Sample centrifugation onto membranes for sequencing

This paper presents a new method for adsorption of proteins in solution onto a polyvinylidene diflouride (PVDF) membrane using centrifugation. The technique uses a low molecular weight cut-off membrane (LMW) placed underneath a PVDF membrane. The paired membranes are placed in a receptacle which in turn fits into a microcentrifuge tube. During sample centrifugation, the LMW acts to increase the amount of protein that is concentrated and adsorbed onto the hydrophobic surface of the PVDF membrane. By alternating between two receptacle sizes, this method can accommodate large (greater than 10 micrograms) and small (less than 10 micrograms) amounts of sample. This paper demonstrates sample recovery for a variety of proteins as quantitated by radioactivity and amino acid analysis after centrifugation onto PVDF. Amino acid and sequence analysis results demonstrate the efficiency with which interfering buffers and sodium dodecyl sulfate are removed as a result of sample centrifugation and washing. Finally, we demonstrate the utility of this technique with samples in the low picomole range to obtain useful sequence information following electrophoretic isolation of cyanogen bromide fragments purified by high performance electrophoresis chromatography.     

Micropurification of Two Human Cerebrospinal Fluid Proteins by High Performance Electrophoresis Chromatography

Abstract: Using C8 reversed-phase HPLC in conjunction with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we have fractionated proteins contained in human CSFs obtained from patients with schizophrenic disorders. When these proteins were electrophoretically blotted onto polyvinylidene difluoride membrane for direct N-terminal amino acid sequencing, several CSF proteins were identified; these included albumin, transferrin, apolipoprotein A-l, β₂-microglobulin, and prealbumin. We have also identified two structurally related human CSF proteins designated cerebrin 28 (M_r 28,000) and cerebrin 30 (M_r 30,000) that have an N-terminal amino acid sequence of NH₂-APPAQVSVQPNF and NH₂-APEAQVSVQPLFXQ, respectively. Comparison of these sequences with existing database at Protein Identification Resource (R 32.0), GenBank (R 72.0), SWISS-PROT (R 22.0), and EMBL (R 31.0) indicated that they are unique proteins. These proteins were subsequently purified by high performance electrophoresis Chromatography (HPEC) using an Applied Biosystems 230A HPEC system. A specific polyclonal antibody was prepared and an ELISA was established for cerebrin 30. It was noted that HPEC is a powerful tool to purify microgram quantities of proteins from human, rabbit, and rat CSFs. Using such a system, we have been able to micropurify as many as 10 proteins simultaneously in a single experiment because the elution of proteins occurred strictly according to their molecular weights. More importantly, we routinely obtained a recovery of >90%. The potential use of this technology for micropurification of proteins was discussed.     

Multiwell in-gel protein digestion and microscale sample preparation for protein identification by mass spectrometry

In-gel peptide digestion has become a widely used technique for characterizing proteins resolved by two-dimensional gel electrophoresis. Peptides generated from gel pieces are frequently contaminated with detergent and salts. Prior to matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis, these contaminants are removed using micro scale C18 sample preparation columns. In this paper, data are presented to demonstrate the application of a solvent resistant MultiScreen 96-well plate with a low peptide binding membrane and ZipTip micropipette based sample preparation. Recoveries of peptides (m/z of 1000 to 5000 Da) derived from standard protein protease digests, were estimated at various stages of the analytical process. An optimized protocol has been established and all the reagents and consumables have been packaged in a ready to use commercial kit. Data will be presented to show the application of this technology package to accelerate the throughput of protein characterization by protease fragmentation.     

Gel absorption-based sample preparation for the analysis of membrane proteome by mass spectrometry

A gel absorption-based sample preparation method for shotgun analysis of membrane proteome has been developed. In this new method, membrane proteins solubilized in a starting buffer containing a high concentration of sodium dodecyl sulfate (SDS) were directly entrapped and immobilized into gel matrix when the membrane protein solution was absorbed by the vacuum-dried polyacrylamide gel. After the detergent and other salts were removed by washing, the proteins were subjected to in-gel digestion and the tryptic peptides were extracted and analyzed by capillary liquid chromatography coupled with tandem mass spectrometry (CapLC-MS/MS). The results showed that the newly developed method not only avoided the protein loss and the adverse protein modifications during gel embedment but also improved the subsequent in-gel digestion and the recovery of tryptic peptides, particularly the hydrophobic peptides, thereby facilitating the identification of membrane proteins, especially the integral membrane proteins. Compared with the conventional tube-gel digestion method, the newly developed method increased the numbers of identified membrane proteins and integral membrane proteins by 25.0% and 30.2%, respectively, demonstrating that the method is of broad practicability in gel-based shotgun analysis of membrane proteome.     

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.     

A modified reaction cartridge for direct protein sequencing on polymeric membranes.

A newly designed reaction vessel implements a vertical cross-flow type reactor with the Applied Biosystems multi-mode reaction cartridge design. This cartridge is designed for sequencing samples on polyvinylidine difluoride-type membranes. The benefits of this design include a reduced reaction chamber volume that results in lower rates of chemical consumption and less risk of sample loss or contamination during sequencing. Visualization of the membrane in the reaction chamber during sequencing facilitates optimization of drying, washing, extraction and transfer times. The cycle modifications described in this report are designed to optimize post-coupling extraction, cleavage and post-cleavage extraction steps during "flow across" conditions for polymeric membranes. Also, efficient washing and drying of membranes allows for a fast cycle time of 30 minutes when using Pulsed Liquid chemistry. Examples of Blott cartridge utility for sequencing polyvinylidine difluoride-bound proteins in the low picomole range are shown by analyzing samples prepared by a two-dimensional purification scheme using the 230A HPEC and sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

A simple and rapid method for the preparation of apolipoproteins for electrophoresis.

A new method is described for the rapid preparation of apolipoproteins for polyacrylamide gel electrophoresis. It is suitable for all serum lipoproteins including chylomicrons. The procedure involves extraction with diethyl ether in the presence of trichloroacetic acid and sodium deoxycholate. The method gives an improved protein recovery, in particular with chylomicrons. In addition, samples do not require dialysis to remove salts (e.g., KBr) prior to processing; as a consequence, the procedure requires only 1 h. Due to this rapidity and the high yields, the procedure is superior to present methods utilizing ethanol-diethyl ether extraction.     

Electrofractionation: a technique for detecting and recovering biomolecules.

Electrofractionation (EF) is a technique that allows electrophoretic materials to be detected and recovered following electrophoresis. The EF apparatus utilizes the resolving power of electrophoresis and the mobile phase of liquid chromatography to create a continuous elution system. Our data indicate that EF is able to detect and recover oligonucleotides, as DNA fragments, proteins, and presumably other material that can be analyzed by electrophoresis. EF shares functional similarities with high-performance electrophoresis chromatography (HPEC) but operates by a different strategy and at a fraction of the cost. Moreover, EF can be constructed largely from standard laboratory equipment. The simplicity, rapid analysis times, low cost, and high recovery yields of EF make this system a practical alternative to the conventional detection and purification methods used for biomolecules.     

Oxazolinone derivative of leucine for GC-MS: a sensitive and robust method for stable isotope kinetic studies of lipoproteins

Stable isotope labeled amino acids are commonly used as endogenous tracers to study the metabolism of lipoproteins. The determination of isotopic enrichment of particular amino acids in apolipoproteins is carried out by gas chromatography mass spectrometry (GC-MS). This report describes a robust and sensitive derivative for analysis of d3-leucine by GC-MS and its utility in studying the metabolism of human lipoproteins. The trifluoromethyloxazolinone (oxazolinone) derivative of leucine was formed in a rapid single step procedure using a mixture of trifluoroacetic anhydride (TFAA) and trifluoroacetic acid (TFA). Analysis of the oxazolinone by negative ion chemical ionization GC-MS gave excellent sensitivity and precision, which enabled accurate determination of low levels of isotopic enrichment from small amounts of protein. For example, enrichments between 0.05% and 100% in 100 pg leucine can be measured with a coefficient of variation of <3%. To demonstrate the utility of this procedure, we measured d3-leucine enrichment in apolipoprotein B (apoB) isolated from VLDL and LDL as well as apoA-I isolated from HDL by gel electrophoresis and western blotting. The derivatization procedure gave excellent enrichment data from a single intravenous bolus dose of 5 mg/kg, from which the fractional catabolic rate and production rate of the lipoproteins were calculated. In conclusion, the oxazolinone derivative provides a robust and simple procedure for the sensitive analysis of isotopic enrichment for metabolic studies of human lipoproteins.     

PURIFICATION OF MYELIN CARBONIC ANHYDRASE

Abstract— A procedure has been developed for the purification of the membrane bound form of carbonic anhydrase from rat brain myelin. The procedure is rapid, requiring only two steps, and can be applied to small amounts of material. Conditions have been established whereby the enzyme can be almost quantitatively solubilized with up to a 60 fold increase in specific activity. Purification by affinity chromatography yields a preparation which is homogeneous by polyacrylamide gel electrophoresis. However, preliminary evidence suggests that activity may be reduced by the removal of lipids during chromatography and subsequent dialysis. The purified preparation is high in dicarboxylic and hydroxyl amino acids and contains only 1×2 cysteine residues. The reduction of cysteine appears to be essential for the full expression of enzymatic activity.     

Purification and characterization of the extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.     

The direct hydrolysis of proteins containing tryptophan on polyvinylidene difluoride membranes by mercaptoethanesulfonic acid in the vapor phase.

A procedure for the amino acid analysis of polypeptides that contain tryptophan on polyvinylidene difluoride membranes is described. Lysozyme, carbonic anhydrase, phytochrome, and ovalbumin were tested. The protein, which was separated from others by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was blotted from the gel onto a polyvinylidene difluoride membrane and directly hydrolyzed by 3 N mercaptoethanesulfonic acid vapor in a vacuum at 176 degrees C for 25 min. The hydrolysate was extracted with 0.1 N HCl and 30% methanol and used for amino acid analysis. The tested proteins were adequately hydrolyzed, and the recovery of tryptophan was very efficient.     

Purification and characterization of a murine cellular retinoic acid-binding protein

A cellular retinoic acid-binding protein from 1-day-old mouse pups has been purified to homogeneity. The isolation procedure included gel filtration on Sephadex G-75, ion exchange chromatography on DEAE cellulose, and chromatofocusing on PBE9-4 ion exchange resin. The chromatofocusing step was most useful in removing the major contaminants, which were otherwise difficult to remove. The binding protein was finally subjected to two cycles of high performance liquid chromatography on a DEAE-5PW column to achieve homogeneity. The protein has an isoelectric point of 4.75 and consists of a single polypeptide, migrating with an apparent Mr of 14,600 in SDS--polyacrylamide gel electrophoresis. Amino-terminal sequence analysis showed that the mouse cellular retinoic acid-binding protein has a high percentage of amino acid identity with other retinoid-binding proteins. However, it is immunologically distinct from the cellular retinol-binding protein.     

Different techniques for urinary protein analysis of normal and lung cancer patients

Many components in urine are useful in clinical diagnosis and urinary proteins are known as important components to define many diseases such as proteinuria, kidney, bladder and urinary tract diseases. In this study, we focused on the comparison of different sample preparation methods for isolating urinary proteins prior to protein analysis of pooled healthy and lung cancer patient samples. Selective method was used for preliminary investigation of some putative urinary protein markers. Urine samples were passed first through a gel filtration column (PD-10 desalting column) to remove high salts and subsequently concentrated. Remaining interferences were removed by ultrafiltration or four precipitation methods. The analysis of urinary proteins by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed many similarities in profiles among preparation methods and a few profiles were different between normal and lung cancer patients. In contrast, the results of two-dimensional gel electrophoresis (2-DE) showed more distinctly different protein patterns. Our finding showed that the sequential preparation of urinary proteins by gel filtration and ultrafiltration could retain most urinary proteins which demonstrated the highest protein spots on 2-D gels and able to identify preliminary urinary protein markers related to cancer. Although sequential preparation of urine samples by gel filtration and protein precipitation resulted in low amounts of proteins on 2-D gels, high Mr proteins were easily detected. Therefore, there are alternative choices for urine sample preparation for studying the urinary proteome and identifying urinary protein markers important for further preclinical diagnostic and therapeutic applications.     

Isolation and characterization of a somatomedin-binding protein from mid-term human amniotic fluid

Human amniotic fluid is rich in a binding protein for somatomedins. This binding protein competes with human placenta membranes for labelled somatomedin A. Consequently, the placenta radioreceptorassay for somatomedin can be used for detection of the binding protein. The protein was isolated from human amniotic fluid by a three-step procedure: First, stepwise ammonium sulphate precipitation; second, hydrophobic chromatography (phenyl-Sepharose); and third, anion-exchange chromatography (fast protein liquid chromatography). The total recovery of binding protein calculated with the placenta radioreceptorassay was 50%. Polyacrylamide gel electrophoresis under native and denaturating conditions of the isolated protein disclosed a single band. The relative molecular mass was 35000, determined by exclusion chromatography, and 32000 under denaturating conditions in sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The isoelectric point was 4.3 according to chromatofocusing and the amino acid composition also disclosed a high content of acidic/amidated residues. The N-terminal amino acid sequence was Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala.     

Structural and immunochemical characterization of human urine arylsulfatase A purified by affinity chromatography

Arylsulfatase A (aryl-sulfate sulfohydrolase, EC 3.1.6.1) was isolated from an ammonium sulfate precipitate of urinary proteins using two different affinity chromatography methods. One method involved the use of concanavalin A-Sepharose affinity chromatography at an early stage of purification, followed by preparative polyacrylamide gel electrophoresis. The other procedure employed arylsulfatase subunit affinity chromatography as the main step and resulted in a remarkably efficient purification. The enzyme had a specific activity of 63 U/mg. The final preparation of arylsulfatase A was homogeneous on the basis of polyacrylamide gel electrophoresis at pH 7.5, and by immunochemical analysis. However, when an enzyme sample obtained by either method of purification was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing or non-reducing conditions, peptide subunits, of 63.5 and 54.5 kDa, were observed. Immunological tests with 125I-labeled enzyme established the presence of a common protein component in both of the electrophoretically separable peptide subunits of human urine arylsulfatase. The amino acid analysis of homogeneous human urine arylsulfatase A showed only a few differences between it and the human liver enzyme. However, immunological cross-reactivity studies using rabbit anti-human urine arylsulfatase revealed immunological difference between the human urine and liver arylsulfatase A enzymes.     

Identification and characterization of glycoproteins after extraction of bovine chromaffin-granule membranes with lithium di-iodosalicylate. Purification of glycoprotein II from the soluble fraction.

Chromaffin-granule membranes were separated into insoluble and soluble fractions after extraction with lithium di-iodosalicylate (LDIS). These fractions were characterized by one- and two-dimensional gel electrophoresis, and glycoproteins were detected after electroblotting with peroxidase-labelled concanavalin A and wheat-germ agglutinin (WGA). The LDIS-insoluble fraction contained components identified as glycoproteins III, H, J and K (carboxypeptidase H). Microsequence analysis indicated that component J is an N-terminally extended form of glycoprotein K. A major glycoprotein, GpII (Mr 80,000-100,000), present in the LDIS-soluble fraction was purified by affinity chromatography on WGA-Sepharose. This was characterized by one- and two-dimensional gel electrophoresis with Coomassie Blue staining, by amino acid analysis and automated N-terminal sequence analysis. Extraction of chromaffin-granule membranes with LDIS is a simple and rapid procedure that facilitates studies concerned with the structure and function of membrane glycoproteins from these and other secretory granules.     

A two-step procedure for purification of papain from extract of papaya latex

A method is presented for purifying papain from extracts of papaya latex. The procedure involves precipitation of the extract of papaya latex with sodium chloride followed by affinity chromatography of the redissolved precipitate. Precipitation of the protein from the latex extract is necessary to separate the papain from material which interferes with the binding of papain to the affinity column. During affinity chromatography, the affinity column is overloaded to insure absence in the final product of impurities which are capable of binding to the affinity column.The papain prepared by this procedure yielded an amino acid analysis and an N-terminal amino acid analysis expected for a sample of pure papain. No Met was detected on amino acid analysis nor was the presence of N-terminal residues other than He detected. On polyacrylamide disc gel electrophoresis at pH 4.3, papain prepared by the method described in this work was indistinguishable from crystalline papain which was prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Both disc gel patterns consisted of a single band and a trailing shadow which was less than 5% of the main band. In routine spectrophotometric assays, the specific activity toward N,α-benzoyl-l-arginine ethyl ester of papain prepared by the procedure described in this work was indistinguishable from crystalline papain prepared by the method of Kimmel and Smith, and further purified by affinity chromatography. Values of 24 sec^?1' and 15 mm were obtained from the turnover number and K_m for the papain-catalyzed hydrolysis of N,α-benzoyl-l-arginine ethyl ester at 25 °C, pH 6.00, $\text{Γ}\text{2 0.30}$ using a pH stat.     

用有限酶切法分析蛋白质的氨基酸序列

报道了一个通过有限酶切蛋白质产生多肽片段的方法.蛋白质经单向SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离和用考马斯亮蓝短暂染色后,切下所需的蛋白质带,将其放入另一个SDS-PAGE凝胶的样品槽内,在电泳过程中该蛋白质被蛋白酶如蛋白酶V8降解,所产生的多肽片段随之被分离.电泳结束后,将多肽片段电印迹至聚偏二氟乙烯(polyvinylidene difluoride,PVDF)膜上.这些多肽片段从PVDF膜上切下后可以直接被用于分析氨基酸序列.该方法能广泛适用于分析一般蛋白质和N端被修饰蛋白质的氨基酸序列.