Comparison of protein precipitation methods for sample preparation prior to proteomic analysis of Chinese hamster ovary cell homogenates

BACKGROUND: Chinese hamster ovary (CHO) cells are the workhorse for obtaining recombinant proteins. Proteomic studies of these cells intend to understand cell biology and obtain more productive and robust cell lines for therapeutic protein production in the pharmaceutical industry. Because of the great importance of precipitation methods for the processing of samples in proteomics, the acetone, methanol-chloroform (M/C), and trichloroacetic acid (TCA)-acetone protocols were compared for CHO cells in terms of protein recovery, band pattern resolution, and presence on SDS-PAGE. RESULTS: Higher recovery and similar band profile with cellular homogenates were obtained using acetone precipitation with ultrasonic bath cycles (104.18 ± 2.67%) or NaOH addition (103.12 ± 5.74%), compared to the other two protocols tested. TCA-acetone precipitates were difficult to solubilize, which negatively influenced recovery percentage (77.91 ± 8.79%) and band presence. M/C with ultrasonic homogenization showed an intermediate recovery between the other two protocols (94.22 ± 4.86%) without affecting protein pattern on SDS-PAGE. These precipitation methods affected the recovery of low MW proteins (< 15 kDa). CONCLUSIONS: These results help in the processing of samples of CHO cells for their proteomic study by means of an easily accessible, fast protocol, with an almost complete recovery of cellular proteins and the capture of the original complexity of the cellular composition. Acetone protocol could be incorporated to sample-preparation workflows in a straightforward manner and can probably be applied to other mammalian cell lines as well.

Analysis of Common Herbicides in Blood by UPLC-HRMS / 法医学杂志

Objective To develop a method to screen and quantify 10 common herbicides (paraquat, diquat, glyphosate, glufosinate, cyanazine, atrazine, metazachlor, acetochlor, chlorsulfuron, and metsulfuron) in blood.Methods With acetonitrile-water solution[V (acetonitrile) ∶V (water) =3∶1]as protein precipitant, 10 common herbicides in blood were detected using ultra-high performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS).Results All the 10 herbicides had good linearity in their linear range (coefficient of determination R2≥0.993), with the recovery rates 67.4％-111.9％, the relative standard deviations 1.5％-10.8％, the accuracies 85.1％-106.1％, intra-day precisions 2.7％-13.5％, and inter-day precisions 3.3％-13.3％.Conclusion This method is easy to operate with high recovery rates.It enables rapid and accurate qualitative screening and quantitative analysis of various herbicides in blood simultaneously.

Research progress on analytical methods for blood steroid hormones / 中草药

Steroid hormone, the component of the endocrine system, plays an extremely important role in body development, immune regulation, and birth control. The accurate determination of steroid hormone is important for the prevention, diagnosis, and treatment of clinical endocrine diseases. In recent years, the determination of endogenous steroid hormones in biological samples has become increasingly widespread in clinical applications. In this article, the commonly used analysis methods of typical blood steroids from 2013 to 2018 were generalized and compared in order to provide more reference for the accurate determination of steroid hormone levels, and also provide more accurate basis for later clinical diagnosis and treatment.

Determination of 5-fluorouracil in serum by direct protein precipitation-ultra performance liquid chromatography / 中国药科大学学报

@#A method based on direct protein precipitation-ultra performance liquid chromatography was established for the determination of trace 5-fluorouracil in serum. The samples were pretreated with 6% perchloric acid as the precipitant and 5-chlorouracil as the internal standard, and the supernatant separated after vortex mixing and high speed centrifugation was used as the test sample. The separation was carried out with an Acquity HSS T3 column and the mobile phase consisting of water and acetonitrile, and the flow rate was 0. 2 mL/min; the injection volume was 5 μL, and the column temperature was 20 °C. The key parameters of this method such as specificity, accuracy, precision and linearity, were validated. Compared with other methods, this method can complete the analysis with significantly reduced time and cost. The established method is useful for therapeutic drug monitoring of 5-fluorouracil plasma concentration and it has been successfully applied in clinical cases.

Bioanalytical method development and validation for determination of metoprolol tartarate and hydrochlorothiazide using HPTLC in human plasma

A simple, sensitive, rapid and economic chromatographic method has been developed for determination of metoprolol tartarate and hydrochlorothiazide in human plasma using paracetamol as an internal standard. The analytical technique used for method development was high-performance thin-layer chromatography. HPTLC Camag with precoated silica gel Plate 60F254 (20 cm×10 cm) at 250 µm thicknesses (E. Merck, Darmstadt, Germany) was used as the stationary phase. The mobile phase used consisted of chloroform: methanol: ammonia (9:1:0.5v/v/v). Densitometric analysis was carried out at a wavelength of 239 nm. The rf values for hydrochlorothiazide, paracetamol and metoprolol tartarate were 0.13±0.04, 0.28±0.05, 0.48±0.04, respectively. Plasma samples were extracted by protein precipitation with methanol. Concentration ranges of 200, 400, 600, 800, 1000, 1200 ng/mL and 2000, 4000, 6000, 8000, 10000, 12000 ng/mL of hydrochlorothiazide and metoprolol tartarate, respectively, were used with plasma for the calibration curves. The percent recovery of metoprolol tartarate and hydrochlorothiazide was found to be 77.30 and 77.02 %, respectively. The stability of metoprolol tartarate and hydrochlorothiazide in plasma were confirmed during three freeze-thaw cycles (-20 ºC) on a bench for 24 hours and post-preparatively for 48 hours. The proposed method was validated statistically and proved suitable for determination of metoprolol tartarate and hydrochlorothiazide in human plasma.

Um método simples, sensível, rápido e econômico empregando a cromatografia em camada delgada de alta eficiência (HPTLC) foi desenvolvido para determinação do tartarato de metoprolol e hidroclorotiazida em plasma humano, usando paracetamol como padrão interno. Placas prontas de sílica-gel 60F254 (20 cm×10 cm), 250 µm de espessura, para HPTLC Camag (E. A Merck, Darmstadt, Alemanha) foramutilizadas como fase estacionária. A fase móvel utilizada consistiu de clorofórmio: metanol: amônia (9:1:0,5 v/v/v). A análise densitométrica foi realizada no comprimento de onda 239 nm. Os valores de Rf de hidroclorotiazida, paracetamol e tartarato de metoprolol foram 0.13±0.04, 0.28±0.05, 0.48±0.04 respectivamente. As proteínas do plasma foram extraídas por precipitação com metanol. Para construção das curvas de calibração, empregaram-se intervalos de concentração de 200, 400, 600, 800, 1000, 1200 ng/mL e 2000, 4000, 6000, 8000, 10000, 12000 ng/mL de hidroclorotiazida e tartarato de metoprolol, respectivamente. Os percentuais de recuperação do tartarato de metoprolol e de hidroclorotiazida foram de 77,30 e 77,02, respectivamente. A estabilidade do tartarato de metoprolol e de hidroclorotiazida no plasma foi confirmada durante três ciclos de congelamento e descongelamento (-20 ºC), durante 24 horas e póspreparação durante 48 horas. O método proposto foi validado estatisticamente, sendo adequado para determinação do tartarato de metoprolol e hidroclorotiazida em plasma humano.

Voltammetric quantification of anti-hepatitis drug Adefovir in biological matrix and pharmaceutical formulation / 药物分析学报

Electrochemical reduction behavior of Adefovir was studied using Hanging Mercury Drop Electrode (HMDE) in Britton-Robinson (BR) buffer solution.Voltammetric study showed one well-defined reduction peak in the potential range -1.2 to -1.4 V (vs.Ag/AgCl) due to reduction of C=N bond of the imidazole ring.Solid-phase extraction and protein-precipitation techniques were employed for extraction of Adefovir from human plasma.The proposed method allows quantification of Adefovir in human plasma over the concentration range 0.50-5.00 μg/mL with the detection limit 0.17 μg/mL,whereas in pharmaceutical formulation 0.25-2.25 μg/mL with the detection limit 0.08 μg/mL.

Padrões eletroforéticos das proteínas de reserva em grãos de sorgo com presença e ausência de tanino / Electrophoretic patterns of grain storage proteins of sorghum with presence and absence of tannin

Muitos métodos para determinar a presença de taninos são descritos na literatura, mas nenhum deles é universalmente aceito como ideal ou utilizado de forma unânime. Alguns métodos colorimétricos não diferenciam taninos de outros compostos fenólicos, outros utilizam substâncias que não são adequadas como padrão. Métodos que utilizam a capacidade dos taninos de precipitar as proteínas podem causar resultados divergentes devido às diferenças na conformação dessas moléculas. Assim, objetivou-se, neste estudo, identificar a presença de taninos em 10 híbridos de sorgo através da análise de padrões proteicos obtidos por eletroforese. O método colorimétrico Azul da Prússia foi utilizado para quantificar os taninos nas amostras. A precipitação das proteínas pelos taninos permitiu identificar os genótipos de sorgo com tanino através dos padrões proteicos das frações albuminas, globulinas e prolaminas. A análise eletroforética das prolaminas mostrou que as bandas produzidas pelo polipeptídeo kafirina, podem ser utilizadas na identificação de sorgo sem tanino no grão.

Several methods are described on the literature to determine the presence of tannin, however none of them is universally accepted as the ideal or even are used in a unanimous way. Some colorimetric methods do not differentiate tannins from others phenolic compounds, others use substances which are not appropriate to use as standard. Methods that use the capacity of tannins to precipitate proteins may end up with divergent results due to differences on the molecule conformation. Thus, the objective of this study was to identify the presence of tannin in 10 hybrids of sorghum throughout analysis of pattern proteins obtained by electrophoresis. The colored method Prussian Blue was used to quantify tannins on the samples. The protein precipitation by tannins permitted to identify the sorghum genotypes with grain tannin through protein standards of fractions of albumin, globulin and prolamins. The electrophoresis analysis of the prolamins showed that the bands produced by the kafirin polypeptide may be used in sorghum identification without the presence of tannin in grain.