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.     

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.     

MACC1 Down-Regulation Inhibits Proliferation and Tumourigenicity of Nasopharyngeal Carcinoma Cells through Akt/β-Catenin Signaling Pathway

The present study was aimed at investigating the expression of metastasis-associated in colon cancer 1 (MACC1) in nasopharyngeal carcinoma (NPC), its relationship with β-catenin, Met expression and the clinicopathological features of NPC, and its roles in carcinogenesis of NPC. Our results showed that MACC1 expression was higher in NPC cells and tissues than that in normal nasopharyngeal cells and chronic inflammation of the nasopharynx tissues, respectively. MACC1 expression was closely related to the clinical stage (p = 0.005) and the N classification (p<0.05) of NPC. Significant correlations between MACC1 expression and Met expression (p = 0.003), MACC1 expression and β-catenin abnormal expression (p = 0.033) were found in NPC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, and colony formation, but induced apoptosis in NPC cells compared with the control group. Furthermore, MACC1 down-regulation inhibited phosphorylated-Akt (Ser473) and β-catenin expression in NPC cells, but phosphorylated-Erk1/2 expression was not altered. Further study showed that phosphotidylinsitol-3-kinase inhibitor downregulated β-catenin and Met expression in NPC cells. There was a significant relationship between MACC1 expression and phosphorylated-Akt expression (p = 0.03), β-catenin abnormal expression and phosphorylated-Akt expression (p = 0.012) in NPC tissue, respectively. In addition, Epstein Barr virus-encoded oncogene latent membrane protein 1 upregulated MACC1 expression in NPC cells. Our results firstly suggest that MACC1 plays an important role in carcinogenesis of NPC through Akt/β-catenin signaling pathway. Targeting MACC1 may be a novel therapeutic strategy for NPC.     

The Shape Trail Test: Application of a New Variant of the Trail Making Test

Objective

The Trail making test (TMT) is culture-loaded because of reliance on the Latin alphabet, limiting its application in Eastern populations. The Shape Trail Test (STT) has been developed as a new variant. This study is to examine the applicability of the STT in a senile Chinese population and to evaluate its potential advantages and disadvantages.

Method

A total of 2470 participants were recruited, including 1151 cognitively normal control (NC), 898 amnestic mild cognitive impairment (aMCI), and 421 mild Alzheimer disease (AD) patients. Besides the STT, the Mini mental state examination and a comprehensive neuropsychological battery involving memory, language, attention, executive function and visuospatial ability were administered to all the participants. In a subgroup of 100 NC and 50 AD patients, both the STT and the Color Trail Test (CTT) were performed.

Results

In NC, the time consumed for Part A and B (STT-A and STT-B) significantly correlated with age and negatively correlated with education (p<0.01). STT-A and B significantly differed among the AD, aMCI and NC. The number that successfully connected within one minute in Part B (STT-B-1 min) correlated well with STT-B (r = 0.71, p<0.01) and distinguished well among NC, aMCI and AD. In the receiver operating characteristic curve analysis, the AUCs (area under the curve) for STT-A, STT-B, and STT-B-1min in identifying AD were 0.698, 0.694 and 0.709, respectively. The STT correlated with the CTT, but the time for completion was longer.

Conclusion

The TMT is a sensitive test of visual search and sequencing. The STT is a meaningful attempt to develop a “culture-fair” variant of the TMT in addition to the CTT.     

Changes in heparan sulfate sulfotransferases and cell-surface heparan sulfate during SKM-1 cells granulocytic differentiation and A549 cells epithelial-mesenchymal transition

Glycoconjugate Journal - Heparan sulfate (HS) with various sulfation patterns is one of important modulators of cancer cell fate through interacting with numerous growth factors. Here we found HS...