Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media.

This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns. Proteins purified by this method could be widely used in biological assays, structure analysis and mass spectrum assay.

On-line determination of sarcosine in biological fluids utilizing dummy molecularly imprinted polymers in microextraction by packed sorbent.

Several years ago, sarcosine received attention as a prostate-cancer marker. Prostate cancer is one of the most widespread types of tumor diseases in men. The prostate-specific antigen is normally used as a marker, and it can only be detected in blood with a sensitivity of approximately 80%. In the present study, dummy molecularly imprinted polymers in microextraction by packed sorbent with on-line liquid chromatography coupled to tandem mass spectrometry was used for the determination of sarcosine in human plasma and urine samples. The polymer network glycine was used for the dummy molecularly imprinted polymers. The selectivity of the method was evaluated using similar prostate-cancer biomarkers. In addition, various parameters affecting the extraction performance were investigated. The method limits of detection and quantification in the plasma and urine were 1.0 and 3.0 ng/mL, respectively. The values of the coefficient of determination were over 0.99 for all runs in the studied concentration range (3.0-10 000 ng/mL). The method recovery was 87 and 89% in plasma and urine, respectively. The intraday and interday precisions of sarcosine in the plasma and urine samples were in the ranges of 4.0-7.1, 3.0-6.3, 2.9-4.7, and 5.0-6.7, respectively.

Preconcentration based on paramagnetic microparticles for the separation of sarcosine using hydrophilic interaction liquid chromatography coupled with coulometric detection.

Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphate buffer pH 9). The optimal mixing ratio was A + B/C 1:1 with a flow rate of 0.80 mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reached a good resolution for separation also with a good LOD (100 nM). In addition, we successfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.

Microfluidic chip coupled with modified paramagnetic particles for sarcosine isolation in urine.

Carcinoma of prostate (CaP) is the second most frequent malignant tumor occurring in men in Europe. Currently there is discussion on a wide range of potential CaP markers.One of them­nonprotein amino acid sarcosine, also known as N-methylglycine was chosen as a challenge for the development of microfluidic system with isolation by modified paramagnetic microparticles. Therefore, the aim of this study was to design a low-cost, simple, and rapid microfluidic system based on sarcosine isolation with modified paramagnetic microparticles and subsequent analysis on the ion exchange LC. We modified Dowex microparticles with Fe2O3 nanoparticles. Our paramagnetic microparticles were able to establish the binding with sarcosine. Moreover, we designed microfluidic device for sarcosine determination. Analysis of samples was carried out with LOD of1 M of a sarcosine that is sufficient because it is similar to concentrations of a sarcosine observed in the CaP patients.Carcinoma of prostate (CaP) is the second most frequent malignant tumor occurring in men in Europe. Currently there is discussion on a wide range of potential CaP markers.One of them­nonprotein amino acid sarcosine, also known as N-methylglycine was chosen as a challenge for the development of microfluidic system with isolation by modified paramagnetic microparticles. Therefore, the aim of this study was to design a low-cost, simple, and rapid microfluidic system based on sarcosine isolation with modified paramagnetic microparticles and subsequent analysis on the ion exchange LC. We modified Dowex microparticles with Fe2O3 nanoparticles. Our paramagnetic microparticles were able to establish the binding with sarcosine. Moreover, we designed microfluidic device for sarcosine determination. Analysis of samples was carried out with LOD of1 M of a sarcosine that is sufficient because it is similar to concentrations of a sarcosine observed in the CaP patients.

A reproducible and high-throughput HPLC/MS method to separate sarcosine from α- and ß-alanine and to quantify sarcosine in human serum and urine.

While sarcosine was recently identified as a potential urine biomarker for prostate cancer, further studies have cast doubt on its utility to diagnose this condition. The inconsistent results may be due to the fact that alanine and sarcosine coelute on an HPLC reversed-phase column and the mass spectrometer cannot differentiate between the two isomers, since the same parent/product ions are generally used to measure them. In this study, we developed a high-throughput liquid chromatography-mass spectrometry (LC-MS) method that resolves sarcosine from alanine isomers, allowing its accurate quantification in human serum and urine. Assay reproducibility was determined using the coefficient of variation (CV) and intraclass correlation coefficient (ICC) in serum aliquots from 10 subjects and urine aliquots from 20 subjects across multiple analytic runs. Paired serum/urine samples from 42 subjects were used to evaluate sarcosine serum/urine correlation. Both urine and serum assays gave high sensitivity (limit of quantitation of 5 ng/mL) and reproducibility (serum assay, intra- and interassay CVs < 3% and ICCs > 99%; urine assay, intra-assay CV = 7.7% and ICC = 98.2% and interassay CV = 12.3% and ICC = 94.2%). In conclusion, this high-throughput LC-MS method is able to resolve sarcosine from α- and ß-alanine and is useful for quantifying sarcosine in serum and urine samples.

Separation of isomers L-alanine and sarcosine in urine by electrospray ionization and tandem differential mobility analysis-mass spectrometry.

Sarcosine, an isomer of L-alanine, has been proposed as a prostate cancer progression biomarker [1]. Both compounds are detected in urine, where the measured sarcosine/alanine ratio has been found to be higher in prostate biopsy-positive group versus controls. We present here preliminary evidence showing that urine samples spiked with sarcosine/alanine can be partially resolved in 3 min via tandem differential mobility analysis-mass spectrometry (DMA-MS). Based on the calibration curves obtained for two mobility peaks, we finally estimate their concentration ratio in urine.

A strategy for identification and quantification of detergents frequently used in the purification of membrane proteins.

A methodology that enables the identification and quantification of detergents frequently used in the purification of membrane proteins has been developed. The procedure consists of detergent separation via thin-layer chromatography, followed by visualization with iodine vapor staining and subsequent quantification with laser densitometry. We demonstrate that a panel of detergents that are frequently used to purify membrane proteins displays distinctive mobilities in a solvent system consisting of chloroform:methanol:ammonium hydroxide (63:35:5), thereby permitting their separation and identification. In addition, we establish with both the nonionic detergent dodecylmaltoside and the anionic detergent sarkosyl that a linear relationship between detergent quantity and optical density is obtained over a wide range of detergent levels. Furthermore, we demonstrate the accuracy and precision of the assay. Moreover, a strategy for determining the intrinsic iodine-staining capacity of a membrane protein following the removal of associated detergent is presented. Finally, we show the utility of this protocol in measuring detergent concentration following detergent exchange via gel filtration chromatography. The efficacy of this approach for characterizing the detergent present in purified membrane protein preparations prior to conducting crystallization trials is discussed.

Exclusion and retention of compensatory kosmotropes by HPLC columns.

With water as the elution solvent, zwitterionic solutes and polyols were retained on HPLC columns, more than was water, by totally hydrophobic packing materials. Relative retentions were systematically affected by oxygen functional groups in the packing material, explicable as specific retention of water. Reproducible elution sequences of 20 solutes at a variety of hydrophobic surfaces (aromatic and both long- and short-alkyl aliphatic surfaces) showed there is a general process, consistent with interactions with hydration water at the surface having solvent properties distinct from bulk water. Early eluting solutes included glycine, sarcosine and taurine. Glycine betaine followed both these and N,N-dimethylglycine. The natural betaines propionobetaine and dimethylsulfoniopropionate also preceded glycine betaine. Dimethylsulfoxide was strongly retained, as (to a lesser extent) was proline betaine. Polyols eluted in the sequence sorbitol, trehalose, glycerol. Changes in the chemical nature of the surface or base material affected relative retentions of water and solutes. The presence of hydrogen-bonding functions increased retention of polyols, as well as water, relative to zwitterionic solutes. Specific effects retention, constraining models based on the formation of low-density water.

Simultaneous determination of betaine and N,N-dimethylglycine in urine.

A method is described for the determination of betaine and its metabolite N,N-dimethylglycine in human urine. The method involves a deamination step and a solid-phase extraction to isolate both substances from urine followed by high performance liquid chromatography (HPLC) separation on a 5-microns SCX column with UV absorbance detection. Limit of detection is about 0.9 micrograms for both substances. Recoveries were > 85%. The method appears to be suitable for monitoring betaine and its metabolite in urine of patients undergoing therapy with betaine.

Serum betaine, N,N-dimethylglycine and N-methylglycine levels in patients with cobalamin and folate deficiency and related inborn errors of metabolism.

Homocysteine and 5-CH3-tetrahydrofolate (5-CH3-THF) are converted to methionine and THF by the CH3-cobalamin (CH3-Cbl)-dependent enzyme methionine synthase. Serum homocysteine levels are elevated in more than 95% of patients with Cbl or folate deficiency and in patients with inborn errors involving the synthesis of 5-CH3-THF or CH3-Cbl. Homocysteine and betaine are converted to methionine and N,N-dimethylglycine by betaine-homocysteine methyltransferase. It requires neither Cbl nor folate, although N,N-dimethylglycine is converted to N-methylglycine and then to glycine in reactions that both involve the formation of 5,10-CH2-THF from THF. Large amounts of betaine are often given orally to patients with inborn errors, even though little is known about its metabolism in normal subjects or these patients. Thus we developed new gas chromatographic-mass spectrometric assays for serum betaine, N,N-dimethylglycine, and N-methylglycine. In 60 blood donors, we found ranges for normal serum of 17.6 to 73.3, 1.42 to 5.27, and 0.60 to 2.67 mumol/L for the three metabolites, respectively, which were normal in the majority of 50 patients with Cbl deficiency, none of whom had increased levels of N-methylglycine. In 25 patients with folate deficiency, serum betaine level was normal in most, but 76% and 60% had elevations of N,N-dimethylglycine and N-methylglycine levels that ranged as high as 343 and 43.2 mumol/L, respectively. All of seven patients on betaine therapy for inborn errors had high values for betaine (167 to 3,900 mumol/L), N,N-dimethylglycine (15.1 to 250 mumol/L), and N-methylglycine (2.93 to 49.3 mumol/L). Serum total homocysteine levels remained very high at 47.2 to 156 mumol/L (normal, 5.4 to 16.2). In patients with cbl C and cbl D mutations, methionine levels remained low or low-normal at 8.3 to 15.6 mumol/L (normal, 13.3 to 42.7) despite betaine treatment. We conclude that (1) betaine levels are maintained in most patients with Cbl and folate deficiency; (2) levels of N,N-dimethylglycine and N-methylglycine are increased in most patients with folate deficiency; and (3) betaine therapy is relatively ineffective in patients with defective synthesis of CH3-Cbl.

Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.

Class separation of methylated free bile acids from bile acids conjugated with taurine and methylglycine was accomplished using a solvent system of 2,2,4-trimethylpentane-absolute ethanol 10:1 (v/v). By developing a silica thin-layer plate two times with solvent in a Brinkmann sandwich tank, the difficult resolution between methyl cholate and methyl glycolithocholate was achieved. Evidence is presented that this separation system may be useful as a preparative step in the analysis of bile acids by gas-liquid chromatography or high pressure liquid chromatography.--Bolt, M. J. G. Separation of methylated free bile acids from their taurine and methyl glycine conjugates by thin-layer chromatography.

High-performance liquid chromatographic separation and quantification of alanopine and strombine in crude tissue extracts.

A method for the separation and quantification of the levels of alanopine and strombine in neutralized, perchloric acid extracts of tissues of marine invertebrates is presented. The method is based on high-performance liquid chromatographic (HPLC) separation, postcolumn derivatization using o-phthaldialdehyde and sodium hypochlorite, and subsequent fluorometric detection. Isocratic separation results in the rapid elution of alanopine and strombine, with elution times of 4.7 and 5.4 min, respectively. The sensitivity of this method is in the range 50-250 pmol. However, the fluorometric detection approach provides the capability for even greater sensitivity.