Salt removal during Off-Gel electrophoresis of protein samples

The Off-Gel technology was recently described for protein fractionation in a solution placed on top of an immobilized pH gradient gel. In addition, this process was found to remove salts from the biological samples to analyze. This desalting effect is studied experimentally in a conductometric prototype cell. A simplified analytical model is developed to understand this process and a good agreement is found with the conductivity measurements. To illustrate the desalting of a biological sample, a 1 mg.mL(-1) solution of beta-lactoglobulin A in 0.1 M NaCl is subjected to electrophoresis in a single compartment Off-Gel cell. The analysis of the resulting sample by ESI-MS demonstrates the effective removal of salt. A finite element diffusion-migration model is also used to illustrate how the nonuniformity of the electric field in the cell, associated with the salt migration, can slow down the desalting process.     

Evaluation of peptide fractionation strategies used in proteome analysis

Peptide fractionation is extremely important for the comprehensive analysis of complex protein mixtures. Although a few comparisons of the relative separation efficiencies of 2‐D methodologies using complex biological samples have appeared, a systematic evaluation was conducted in this study. Four different fractionation methods, namely strong‐cation exchange, hydrophilic interaction chromatography, alkaline‐RP and solution isoelectric focusing, which can be used prior to LC‐MS/MS analysis, were compared. Strong‐cation exchange × RPLC was used after desalting the sample; significantly more proteins were identified, compared with the nondesalted sample (1990 and 1375). We also found that the use of a combination of analytical methods resulted in a dramatic increase in the number of unique peptides that could be identified, compared with only a small increase in protein levels. The increased number of distinct peptides that can be identified is especially beneficial, not only for unequivocally identifying proteins but also for proteomic studies involving posttranslational modifications and peptide‐based quantification approaches using stable isotope labeling. The identification and quantification of more peptides per protein provide valuable information that improves both the quantification of, and confidence of protein identification.     

On-line desalting-mass spectrometry system for the structural determination of hydrophilic metabolites, using a column switching technique and a volatile ion-pairing reagent

A novel desalting method, using a column switching technique and a volatile ion-pairing reagent, pentadecafluorooctanoic acid, was developed. This system allows hydrophilic and cationic compounds in a nonvolatile buffer to be directly introduced into a mass spectrometer for structural elucidation. The desalting procedure consists of four steps: (1) the fractionation of a target compound from a separation column, (2) the removal of salts with pentadecafluorooctanoic acid on the trap column, (3) the desorption of the compound from the trap column, and (4) the re-equilibration of the trap column with a pentadecafluorooctanoic acid solution. In this procedure, we investigated the methods for optimizing the desalting and re-equilibration steps. Various amino acids, including branched chain amino acids, aromatic amino acids, basic amino acids and methionine, after separation with phosphate buffer on a cation-exchange column, were successively desalted by this method, and were observed as protonated ions by mass spectrometry. This desalting system could be useful for the structural elucidation of unknown hydrophilic compounds eluted by conventional high-performance liquid chromatography methods, such as ion-exchange chromatography, with mobile phases containing nonvolatile salts. As an example, we present the structural elucidation of unknown metabolites in bovine serum.     

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis

Protein samples should be free of salt and other disturbing agents and have an appropriate concentration to be suitable for two-dimensional (2D) electrophoresis, the principal step of proteomics. To find the most efficient method for sample preparation, we used human plasma and compared four widely applied precipitation methods, using trichloroacetic acid (TCA), acetone, chloroform/methanol and ammonium sulfate, as well as ultrafiltration. Precipitation with TCA and acetone and ultrafiltration resulted in an efficient sample concentration and desalting. We also found that ammonium sulfate fractionation can efficiently remove albumin, which represents more than 50% of plasma proteins.     

High-throughput biopolymer desalting by solid-phase extraction prior to mass spectrometric analysis.

In the last 10 years mass spectrometry (MS) has become an important method for analysis of peptides, proteins and DNA. It was recently utilized for accurate high-throughput protein identification, sequencing and DNA genotyping. The presence of non-volatile buffers compromises sensitivity and accuracy of MS biopolymer analysis; it is essential to remove sample contaminants prior to analysis. We have developed a fast and efficient method for desalting of DNA oligonucleotides and peptides using 96-well solid-phase extraction plates packed with 5 mg of Waters Oasis HLB sorbent (Waters, Milford, MA, USA). This reversed-phase sorbent retains the biopolymer analytes, while non-retained inorganic ions are washed out with pure deionized water. DNA oligonucleotides or peptides are eluted using a small amount (20-100 microl) of acetonitrile-water (70:30, v/v) solution. The SPE desalting performance meets the requirements for MS applications such as protein digest analysis and DNA genotyping.     

In situ alkylation of cysteine residues in a hydrophobic membrane protein immobilized on polyvinylidene difluoride membranes by electroblotting prior to microsequence and amino acid analysis.

For identification of cysteine residues on microsequence analysis it is crucial to derivatize the sulfhydryl groups. This reaction requires a desalting step which often represents a major obstacle, especially if the sample consists of limited amounts of a hydrophobic membrane protein. An alkylation procedure is described, allowing efficient derivatization (greater than 90%) of cysteines and cystines even in low microgram quantities, as revealed by test analyses with lysozyme and a hydrophobic membrane protein. The modified protein is recovered in high yields in a form suitable for both microsequence analysis and amino acid analysis. The method involves electrophoretic desalting by miniaturized Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in situ alkylation after electro-transfer onto polyvinylidene difluoride membranes. Precautions against NH2-terminal blocking during sample preparations are provided. The general applicability of the method is illustrated by the structural characterization of the low abundance membrane receptor for human urokinase plasminogen activator.     

Isolation of nonprotamine proteins from the nuclei of the gonad cells of the Russian sturgeonAcipenser güldenstadti

Eight nonprotamine proteins have extracted from the nuclei of the cells of the gonads of the Russian sturgeon with 0.35 M sodium chloride solution followed by fractionation of carboxymethyl-Sephadex G-25 and desalting on Bio-Gel P-2, and their amino acid compositions have been determined.     

Fractionation of Regenerated Silk Fibroin and Characterization of the Fractions

The molecular weight (MW) of regenerated silk fibroin (RSF) decreases during degumming and dissolving processes. Although MW and the MW distribution generally affect polymer material processability and properties, few reports have described studies examining the influences of MW and the distribution on silk fibroin (SF) material. To prepare different MW SF fractions, the appropriate conditions for fractionation of RSF by ammonium sulfate (AS) precipitation process were investigated. The MW and the distribution of each fraction were found using gel permeation chromatography (GPC) and SDS-polyacrylamide electrophoresis (SDS-PAGE). After films of the fractionated SFs formed, the secondary structure, surface properties, and cell proliferation of films were evaluated. Nanofiber nonwoven mats and 3D porous sponges were fabricated using the fractionated SF aqueous solution. Then, their structures and mechanical properties were analyzed. The results showed AS precipitation using a dialysis membrane at low temperature to be a suitable fractionation method for RSF. Moreover, MW affects the nanofiber and sponge morphology and mechanical properties, although no influence of MW was observed on the secondary structure or crystallinity of the fabricated materials.     

Quick screening of major constituents in plant tissue by temperature programmed fractionation mass spectrometry

Temperature programmed fractionation mass spectrometry can be used to quickly screen for the major constituents in plant and herbal tissues. The method is particularly useful if one is searching for a good plant source of a desired compound. It is also shown that simple extraction followed by mass spectrometry has no advantage over the quick screening method. Limitations and possible improvements of the method are discussed.     

Efficient enrichment and desalting of protein digests using magnetic mesocellular carbon foams in matrix-assisted laser desorption/ionization mass spectrometry

We demonstrate that magnetic mesocellular carbon foams (Mag-MCF-C) can be effectively used for enrichment and desalting of protein digests or peptides in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The large mesocellular pores and surface area of Mag-MCF-C are likely to mainly contribute to high efficiency in enrichment and desalting of protein digests. The magnetic property of Mag-MCF-C enabled easy and simple enrichment and desalting process comprising adsorption, washing, and separation steps by using an external magnet. Following elution from Mag-MCF-C by using a matrix solution (CHCA in 70% ACN/0.1% TFA), the peptides were subjected to MALDI-MS analysis. As a result, MALDI mass spectra of peptides or tryptic protein digests were distinct even at a peptide concentration as low as 50 pM. The use of Mag-MCF-C resulted in significantly improved sequence coverage for protein identification when compared to other conventional methods. Mag-MCF-C will find applications in mass spectrometric analysis of low abundance peptides or protein digests with high sensitivity.     

Removal of sodium dodecyl sulfate from protein samples prior to matrix-assisted laser desorption/ionization mass spectrometry

Sodium dodecyl sulfate (SDS) is widely used for protein solubilization and for separation of proteins by SDS polyacrylamide gel electrophoresis (SDS-PAGE). However, SDS interferes with other techniques used for characterization of proteins, such as mass spectrometry (MS) and amino acid sequencing. In this paper, we have compared three procedures to remove SDS from proteins, including chloroform/methanol/water extraction (C/M/W), cold acetone extraction and desalting columns, in order to find a rapid and reproducible procedure that provides sufficient reduction of SDS and high recovery rates for proteins prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). A 1000-fold reduction of SDS concentration and a protein recovery at approximately 50% were obtained with the C/M/W procedure. The cold acetone procedure gave a 100-fold reduction of SDS and a protein recovery of approximately 80%. By using desalting columns, the removal of SDS was 100-fold, with a protein recovery of nearly 50%. Both the C/M/W and the cold acetone methods provided sufficient reduction of SDS, high recovery rates of protein and allowed the acquisition of MALDI spectra. The use of n-octyl-beta-D-glucopyranoside in the protein sample preparation enhanced the MALDI signal for protein samples containing more than 2 10(-4)% SDS, after the C/M/W extraction. Following the cold acetone procedure, the use of n-octylglucoside was found to be necessary in order to obtain spectra, but they were of lower quality than those obtained with the C/M/W method, probably due to higher residual amounts of SDS.     

适用于质谱分析的在线固相萃取除盐方法

建立了一个简单、快速、有效的适用于质谱或液相色谱-质谱联用的在线固相萃取(SPE)高通量除盐方法。方法分为单柱和双柱模式,借助于包含双梯度泵(上样泵/分析泵)、自动进样器和配有十通切换阀的柱温箱的高效液相色谱系统,完成样品的自动化在线除盐。单柱模式通过上样泵实现在SPE柱上进样和除盐,被分析物则保留在SPE柱上;除盐完成后,通过阀切换利用分析泵洗脱富集在SPE柱上的被分析物。双柱模式则在单柱模式基础上增加了1根SPE柱,在色谱管理软件控制下2根SPE柱轮流工作,高效率完成样品的在线除盐。该方法在结合质谱分析蛋白质、多肽等领域具有较好的应用前景。     

On-target sample preparation of 4-sulfophenyl isothiocyanate-derivatized peptides using AnchorChip Targets

De novo sequencing of tryptic peptides by post source decay (PSD) or collision induced dissociation (CID) analysis using MALDI TOF-TOF instruments is due to the easy interpretation facilitated by the introduction of N-terminal sulfonated derivatives. Recently, a stable and cheap reagent, 4-sulfophenyl isothiocyanate (SPITC), has been successfully used for N-terminal derivatization. Previously described methods have always used desalting and concentration by reverse-phase chromatography prior to mass spectrometric analysis. Here we present an on-target sample preparation method based on AnchorChip target technology. The method was optimized for reduction of by-products and sensitivity with SPITC-derivatized tryptic BSA peptides, and successfully applied to protein identification from silver-stained two-dimensional electrophoretic gels of fish liver extracts. The method is simple and sensitive and allowed protein identification based on de novo sequencing and BLAST search from species with limited sequence information.     

Fast C18 solid-phase desalting/delipidation of the human serum apolipoproteins for matrix-assisted laser desorption ionization and electrospray ionization mass spectrometric analysis

A new method for the delipidation of human serum lipoproteins involving the use of a reversed-phase C₁₈ solid-phase extraction (SPE) cartridge is introduced for use with matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. This method is compared with two other methods of lipoprotein delipidation. The SPE method of delipidation produces a higher and more reproducible protein yield than the conventional liquid–liquid methanol–diethyl ether delipidation technique. Furthermore, the SPE method implements a fast, sequential, desalting and delipidation of the lipoproteins for subsequent mass spectrometric analysis providing high quality spectra.     

Monitoring and evaluation of dechlorination processes using compound-specific chlorine isotope analysis

A simple, quick and sensitive method for the compound-specific stable chlorine isotope analysis of chlorinated solvents by conventional quadrupole gas chromatography/mass spectrometry (GC/MS) is presented. With this method, compound-specific stable chlorine isotope ratios of typical chlorinated solvents like tetrachloroethene (PCE) and trichloroethene (TCE) can be determined quantitatively within 30 min by direct injection. The chlorine isotope ratios of target substances are calculated from the peak areas of several selected molecular ions and fragment ions of the substances, using a set of unique mathematical equations. The precision of the method was demonstrated through reproducibility tests. An internal precision of +/-0.4 per thousand to +/-1.1 per thousand was obtained when analyzing PCE and TCE in the 10-1000 pmol range. The validity of the method was further demonstrated by determining the chlorine isotopic fractionation factor during the reductive dechlorination of TCE in a batch experiment using zero-valent iron. The chlorine isotopic fractionation factor was calculated as 0.9976 +/- 0.0011 with a correlation coefficient of 0.9469 (n = 38). The high correlation coefficient indicates that compound-specific stable chlorine isotope analysis can be performed with sufficient accuracy using conventional quadrupole GC/MS when significant fractionation takes place during a reaction. For the first time, the chlorine isotope fractionation factor of TCE during an abiotic anaerobic dechlorination process was determined using quadrupole GC/MS, without offline sample preparation.     

Recovery of protein by coomassie brilliant blue precipitation prior to electrophoresis.

The interaction of protein with Coomassie Brilliant Blue G-250 results in formation of an insoluble protein-dye complex which can be recovered by centrifugation and redissolved for electrophoretic analysis. The precipitated protein can be washed in acetone to remove excess dye in order to enhance resolution. The residual dye becomes dissociated from the proteins on electrophoresis and can be exploited as a "dye front". The method allows simultaneous protein assay and recovery of microgram amounts of protein from dilute solution and could be widely applied for conserving, concentrating and desalting minute amounts of valuable sample prior to electrophoretic analysis.     

Identification of proteins by combination of size-exclusion chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and comparison of some desalting procedures for both intact proteins and their tryptic digests

Separation of a protein mixture by size-exclusion chromatography (SEC) was combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Identification of proteins in the collected fractions was performed both as intact proteins by MALDI-TOFMS and using peptide mass fingerprinting (PMF) after their digestion with trypsin. The presence of salts mostly disturbs the MALDI-TOFMS signal and, therefore, proper purification or desalting procedures must be employed. Four desalting procedures (desalting column packed with Sephadex G-100, on-target washing, centrifugal filter devices and ZipTip C(18)) for purification of fractions of proteins separated by SEC and their tryptic digests prior to determination of their exact molecular masses by MALDI-TOFMS were compared. In the case of intact proteins, the experiments showed that the best desalting procedures are the use of ZipTip C(18) pipette tips and Ultrafree CL centrifugal filter devices. The peptide digests can be purified by using ZipTip C(18) pipette tips or on-target washing when both of these procedures provide similar results. On-target washing can be used as a simple procedure to improve the mass spectra of salt-containing samples. Analyses of the droplets collected after the on-target washing show losses of sample and matrix caused by dissolution of these compounds during this procedure. Further, it was found that protein identification based on PMF is more sensitive than analyses of intact proteins and that multiple on-target washing is very advantageous for analyses of peptide mixtures with a high content of salts.     

Ionic‐liquid‐assisted desorption of DNA from polyamidoamine‐grafted silica nanoparticles surface by a low‐salt solution

We studied the influences of imidazolium‐based ionic liquids as additives in low ionic strength phosphate solution on releasing DNA from polyamidoamine dendrimer‐grafted silica nanoparticle surfaces. The effects of the side‐chain length of the imidazolium group, the anion and the concentration of the ionic liquid, the generation of the dendrimer, and the pH and the concentration of the release solution were investigated. It was found that addition of 4 mM 1‐hexyl‐3‐methylimidazolium bromide to 5 mM phosphate at pH 11 could markedly promote the desorption of DNA fragments, with a desorption efficiency of 99.0%. Compared with the conventional strategies employing high‐salt solutions or elevated temperature for acceptable recoveries, the method described here enabled quick release of DNA fragments that permitted direct, accurate analysis, and further treatment without desalting.     

Quantitative magnetic resonance imaging of urea and lysozyme in protein chromatography

Magnetic resonance imaging (MRI) techniques have been implemented to enable quantitative imaging of protein and urea within a 5 ml HiTrap size-exclusion chromatography desalting column, without introduction of contrast agents. One-, two- and three-dimensional images of urea injected at concentrations of 2, 4, 6 and 8 M were acquired. One-dimensional profiles of lysozyme at concentrations between 5 and 25 mg ml(-1) were also obtained. All data were accurate to within +/- 15% when compared to the known amount injected. Quantitative MRI elution profiles of both urea and lysozyme were then obtained in real-time during a desalting separation.     

亚微米电喷雾喷针在非变性质谱分析中的研究进展

非变性质谱技术已成为表征蛋白质结构的重要工具之一。与传统的电喷雾喷针(Electrospray emitter,ESI emitter)相比,亚微米电喷雾喷针具有改变离子电荷态分布和降低盐离子加合等多种特性,可在生理环境下直接解析蛋白质的结构。该文综述了亚微米电喷雾喷针的特性及其在非变性质谱分析中的应用,并对其未来的发展趋势进行了展望。