Absolute quantification of proteins in the fatty acid biosynthetic pathway using protein standard absolute quantification

With worldwide attention on renewable energy and climate change,metabolic engineering of the fatty acid biosynthetic pathway has become an active area of research,with a view to enhance production of biofuels.Indeed,this pathway has already been extensively studied in Escherichia coli.Nevertheless,little is known about the absolute abundance of the enzymes involved,information that may be valuable for engineering,such as the optimal molar ratios of different proteins.In this study,we use protein standard absolute quantification(PSAQ)to measure the absolute abundance of proteins that catalyze fatty acid biosynthesis in E.coli.In addition,the changes of protein abundance were analyzed by comparing the differences between high-yield and the background strain.Our work highlights opportunities to enhance fatty acid production by measuring protein molar ratios and identifying catalytic and regulatory bottlenecks.More importantly,our results provide evidence that PSAQ is a generally valuable tool to investigate metabolic pathways.

Host cell protein quantification workflow using optimized standards combined with data-independent acquisition mass spectrometry

Monitoring of host cell proteins(HCPs)during the manufacturing of monoclonal antibodies(mAb)has become a critical requirement to provide effective and safe drug products.Enzyme-linked immunosorbent assays are still the gold standard methods for the quantification of protein impurities.However,this technique has several limitations and does,among others,not enable the precise identification of proteins.In this context,mass spectrometry(MS)became an alternative and orthogonal method that delivers qualitative and quantitative information on all identified HCPs.However,in order to be routinely implemented in biopharmaceutical companies,liquid chromatography-MS based methods still need to be standardized to provide highest sensitivity and robust and accurate quantification.Here,we present a promising MS-based analytical workflow coupling the use of an innovative quantification standard,the HCP Profiler solution,with a spectral library-based data-independent acquisition(DIA)method and strict data validation criteria.The performances of the HCP Profiler solution were compared to more conventional standard protein spikes and the DIA approach was benchmarked against a classical datadependent acquisition on a series of samples produced at various stages of the manufacturing process.While we also explored spectral library-free DIA interpretation,the spectral library-based approach still showed highest accuracy and reproducibility(coefficients of variation<10%)with a sensitivity down to the sub-ng/mg mAb level.Thus,this workflow is today mature to be used as a robust and straightforward method to support mAb manufacturing process developments and drug products quality control.

Absolute quantitative detection of genetically modified soybean MON87708×MON89788 with stacked traits by digital polymerase chain reaction

The main advantage of digital PCR(dPCR) is that it facilitates absolute quantification of the target without reference to the standard/calibration curve.Crystal droplet dPCR has a three-color staining detection function,which enables multiplex PCR reaction.In this study,this technique was used to establish triple dPCR detection for the genetically modified soybean MON87708 × MON89788 with stacked traits.Specific absolute quantitative detection was accomplished for the genomic DNA extracted from the homogenized seeds of GM stack MON87708× MON89788 soybean.Our results can serve as a reference for the absolute quantitative detection of stacked events of genetically modified crops.

Quantification of CP4-EPSPS in genetically modified Nicotiana tabacum leaves by LC-MS/MS with ^(18)O-labeling

The CP4-EPSPS gene is widely used in herbicide-tolerant plants/crops all over the world. In this study, a method was developed by coupling liquid chromatography with high sensitivity to tandem mass spectrometry to quantify the amount of CP4-EPSPS expression in Nicotiana tabacum leaves. The quantification of protein was converted to measure the unique peptide of CP4-EPSPS protein. One peptide unique to CP4-EPSPS was synthesized and labeled with.H2^18O to get 180 stable isotope labeled peptide. The peptide served as the internal standard. The validated method had good specificity and linearity. The intra-and inter-day precisions and accuracy for all samples were satisfactory. The results demonstrated that the novel method was sensitive and selective to quantify CP4- EPSPS in the crude extract without time-consuming pre-separation or.the purification procedures.

COMPOSITION DETERMINATION OF BINARY POLYMER MIXTURES BY SIZE EXCLUSION CHROMATOGRAPHY WITH LIGHT SCATTERING DETECTION

Base on the principle of absolute quantification of size exclusion chromatography （SEC）, a light scattering （LS） detector coupled with a concentration detector （refractive index detector） is utilized to determine the compositions of complicated binary mixtures. A theoretical analysis predicts that the response factors for both LS and RI detectors are linear functions with the composition of any specified polymer mixtures in the binary polymer mixtures. Two pairs of complicated binary mixtures were used to test the theory mentioned in the present paper, and the experimental results show an excellent accordance with the theory.

Depletion of MRPL35 inhibits gastric carcinoma cell proliferation by regulating downstream signaling proteins

BACKGROUND Gastric carcinoma(GC)is a digestive system disease with high morbidity and mortality.However,early clinical detection is difficult,and the therapeutic effect for advanced disease is not satisfactory.Thus,finding new tumor markers and therapeutic targets conducive to the treatment of GC is imperative.MRPL35 is a member of the large subunit family of mitochondrial ribosomal protein.MRPL35 shows the characteristic of oncogene in colorectal cancer and esophageal cancer,which promotes the exploration of the correlation between MRPL35 and GC.We proposed that the expression of MRPL35 might be critical in GC.AIM To study the effect of MRPL35 knockdown on GC cell proliferation.METHODS The expression of MRPL35 in GC was evaluated based on data from the publictumor database UALCAN(www.ualcan.path.uab.edu).The effect of theexpression of MRPL35 on the prognosis was evaluated with KMplot(www.kmplot.com).The expression of MRPL35 was assessed on the tissuemicroarray by immunohistochemistry and the level of MRPL35 mRNA in 25 pairsof clinical GC tissues and matched adjacent tissues was detected by quantitativereverse transcription-polymerase chain reaction.Celigo cell count assay,colonyformation assay,and flow cytometry were used to assess the role of MRPL35 inGC cell proliferation and apoptosis in vitro.Additionally,tumor formationexperiment in BALB/c nude mice was utilized to determine the effect of MRPL35on GC cell proliferation.After knockdown of MRPL35,related proteins wereidentified by isobaric tags for relative and absolute quantification analysis,andthe expression of related proteins was detected by Western blot.RESULTSThe expression of MRPL35 was up-regulated in GC(P=1.77×10^(-4)).The Kaplan-Meier plots of the overall survival indicated that high expression of MRPL35 wasassociated with a poor survival in GC.Compared with adjacent tissues,theexpression of MRPL35 in GC tissues was increased,which was related to age(P=0.03),lymph node metastasis(P=0.007),and pathological tumor-node-metastasisstage(P=0.024).Knockdown of MRPL35 inhibited GC cell proliferation andcolony formation and induced apoptosis.Animal experiment results showed thatknockdown of MRPL35 inhibited tumor formation in BALB/c nude mice.Westernblotting analysis showed that after knockdown of MRPL35,the expression ofPICK1 and BCL-XL proteins decreased,and that of AGR2 protein increased.CONCLUSIONCollectively,our findings demonstrate that knockdown of MRPL35 inhibits GCcell proliferation through related proteins including PICK1,BCL-XL,and AGR2.

iTRAQ-based quantitative proteome characterization of wheat grains during filling stages

Using isobaric tags for relative and absolute quantification （iTRAQ） and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins （DEPs） includind high-molecular weight giutenin subunit （W5AIU1）, low-molecular weight glutenin subunit （QSW3V4）, gliadin/avenin-like seed protein （D2KFG9）, and avenin-like protein （W5DVL2）, all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Quantitative analysis of glutamate compounds in the swine brain following central analgesics nasal spray using proton magnetic resonance spectroscopy and linear combination model

BACKGROUND： In localized brain proton magnetic resonance spectroscopy （^1H-MRS）, metabolite levels are often expressed as ratios, rather than absolute concentrations. Frequently, the denominator is creatine, which is assumed to be stable in normal, as well as many pathological, states. However, in vivo creatine levels do not remain constant. Therefore, absolute metabolite measurements, which provide the precise concentrations of certain chemical compounds, are superior to metabolite ratios for determining pathological and evolutional changes. OBJECTIVE： To investigate the feasibility of quantification analysis of brain metabolite changes caused by central analgesics nasal spray using the ^1H-MRS and linear combination model （LCModel） methods. DESIGN, TIME AND SETTING： This neuroimaging, observational, animal study was performed at the Laboratory of the Department of Medical Imaging, Second Affiliated Hospital, Medical College, Shantou University, China from July to December 2007. MATERIALS： Butorphanol tartrate nasal spray, as a mixed agonist-antagonist opioid analgesic, was purchased from Shanghai Hengrui Pharmacy, China. A General Electric Signa 1.5T System （General Electric Medical Systems, Milwaukee, WI, USA） and LCModel software （Stephen Provencher, Oakville, Ontario, Canada） were used in this study. METHODS： MRS images were acquired in ten healthy swine aged 2 weeks using single-voxel point-resolved spectroscopic sequence. A region of interest （2 cm × 2 cm × 2 cm） was placed in the image centers of maximum brain parenchyma. Repeated MRS scanning was performed 15-20 minutes after intranasal administration of 1 mg of butorphanol tartrate. Three settings of repetition time/echo time were selected before and after nasal spray administration 3 000 ms/30 ms,1 500 ms/30 ms, and 3 000 ms/50 ms. Metabolite concentrations were estimated by LCModel software. MAIN OUTCOME MEASURES： ^1H-MRS spectra was obtained using various repetition time/echo time settings. Concentrations of glutamate compounds （glutamate ＋ glutamine）, N-acetyl aspartate, and choline were detected in swine brain prior to and following nasal spray treatment. RESULTS： The glutamate compounds curve was consistent with original spectra, when a repetition time/echo time of 3 000 ms/30 ms was adopted. Concentrations of glutamate compounds, N-acetyl aspartate, and choline decreased following administration. The most significant reduction was observed in glutamate compound concentrations from （9.28 ± 0.54） mmol/kg to （7.28 ± 0.54） mmol/kg （P 〈 0.05）. CONCLUSION： ^1H-MRS and LCModel software were effectively utilized to quantitatively analyze and measure brain metabolites. Glutamate compounds might be an important neurotransmitter in central analgesia.

Effect of Drought Stress on Proteome of Maize Grain during Grain Filling

Based on isobaric tags for relative and absolute quantification(iTRAQ)technology,the proteome of grains of a maize cultivar Huangzao 4 under drought stress at grain filling stage was analyzed.The results show that under drought stress,438 proteins were differentially expressed in the maize grains during grain filling.Among them,200 were up-regulated and 238 were down-regulated.The gene ontology(GO)analysis shows that the biological processes in which differential proteins are more involved are cellular processes,metabolic processes and single biological processes;proteins in the cell component category are mainly distributed in cells,cell parts and organelles;and the proteins the molecular function category mainly possess catalytic activity and binding function.Differentially expressed proteins classified by COG are mainly involved in protein post-translational modification and transport,molecular chaperones,general functional genes,translation,ribosomal structure,biosynthesis,energy production and transformation,carbohydrate transport and metabolism,amino acid transport and metabolism,etc.The subcellular structure of the differentially expressed proteins is mainly located in the cell chloroplast and cytosol.The proportions are 35.01%and 30.21%respectively.KEGG metabolic pathway enrichment analysis shows that the differentially expressed proteins are mostly involved in antibiotic biosynthesis,microbial metabolism in different environments,and endoplasmic reticulum protein processing;the metabolic pathways with higher enrichment are the carbon fixation pathway and estrogen signaling pathway of prokaryotes;and the higher enrichment and greater significance are in the tricarboxylic acid cycle,carbon fixation of photosynthetic organisms and proteasome.The results of this study preliminarily reveal the adaptive mechanism of maize grains in response to drought stress during grain filling,providing a theoretical reference for maize drought-resistant molecular breeding.

Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Characterization of genetically modified organisms through determina­tion of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained.Southern blot,fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and,with the exception of mating,give ambiguous results.With the emergence of the real-time quantitative PCR technology,different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis.However,the accuracy in quantitation by this method can be influenced in all steps of analysis.This review focuses on the aspects that influence pre-analytical steps(DNA extraction and DNA quantification methods),quantification strategies and data analysis in quantification of copy num­ber and zygosity in transgenic animals.

Designing specific bacterial 16S primers to sequence and quantitate plant endo-bacteriome

Plant endophytic bacteria colonize the internal tissues of plants and interact with plants closely.The past two decades have witnessed the increasing application of next-generation 16S r RNA gene sequencing in the investigation of bacterial communities.However,deciphering plant endo-bacterial communities by this method is difficult because of the co-amplification of massive plant organellar DNAs with bacterial 16S.Here,we designed polymerase chain reaction(PCR) primer sets,including799F/1107R,322F/796R,and 322F-Dr/796Rs(primer pair 322F/796R with a penultimate-base substitution in 322F),that can specifically amplify bacterial 16S from plant total DNAs.We computationally and experimentally evaluated the specificity,coverage,and accuracy of the newly designed primer sets.Both 799F/1107R and 322F-Dr/796Rs produced plant DNA-free 16S amplicon libraries or reduced plant DNA contamination to lower than 5% for the plant materials with extremely-low-abundance bacterial communities.The primer set 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of rice leaf or root endo-bacteriome,which revealed 10^(6)–10^(7) and 10^(9)–10^(10) bacteria per gram fresh weight,respectively.These 16S primer sets and amplification methods enable the simple and inexpensive next-generation sequencing and quantification of plant endo-bacteriome,which will significantly advance studies on the plant-related microbiome.

Design and use of group-specific primers and probes for real-time quantitative PCR

Real-time quantitative polymerase chain reaction(qPCR)has gained popularity as a technique to detect and quantify a specific group of target microorganisms from various environmental samples including soil,water,sediments,and sludge.Although qPCR is a very useful technique for nucleic acid quantification,accurately quantifying the target microbial group strongly depends on the quality of the primer and probe used.Many aspects of conducting qPCR assays have become increasingly routine and automated;however,one of the most important aspects,designing and selecting primer and probe sets,is often a somewhat arcane process.In many cases,failed or non-specific amplification can be attributed to improperly designed primer-probe sets.This paper is intended to provide guidelines and general principles for designing group-specific primers and probes for qPCR assays.We demonstrate the effectiveness of these guidelines by reviewing the use of qPCR to study anaerobic processes and biologic nutrient removal processes.qPCR assays using group-specific primers and probes designed with this method,have been used to successfully quantify 16S ribosomal Ribonucleic Acid(16S rRNA)gene copy numbers from target methanogenic and ammoniaoxidizing bacteria in various laboratory-and full-scale biologic processes.Researchers with a good command of primer and probe design can use qPCR as a valuable tool to study biodiversity and to develop more efficient control strategies for biologic processes.

Cell-free synthesis of stable isotope-labeled internal standards for targeted quantitative proteomics

High-sensitivity mass spectrometry approaches using selected reaction monitoring(SRM)or multiple reaction monitoring(MRM)methods are powerful tools for targeted quantitative proteomics-based investigation of dynamics in specific biological systems.Both high-sensitivity detection of lowabundance proteins and their quantification using this technique employ stable isotope-labeled peptide internal standards.Currently,there are various ways for preparing standards,including chemical peptide synthesis,cellular protein expression,and cell-free protein or peptide synthesis.Cell-free protein synthesis(CFPS)or in vitro translation(IVT)systems in particular provide high-throughput and low-cost preparation methods,and various cell types and reconstituted forms are now commercially available.Herein,we review the use of such systems for precise and reliable protein quantification.

Quantitative analysis of serum protein in patients with acute coronary syndrome

Background The early diagnosis of acute coronary syndromes(ACS)is of great significance for saving patient′s life. The high-resolution and high-throughput proteomic techniques provide a new means for the discovery of disease-specific biomarkers.We screen serum protein molecules associated with ACS using differential proteomics methods with isotope-labeled relative and absolute quantification(iTRAQ). Method Coronary sinus serum(10 in each group)was collected from ACS patients and healthy controls with the same Genetic background. iTRAQ analysis was performed to screen for significantly different proteins between ACS and healthy controls after pretreatment with high-abundance protein. Result In the iTRAQ analysis of the serum samples from two groups,92 proteins with a mean difference of more than 1.5 times were screened in serum of patients with ACS compared with healthy control serum. The functions of these proteins are involved in stress response,transport,immune response and signal transduction. Conclusions ACS is associated with altered expression of many proteins which may serve as the potential markers of ACS in serum.