Determination Of Protein Abundance Research Articles

Effect of pH and lipopolysaccharide on tight junction regulators and inflammatory markers in intestinal cells as an experimental model of weaning transition in dairy calves

Acidic conditions combined with the presence of lipopolysaccharide (LPS) may increase the permeability of gastrointestinal epithelium. Feeding starch-rich starter to dairy calves is associated with ruminal acidosis and decreases the pH of other segments of the gastrointestinal tract, and that affects the tight junction regulator. The objective was to evaluate the effect of the combination of different pH (7.4 vs. 6.0) and LPS concentrations (0, 0.5, 10 ng/mL) in intestinal cells on tight junction regulators, inflammatory markers, and permeability. The human colon carcinoma Caco-2 cell line was used with the main treatment of pH and LPS in a 2 × 3 factorial arrangement. The pH was acidic (pH 6.0) or physiologic (pH 7.4), whereas LPS was 0, 0.5, or 10 ng/mL. After cells reached 70%–80% of confluence, the media were replaced with each respective treatment medium. Cells were treated for 3 h for mRNA abundance analysis, 3 and 6 h for protein abundance determination, and 3, 6, 12, and 24 h for permeability determination. Protein abundance of the myosin light-chain kinase (MYLK) and toll-like receptor 4 (TLR4) were measured by western blot. The mRNA abundance of IL-8, MYLK, peroxisome proliferator activated receptor gamma, and nuclear factor kappa B (Nfkb1) was determined by real-time, quantitative PCR. Paracellular permeability was determined with Lucifer yellow after 21 d of incubation. Cell culture was performed in biological triplicate; each biological replicate for real-time, quantitative PCR had 2 technical replicates, and for protein abundance and permeability assay had one technical replicate. The MIXED procedure of SAS (SAS Institute Inc.) was used with LPS, pH, and pH × LPS as fixed effects. Significance was declared at P ≤ 0.05 and tendencies when 0.05 <P ≤ 0.10. Increasing LPS doses did not affect the protein abundance of MYLK and TLR4, nor mRNA abundance of MYLK and PPRG. The LPS tended to increase mRNA abundance of IL-8 while pH × LPS interactively increased mRNA abundance of Nfkb1, where mRNA abundance of Nfkb1 was lower in cells exposed to pH 6.0 when combined with 0 and 10 ng/mL of LPS. Contrary to expectations, LPS did not affect the permeability of Caco-2 cells. The mRNA abundance of MYLK was greater at pH 6.0 versus pH 7.4. Also, protein abundance of TLR4 was lower at pH 6.0 than pH 7.4, and it decreased when exposure increased to 6 h. In addition, mRNA abundance of IL-8 was lower at pH 6.0 versus pH 7.4. Permeability was greater at pH 6.0 versus 7.4 after 6, 12, and 24 h of treatment. In summary, the effect of LPS and its interaction with pH showed less impact than expected on dependent variables measured, which might be attributed to the adopted clinically achievable LPS doses likely not being high enough to draw a strong response as observed in the literature. On the other hand, pH was far more relevant, modulating mRNA abundance of inflammatory markers, tight junction regulators, and permeability in in vitro colon cell models.

Effect of sample fractionation and normalization when immunoblotting for human muscle Na+/K+-ATPase subunits and glycogen synthase

Immunoblotting is widely used in muscle physiology to determine protein regulation and abundance. However, research groups use different protocols, which may result in differential outcomes. Herein, we investigated the effect of various homogenization procedures on determination of protein abundance in human m. vastus lateralis biopsies. Furthermore, we investigated differences in abundance between young healthy males (n = 12) and type-2 diabetics (n = 4), and the effect of data normalization. Fractionated lysates had the lowest variation in total protein determination as compared to non-fractionated homogenates. Abundance of NKAα2, NKAβ1, FXYD1, and glycogen synthase was higher (P < 0.05) in young healthy than in type-2 diabetics determined in both fractionated and non-fractionated samples for which normalization to the stain-free signal and/or standard curve did not affect outcomes. Precision and reliability of protein abundance determination between sample types showed a moderate to good reliability for these proteins, whereas the commonly used house-keeping protein, actin, showed poor reliability. In conclusion, fractionated and non-fractionated immunoblotting samples yield similar data for several sarcolemmal and cytosolic proteins, except for actin, which, therefore appears inappropriate for data normalization in immunoblotting of human skeletal muscle. Thus, fractionation does not seem to be a major source of bias when immunoblotting for NKA subunits and GS.

1,25-Dihydroxyvitamin D3 Attenuates the Effects of Lipopolysaccharide by Causing ADAM10-Dependent Ectodomain Shedding of Toll-Like Receptor 4

Background/Aims: We investigated how 1,25-dihydroxyvitamin D₃ (1,25D₃) inhibits the effects of lipopolysaccharide (LPS) in human aortic endothelial cells. Methods: Cellular signaling was explored by determination of protein abundance with Western blot, measurement of cytosolic Ca²⁺ concentration and immunofluorescence staining for a disintegrin and metalloprotease 10 (ADAM10). Results: LPS stimulated the expression of intercellular adhesion molecule 1 (ICAM-1) through toll-like receptor 4 (TLR4) and subsequent activation of p38 mitogen-activated protein kinase (p38 MAPK). Pretreatment with 1,25D₃ attenuated LPS-induced p38 MAPK activation and ICAM-1 expression by causing ectodomain shedding of TLR4. This effect of 1,25D₃ depended on its ability to induce a rapid extracellular Ca²⁺ influx through L-type calcium channels because the ectodomain shedding was prevented by the absence of extracellular Ca²⁺ or the presence of verapamil. TLR4 ectodomain shedding was also induced by Bay K8644 (L-type calcium channel agonist). Both 1,25D₃ and Bay K8644 caused extracellular Ca²⁺ influx-dependent ADAM10 translocation to the cell surface. Depletion of ADAM10 by siRNA transfection prevented 1,25D₃- and Bay K8644-induced ectodomain shedding of TLR4, and abolished the inhibitory effect of 1,25D₃ on LPS-induced ICAM-1 expression. Conclusion: 1,25D₃ causes ectodomain shedding of TLR4 and thereby decreases the responsiveness of cells to LPS. ADAM10, activated by extracellular Ca²⁺ influx, was implicated in the ectodomain cleavage of TLR4.

PQuant improves quantitation by keeping out interfering signals and evaluating the accuracy of calculated ratios.

In relative protein abundance determination from peptide intensities recorded in full mass scans, a major complication that affects quantitation accuracy is signal interference from coeluting ions of similar m/z values. Here, we present pQuant, a quantitation software tool that solves this problem. pQuant detects interference signals, identifies for each peptide a pair of least interfered isotopic chromatograms: one for the light and one for the heavy isotope-labeled peptide. On the basis of these isotopic pairs, pQuant calculates the relative heavy/light peptide ratios along with their 99.75% confidence intervals (CIs). From the peptides ratios and their CIs, pQuant estimates the protein ratios and associated CIs by kernel density estimation. We tested pQuant, Census and MaxQuant on data sets obtained from mixtures (at varying mixing ratios from 10:1 to 1:10) of light- and heavy-SILAC labeled HeLa cells or (14)N- and (15)N-labeled Escherichia coli cells. pQuant quantitated more peptides with better accuracy than Census and MaxQuant in all 14 data sets. On the SILAC data sets, the nonquantified "NaN" (not a number) ratios generated by Census, MaxQuant, and pQuant accounted for 2.5-10.7%, 1.8-2.7%, and 0.01-0.5% of all ratios, respectively. On the (14)N/(15)N data sets, which cannot be quantified by MaxQuant, Census and pQuant produced 0.9-10.0% and 0.3-2.9% NaN ratios, respectively. Excluding these NaN results, the standard deviations of the numerical ratios calculated by Census or MaxQuant are 30-100% larger than those by pQuant. These results show that pQuant outperforms Census and MaxQuant in SILAC and (15)N-based quantitation.

Protein abundance in multiplexed samples (PAMUS) for quantitation of Trichoderma reesei secretome

Protein abundance determination across multiple samples proved to be a daunting task and far fewer methods have been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest. Herein, we introduce a new method for estimation of protein abundances in multiplexed samples (PAMUS). Protein abundance in the multiplexed sample comprising of the eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in four different carbon sources, namely glucose, cellulose, starch, and a mixture of starch and cellulose was determined. For protein abundance in the multiplexed sample, exponentially modified protein abundance index (emPAI) was used. Using the PAMUS method, we estimated the abundance of extracellular lignocellulolytic proteins secreted by two T. reesei strains in response to various carbon sources. The results reveal that cellulose induces biosynthesis of cellulases. PAMUS analysis of the secretomes implicates T. reesei Rut C30 as a hyper cellulolytic strain and further revealed the optimum concentrations of each secreted enzyme during cellulosic substrate utilization. Our study demonstrates the plausible use of the PAMUS method for designing enzyme cocktails for optimum cellulose hydrolysis, and its potential applications in future studies involving other multiplexed biological samples. Relative protein quantitation across multiple complex biological samples dominates the field of quantitative proteomics. Protein abundance determination across multiple samples, on the other hand, proves to be a daunting task and far fewer methods have been successfully devised for this purpose. Despite the technical challenges faced, protein abundance determination over multiple samples is still an area of interest as it provides unique information about the biological processes, and physiological states of particular disease, or that of microbes. This study introduces a new method of estimation of protein abundance in multiplexed samples (PAMUS) which is applied to study eight complex secretomes by Trichoderma reesei QM6a and Rut C30 grown in different carbon sources. The expression levels of proteins in the multiplexed eight complex secretome samples were determined by isobaric tags for relative and absolute quantitation (iTRAQ) reagents coupled with exponentially modified protein abundance index (emPAI) to calculate the abundance of each identified protein in eight conditions. This method with microbial secretome as an example facilitated a direct comparison of the abundance of proteins. This PAMUS could be applied for any multiple biological samples, for example, to study human disease to evaluate dynamic expression of proteins during disease progressions.

Quantitative Time-course Profiling of Parasite and Host Cell Proteins in the Human Malaria Parasite Plasmodium falciparum

Studies of the Plasmodium falciparum transcriptome have shown that the tightly controlled progression of the parasite through the intra-erythrocytic developmental cycle (IDC) is accompanied by a continuous gene expression cascade in which most expressed genes exhibit a single transcriptional peak. Because the biochemical and cellular functions of most genes are mediated by the encoded proteins, understanding the relationship between mRNA and protein levels is crucial for inferring biological activity from transcriptional gene expression data. Although studies on other organisms show that <50% of protein abundance variation may be attributable to corresponding mRNA levels, the situation in Plasmodium is further complicated by the dynamic nature of the cyclic gene expression cascade. In this study, we simultaneously determined mRNA and protein abundance profiles for P. falciparum parasites during the IDC at 2-hour resolution based on oligonucleotide microarrays and two-dimensional differential gel electrophoresis protein gels. We find that most proteins are represented by more than one isoform, presumably because of post-translational modifications. Like transcripts, most proteins exhibit cyclic abundance profiles with one peak during the IDC, whereas the presence of functionally related proteins is highly correlated. In contrast, the abundance of most parasite proteins peaks significantly later (median 11 h) than the corresponding transcripts and often decreases slowly in the second half of the IDC. Computational modeling indicates that the considerable and varied incongruence between transcript and protein abundance may largely be caused by the dynamics of translation and protein degradation. Furthermore, we present cyclic abundance profiles also for parasite-associated human proteins and confirm the presence of five human proteins with a potential role in antioxidant defense within the parasites. Together, our data provide fundamental insights into transcript-protein relationships in P. falciparum that are important for the correct interpretation of transcriptional data and that may facilitate the improvement and development of malaria diagnostics and drug therapy.

Identification and differential induction of the expression of aquaporins by salinity in broccoli plants

Plant aquaporins belong to a large superfamily of conserved proteins called the major intrinsic proteins (MIPs). There is limited information about the diversity of MIPs and their water transport capacity in broccoli (Brassica oleracea) plants. In this study, the cDNAs of isoforms of Plasma Membrane Intrinsic Proteins (PIPs), a class of aquaporins, from broccoli roots have been partially sequenced. Thus, sequencing experiments led to the identification of eight PIP1 and three PIP2 genes encoding PIPs in B. oleracea plants. The occurrence of different gene products encoding PIPs suggests that they may play different roles in plants. The screening of their expression as well as the expression of two specific PIP2 isoforms (BoPIP2;2 and BoPIP2;3), in different organs and under different salt-stress conditions in two varieties, has helped to unravel the function and the regulation of PIPs in plants. Thus, a high degree of BoPIP2;3 expression in mature leaves suggests that this BoPIP2;3 isoform plays important roles, not only in root water relations but also in the physiology and development of leaves. In addition, differences between gene and protein patterns led us to consider that mRNA synthesis is inhibited by the accumulation of the corresponding encoded protein. Therefore, transcript levels, protein abundance determination and the integrated hydraulic architecture of the roots must be considered in order to interpret the response of broccoli to salinity.

A Quantitative Results-driven Approach to Analyzing Multisite Protein Phosphorylation

Multisite protein phosphorylation appears to be quite common. Nevertheless our understanding of how multiple phosphorylation events regulate the function of a protein is limited in many cases. The ability to measure temporal changes in the site-specific phosphorylation profile of a protein in response to a given stimulus or cellular activity would provide an immediate indication of the functional significance of any phosphorylation site to a given process. Here we describe a mass spectrometry-based method to identify functionally relevant phosphorylation sites on a protein. It combines stable isotope labeling with a highly selective mass spectrometry analysis to detect and quantitate phosphorylation sites in response to a cellular signal. This approach requires no a priori knowledge of the phosphorylation state of the protein, does not require purification of phosphopeptides, and reliably detects substoichiometric levels of phosphorylation. Following a review of the quantitative results, only those phosphorylation sites that show a change in relative abundance are selected for identification and further study. We used this results-driven approach to study phosphorylation of the budding yeast transcription factor Pho4 in response to phosphate starvation. Phosphorylation of Pho4 on five cyclin-dependent kinase (Cdk) consensus sites has been shown to regulate the transcriptional activity of Pho4 in response to changes in environmental phosphate levels. Here we show that in phosphate-rich medium Pho4 is phosphorylated on at least 15 distinct sites including the five Cdk sites described previously. In excellent agreement with the known mechanism for regulation of Pho4 we found that phosphorylation at all five of the Cdk sites was repressed in phosphate-depleted medium. In addition to these five sites, we identified four novel phosphorylation sites that were also responsive to changes in phosphate availability. Selecting a limited number of Pho4 phosphorylation sites, we performed a more detailed kinetic analysis using an isotope-free strategy. We used LC-MS with selected reaction monitoring to greatly improve the accuracy, sensitivity, and dynamic range of the subsequent experiments. A detailed analysis of the cell-based phosphorylation at the selected Pho4 sites confirmed an apparent site preference for the Pho80-Pho85 cyclin-cyclin-dependent kinase complex.