A Fast Workflow for Identification and Quantification of Proteomes

The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.The performance of mass spectrometry has been improved tremendously over the last few years (1–3), making mass spectrometry-based proteomics a viable approach for large-scale protein analysis in biological research. Scientists around the world are striving to fulfill the promise of identifying and quantifying almost all gene products expressed in a cell line or tissue. This would make mass spectrometry-based protein analysis an approach that is compatible to the second-generation mRNA deep-seq technique (4, 5).Two liquid chromatography (LC)-MS strategies have been employed to achieve deep proteome coverage. One is a single run with a long chromatography column and gradient to take advantage of the resolving power of HPLC to reduce the complexity of peptide mixtures; the other is a sequential run with two-dimensional separation (typically ion-exchange and reverse phase) to reduce peptide complexity. It was reported by two laboratories that 2761 and 4500 proteins were identified with a 10 h chromatography gradient on a dual pressure linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap Velos)(6–8). Similarly, 3734 proteins were identified using a 8 h gradient on a 2 m long column with a hybrid triple quadrupole - time of flight (Q-TOF, AB sciex 5600 Q-TOF)(9) mass spectrometer. The two-dimensional approach has yielded more identification with longer time. For example, 10,006 proteins (representing over 9000 gene products, GPs)¹ were identified in U2OS cell (10), and 10,255 proteins (representing 9207 GPs) from HeLa cells (11). It took weeks (for example, 2–3 weeks) of machine running time to achieve such proteome coverage, pushing proteome analysis to the level that is comparable to mRNA-seq. With the introduction of faster machines, human proteome coverage now has reached the level of 7000–8500 proteins (representing 7000–8000 GPs) in 3 days (12). Notwithstanding the impressive improvement, the current approach using long column and long gradient suffers from inherent limitations: it takes long machine running time and it is challenging to keep reproducibility among repeated runs. Thus, current throughput and reproducibility have hindered the application of in-depth proteomics to traditional biological researches. A timesaving approach is in urgent need.In this study, we used the first-dimension (1D) short pH 10 RP prefractionation to reduce the complexity of the proteome (13), followed by sequential 30 min second-dimension (2D) short pH 3 reverse phase-(RP)-LC-MS/MS runs for protein identification (14). The results demonstrated that it is possible to identify 8000 gene products from mammalian cells within 12 h of total MS measurement time by applying this dual-short 2D-RPLC-MS/MS strategy (Fast sequencing, Fast-seq). The robustness of the strategy was revealed by parallel testing on different MS systems including quadrupole orbitrap mass spectrometer (Q-Exactive), hybrid Q-TOF (Triple-TOF 5600), and dual pressure linear ion-trap orbitrap mass spectrometer (LTQ-Orbitrap Velos), indicating the inherent strength of the approach as to merely taking advantage of the better MS instruments. This strategy increases the efficiency of MS sequencing in unit time for the identification of proteins. We achieved identification of 2200 proteins/30 mins on LTQ-Orbitrap Velos, 2800 proteins/30 mins on Q-Exactive and Triple-TOF 5600 respectively. We further optimized Fast-seq and worked out a quantitative-version of the Fast-seq workflow: Fast-quantification (Fast-quan) and applied it for protein abundance quantification in HUVEC cell that was treated with a drug candidate MLN4924 (a drug in phase III clinical trial). We were able to quantify > 6700 GPs in 1 day of MS running time and found 99 proteins were up-regulated with high confidence. We expect this efficient alternative approach for in-depth proteome analysis will make the application of MS-based proteomics more accessible to biological applications.     

Rational design of microRNA–siRNA chimeras for multifunctional target suppression

MicroRNAs (miRNAs) are involved in a variety of human diseases by simultaneously suppressing many gene targets. Thus, the therapeutic value of miRNAs has been intensely studied. However, there are potential limitations with miRNA-based therapeutics such as a relatively moderate impact on gene target regulation and cellular phenotypic control. To address these issues, we proposed to design new chimeric small RNAs (aiRNAs) by incorporating sequences from both miRNAs and siRNAs. These aiRNAs not only inherited functions from natural miRNAs, but also gained new functions of gene knockdown in an siRNA-like fashion. The improved efficacy of multifunctional aiRNAs was demonstrated in our study by design and testing of an aiRNA that inherited the functions of both miR-200a and an AKT1-targeting siRNA for simultaneous suppression of cancer cell motility and proliferation. The general principles of aiRNA design were further validated by engineering new aiRNAs mimicking another miRNA, miR-9. By regulating multiple cellular functions, aiRNAs could be used as an improved tool over miRNAs to target disease-related genes, thus alleviating our dependency on a limited number of miRNAs for the development of RNAi-based therapeutics.     

Cytosolic phospholipase A2α sustains pAKT,pERK and AR levels in PTEN-null/mutated prostate cancer cells

Constitutive phosphorylation of protein kinase B (AKT) is a common feature of cancer caused by genetic alteration in the phosphatase and tensin homolog (PTEN) gene and is associated with poor prognosis. This study determined the role of cytosolic phospholipase A₂α (cPLA₂α) in AKT, extracellular signal-regulated kinase (ERK) and androgen receptor (AR) signaling in PTEN-null/mutated prostate cancer cells. Doxycycline (Dox)-induced expression of cPLA₂α led to an increase in pAKT, pGSK3β and cyclin D1 levels in LNCaP cells that possess a PTEN frame-shift mutation. In contrast, silencing cPLA₂α expression with siRNA decreased pAKT, pGSK3β and cyclin D1 levels in both PC-3 (PTEN deletion) and LNCaP cells. Silencing of cPLA₂α decreased pERK and AR protein levels. The inhibitory effect of cPLA₂α siRNA on pAKT and AR protein levels was reduced by the addition of arachidonic acid (AA), whereas the stimulatory effect of AA on pAKT, pERK and AR levels was decreased by an inhibitor of 5-hydroxyeicosatetraenoic acid production. Pharmacological blockade of cPLA₂α with Efipladib reduced pAKT and AR levels with a concomitant inhibition of PC-3 and LNCaP cell proliferation. These results demonstrate an important role for cPLA₂α in sustaining AKT, ERK and AR signaling in PTEN-null/mutated prostate cancer cells and provide a potential molecular target for treating prostate cancer.     

Effects of seasonal snow on the growing season of temperate vegetation in China

Variations in seasonal snowfall regulate regional and global climatic systems and vegetation growth by changing energy budgets of the lower atmosphere and land surface. We investigated the effects of snow on the start of growing season (SGS) of temperate vegetation in China. Across the entire temperate region in China, the winter snow depth increased at a rate of 0.15 cm yr^?1 (P = 0.07) during the period 1982–1998, and decreased at a rate of 0.36 cm yr^?1 (P = 0.09) during the period 1998–2005. Correspondingly, the SGS advanced at a rate of 0.68 day yr^?1 (P < 0.01) during 1982–1998, and delayed at a rate of 2.13 day yr^?1 (P = 0.07) during 1998–2005, against a warming trend throughout the entire study period of 1982–2005. Spring air temperature strongly regulated the SGS of both deciduous broad‐leaf and coniferous forests, whereas the winter snow had a greater impact on the SGS of grassland and shrubs. Snow depth variation combined with air temperature contributed to the variability in the SGS of grassland and shrubs, as snow acted as an insulator and modulated the underground thermal conditions. In addition, differences were seen between the impacts of winter snow depth and spring snow depth on the SGS; as snow depths increased, the effect associated went from delaying SGS to advancing SGS. The observed thresholds for these effects were snow depths of 6.8 cm (winter) and 4.0 cm (spring). The results of this study suggest that the response of the vegetation's SGS to seasonal snow change may be attributed to the coupling effects of air temperature and snow depth associated with the underground thermal conditions.     

Metabolic engineering of Corynebacterium glutamicum for increasing the production of l-ornithine by increasing NADPH availability

The experiments presented here were based on the conclusions of our previous proteomic analysis. Increasing the availability of glutamate by overexpression of the genes encoding enzymes in the l-ornithine biosynthesis pathway upstream of glutamate and disruption of speE, which encodes spermidine synthase, improved l-ornithine production by Corynebacterium glutamicum. Production of l-ornithine requires 2 moles of NADPH per mole of l-ornithine. Thus, the effect of NADPH availability on l-ornithine production was also investigated. Expression of Clostridium acetobutylicum gapC, which encodes NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, and Bacillus subtilis rocG, which encodes NAD-dependent glutamate dehydrogenase, led to an increase of l-ornithine concentration caused by greater availability of NADPH. Quantitative real-time PCR analysis demonstrates that the increased levels of NADPH resulted from the expression of the gapC or rocG gene rather than that of genes (gnd, icd, and ppnK) involved in NADPH biosynthesis. The resulting strain, C. glutamicum ΔAPRE::rocG, produced 14.84 g l^?1 of l-ornithine. This strategy of overexpression of gapC and rocG will be useful for improving production of target compounds using NADPH as reducing equivalent within their synthetic pathways.     

Defence responses regulated by jasmonate and delayed senescence caused by ethylene receptor mutation contribute to the tolerance of petunia to Botrytis cinerea

Ethylene and jasmonate (JA) have powerful effects when plants are challenged by pathogens. The inducible promoter‐regulated expression of the Arabidopsis ethylene receptor mutant ethylene‐insensitive1‐1 (etr1‐1) causes ethylene insensitivity in petunia. To investigate the molecular mechanisms involved in transgenic petunia responses to Botrytis cinerea related to the ethylene and JA pathways, etr1‐1‐expressing petunia plants were inoculated with Botrytis cinerea. The induced expression of etr1‐1 by a chemical inducer dexamethasone resulted in retarded senescence and reduced disease symptoms on detached leaves and flowers or intact plants. The extent of decreased disease symptoms correlated positively with etr1‐1 expression. The JA pathway, independent of the ethylene pathway, activated petunia ethylene response factor (PhERF) expression and consequent defence‐related gene expression. These results demonstrate that ethylene induced by biotic stress influences senescence, and that JA in combination with delayed senescence by etr1‐1 expression alters tolerance to pathogens.     

Exendin-4 Protects Mitochondria from Reactive Oxygen Species Induced Apoptosis in Pancreatic Beta Cells

Objective

Mitochondrial oxidative stress is the basis for pancreatic β-cell apoptosis and a common pathway for numerous types of damage, including glucotoxicity and lipotoxicity. We cultivated mice pancreatic β-cell tumor Min6 cell lines in vitro and observed pancreatic β-cell apoptosis and changes in mitochondrial function before and after the addition of Exendin-4. Based on these observations, we discuss the protective role of Exendin-4 against mitochondrial oxidative damage and its relationship with Ca²⁺-independent phospholipase A2.

Methods

We established a pancreatic β-cell oxidative stress damage model using Min6 cell lines cultured in vitro with tert-buty1 hydroperoxide and hydrogen peroxide. We then added Exendin-4 to observe changes in the rate of cell apoptosis (Annexin-V-FITC-PI staining flow cytometry and DNA ladder). We detected the activity of the caspase 3 and 8 apoptotic factors, measured the mitochondrial membrane potential losses and reactive oxygen species production levels, and detected the expression of cytochrome c and Smac/DLAMO in the cytosol and mitochondria, mitochondrial Ca₂-independent phospholipase A2 and Ca²⁺-independent phospholipase A2 mRNA.

Results

The time-concentration curve showed that different percentages of apoptosis occurred at different time-concentrations in tert-buty1 hydroperoxide- and hydrogen peroxide-induced Min6 cells. Incubation with 100 µmol/l of Exendin-4 for 48 hours reduced the Min6 cell apoptosis rate (p<0.05). The mitochondrial membrane potential loss and total reactive oxygen species levels decreased (p<0.05), and the release of cytochrome c and Smac/DLAMO from the mitochondria was reduced. The study also showed that Ca²⁺-independent phospholipase A2 activity was positively related to Exendin-4 activity.

Conclusion

Exendin-4 reduces Min6 cell oxidative damage and the cell apoptosis rate, which may be related to Ca²-independent phospholipase A2.     

Oncogenic Fibulin-5 Promotes Nasopharyngeal Carcinoma Cell Metastasis through the FLJ10540/AKT Pathway and Correlates with Poor Prognosis

Background

Nasopharyngeal carcinoma (NPC) is known for its high metastatic potential and locoregional recurrence, although the molecular alterations that are driving NPC metastasis remain unclear at this time. This study aimed to examine the expression of fibulin-5 in NPC, correlate the results with clinicopathological variables and survival, and to investigate the role of fibulin-5 in human NPC cell lines.

Material and Methods

Standard semi-quantitative-RT-PCR, quantitative-RT-PCR, immunoblotting, and immunohistochemistry were used to investigate the mRNA and protein expression profiles of fibulin-5 in normal and NPC tissues. Immunohistochemistry of fibulin-5 was correlated with clinicopathological characteristics by univariate analyses. NPC cells overexpressing fibulin-5 or fibulin-5-siRNA cells were generated by stable transfection to characterize the molecular mechanisms of fibulin-5-elicited cell growth and metastasis.

Results

Our results demonstrated that fibulin-5 overexpression in NPC specimens and significantly correlated with advanced tumor metastasis indicating a poor 5-year overall survival. Fibulin-5 was mainly expressed in the nucleus in human NPC specimens and cell lines. Functionally, fibulin-5 overexpression yielded fast growth in NPC cells. In addition, fibulin-5 promotes cell metastasis in NPC cells through increased FLJ10540 and phosphor-AKT activity. In contrast, siRNA depletion of fibulin-5 suppressed FLJ10540 expression and phosphor-AKT activity. Suppression of either fibulin-5 or FLJ10540 can cause significant inhibition with regards to cell motility in NPC cells. Finally, immunohistochemical analysis of human aggressive NPC specimens showed a significant and positive correlation between fibulin-5 and FLJ10540 expression.

Conclusion

Higher fibulin-5 expression is not only an important indicator of poor survival, but also contributes to the development of new therapeutic strategies in the FLJ10540/AKT pathway for NPC treatment.