. All MS data have been deposited in the ProteomeXchange with identifier PXD000923 (http://proteomecentral.proteomexchange.org/dataset/PXD000923). This study will help us to understand pistil development and pollination on the posttranslational level.

Comparative proteomic analysis of hearts of adult SCNT Bama miniature pigs (Sus scrofa)

This study aims to determine the effects of SCNT on cardiac development of SCNT pigs through proteomic methods. Heart proteins from three adult SCNTs and two normal reproductive Bama miniature pigs were extracted, separated, and identified via comparative proteomic methods, including two-dimensional gel electrophoresis, mass spectrometry, and Western blot. Eleven differentially expressed spots were identified as differentially expressed proteins, of which five spots were upregulated proteins such as cardiac myosin heavy chain, cathepsin D, and heat shock protein beta-1 (HSP27). By contrast, six spots were downregulated proteins such as alpha skeletal muscle and actin. The results also demonstrated that nuclear transfer might result in abnormal expression of some important proteins in hearts from SCNT pigs, and affect the cardiac development in SCNT pigs' survival.

Profiling the potential tumor markers of pancreatic ductal adenocarcinoma using 2D-DIGE and MALDI-TOF-MS: Up-regulation of Complement C3 and alpha-2-HS-glycoprotein

Background/aimsPancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with an increasing incidence worldwide. Due to lack of early diagnosis and poor prognosis, it is rather critical to improve the early diagnosis of PDAC. A comparative proteomic method was used to analyze serum proteins to find a new potential specific marker. MethodsComparative analysis of the pancreatic peripheral blood protein profiling from 40 pancreatic cancer patients, 10 pancreatic benign tumor patients, 10 chronic pancreatitis patients and 40 cancer-free controls. The samples were carried out by 2D-differential gel electrophoresis (2D-DIGE) and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Two up-regulated proteins were further validation by real time RT-PCR, Western blot analysis and Immunohistochemistry (IHC). ResultsWe identified fourteen differently expressed proteins in PDAC group compared with cancer-free control group, including 9 up-regulation and 5 down-regulation proteins. Increased Complement C3 and alpha-2-HS-glycoprotein (AHSG) were further confirmed by real time RT-PCR, Western blot analysis and IHC. The expressions of Complement C3 and AHSG were higher in PDAC than that in other groups. ConclusionsThese results suggest that Complement C3 and AHSG might be the potential tumor markers in PDAC screening and diagnosis. The finding of inflammation mediated factor Complement C3 revealed that inflammation might be closely related with the occurrence and development process of PDAC.

Comparative Proteomic Analysis of Human SH-SY5Y Neuroblastoma Cells under Simulated Microgravity

Microgravity is one of the most important features in spaceflight. Previous evidence has shown that neurophysiological impairment signs occurred under microgravity. The present study was undertaken to explore the change in protein abundance in human SH-SY5Y neuroblastoma cells that were grown in a microgravity environment. The comparative proteomic method based on the (18)O labeling technique was applied to investigate the up-regulated proteins and down-regulated proteins in SH-SY5Y under simulated microgravity. Twenty-two differentially abundant proteins were quantified in human SH-SY5Y neuroblastoma cells. The cell microfilament network was disrupted under simulated microgravity, which was determined by the immunocytochemistry. The concentration of reactive oxygen species, malondialdehyde, and free Ca2+ ion significantly increased, and the level of ATP significantly decreased under simulated microgravity. However, there was no obvious cell apoptosis observed under simulated microgravity. These results provide new molecular evidence for the change in protein abundance in SH-SY5Y cells under simulated microgravity, which might unfold biological mechanisms and the development of effective countermeasures to deal with microgravity-related neurological problems. We believe that the state-of-the-art proteomic assay may be a means by which aerospace scientists will begin to understand the underlying mechanisms of space life activities at the protein level.

Exploring Pathogenic Mechanisms of Botrytis cinerea Secretome under Different Ambient pH Based on Comparative Proteomic Analysis

Botrytis cinerea causes gray mold rot on over 200 plant species worldwide, resulting in great economic loss every year. Cooperation of proteins secreted by B. cinerea plays an important role in its successful infection to host plants. The ambient pH, as one of the most important environmental parameters, can regulate expression of secreted proteins in various fungal pathogens. In the present study, we mainly investigated the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. Distinct differences in secretome of B. cinerea were found between pH 4 and 6 treatments, and 47 differential spots, corresponding to 21 unique proteins, were identified using MALDI-TOF/TOF. At pH 4, more proteins related to proteolysis were induced, whereas most of up-accumulated proteins were cell wall degrading enzymes at pH 6. Analysis of gene expression using quantitative real-time PCR suggests that production of most of these proteins was regulated at the level of transcription. These findings indicate that B. cinerea can adjust protein profile of secretome responding to different ambient pH values and provide evidence to deeply understand the complicated infecting mechanisms of B. cinerea on a wide range of plant hosts.

Acquisition of Resistance of Pancreatic Cancer Cells to 2-Methoxyestradiol Is Associated with the Upregulation of Manganese Superoxide Dismutase

Acquired resistance of cancer cells to anticancer drugs or ionizing radiation (IR) is one of the major obstacles in cancer treatment. Pancreatic cancer is an exceptional aggressive cancer, and acquired drug resistance in this cancer is common. Reactive oxygen species (ROS) play an essential role in cell apoptosis, which is a key mechanism by which radio- or chemotherapy induce cell killing. Mitochondria are the major source of ROS in cells. Thus, alterations in the expression of mitochondrial proteins, involved in ROS production or scavenging, may be closely linked to the resistance of cancer cells to radio- or chemotherapy. In the present study, we generated a stable cell line by exposing pancreatic cancer cells to increasing concentrations of ROS-inducing, anticancer compound 2-methoxyestradiol (2-ME) over a 3-month period. The resulting cell line showed strong resistance to 2-ME and contained an elevated level of ROS. We then used a comparative proteomics method to profile the differential expression of mitochondrial proteins between the parental and the resistant cells. One protein identified to be upregulated in the resistant cells was manganese superoxide dismutase (SOD2), a mitochondrial protein that converts superoxide radicals to hydrogen peroxides. Silencing of SOD2 resensitized the resistant cells to 2-ME, and overexpression of SOD2 led the parental cells to 2-ME resistance. In addition, the 2-ME-resistant cells also showed resistance to IR. Our results suggest that upregulation of SOD2 expression is an important mechanism by which pancreatic cancer cells acquire resistance to ROS-inducing, anticancer drugs, and potentially also to IR.

The orthologue of Sjögren's syndrome nuclear autoantigen 1 (SSNA1) in Trypanosoma brucei is an immunogenic self-assembling molecule.

Primary Sjögren's Syndrome (PSS) is a highly prevalent autoimmune disease, typically manifesting as lymphocytic infiltration of the exocrine glands leading to chronically impaired lacrimal and salivary secretion. Sjögren's Syndrome nuclear autoantigen 1 (SSNA1 or NA14) is a major specific target for autoantibodies in PSS but the precise function and clinical relevance of this protein are largely unknown. Orthologues of the gene are absent from many of the commonly used model organisms but are present in Chlamyodomonas reinhardtii (in which it has been termed DIP13) and most protozoa. We report the functional characterisation of the orthologue of SSNA1 in the kinetoplastid parasite, Trypanosoma brucei. Both TbDIP13 and human SSNA1 are small coiled-coil proteins which are predicted to be remote homologues of the actin-binding protein tropomyosin. We use comparative proteomic methods to identify potential interacting partners of TbDIP13. We also show evidence that TbDIP13 is able to self-assemble into fibril-like structures both in vitro and in vivo, a property which may contribute to its immunogenicity. Endogenous TbDIP13 partially co-localises with acetylated α-tubulin in the insect procyclic stage of the parasite. However, deletion of the DIP13 gene in cultured bloodstream and procyclic stages of T. brucei has little effect on parasite growth or morphology, indicating either a degree of functional redundancy or a function in an alternative stage of the parasite life cycle.

Comparative Proteomic Analysis of Changes in the Bovine Whey Proteome during the Transition from Colostrum to Milk

Bovine whey protein expression patterns of colostrum are much different from that of milk. Moreover, bovine colostrum is an important source of protective, nutritional and developmental factors for the newborn. However, to our knowledge, no research has been performed to date using a comparative proteomic method on the changes in the bovine whey proteome during the transition from colostrum to milk. This study therefore separated whey protein of days 1, 3, 7 and 21 after calving using two dimension electrophoresis. Differentially expressed proteins at different collection times were identified using high-performance liquid chromatography in tandem with mass spectrometry (LC/MS) and validated by enzyme-linked immunosorbent assay (ELISA) in order to understand the developmental changes in the bovine whey proteome during the transition from colostrum to milk. The expression patterns of whey protein of days 1 and 3 post-partum were similar except that immunoglobulin G was down-regulated on day 3, and four proteins were found to be down-regulated on days 7 and 21 compared with day 1 after delivering, including immunoglobulin G, immunoglobulin M, albumin, and lactotransferrin, which are involved in immunity and molecule transport. The results of this study confirm the comparative proteomic method has the advantage over other methods such as ELISA and immunoassays in that it can simultaneously detect more differentially expressed proteins. In addition, the difference in composition of milk indicates a need for adjustment of the colostrum feeding regimen to ensure a protective immunological status for newborn calves.

Intrauterine growth restriction and postnatal high-protein diet affect the kidneys in adult rats

Objective Intrauterine growth restriction (IUGR) is associated with hypertension and chronic kidney disease in adulthood. Postnatal overnutrition after IUGR may be of pathogenic importance for the development of diabetes and cardiovascular disease. This study was to identify the effects of IUGR and a postnatal high-protein diet on the kidneys in adult rats. Methods Intrauterine growth restriction was induced in Sprague-Dawley rats by isocaloric protein restriction in pregnant dams. IUGR pups were divided into two groups that were a standard-protein diet (IUGR group) or a high-protein diet (HP group). A comparative proteomic method was used to study the differences of protein expression profiles between normal adult rats and adult rats with IUGR and the effects of a postnatal high-protein diet on the protein expression profiles of the kidneys. Results The IUGR adults had higher urinary excretion of protein and blood pressure than controls and the HP diet caused more severe hypertension and proteinuria than IUGR itself. The differential proteomic expression analysis found 12 proteins that had significantly differential expression between the IUGR and control groups, which were transcription regulators and structural molecules. The differential proteomic expression analysis between the HP and control groups found 13 proteins that had significantly differential expression and were involved primarily in body metabolism, oxidation reduction, and apoptosis regulation. Conclusion An HP diet intervention after IUGR worsens the severity of hypertension and proteinuria, and this study may provide valuable experimental evidence of proteins involved in the pathogenesis of kidney disease in IUGR and the effect of postnatal overnutrition.

Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.