Efficient Protein Separation Research Articles

Low-mass proteome analysis based on liquid chromatography fractionation, nanoliter protein concentration/digestion, and microspot matrix-assisted laser desorption ionization mass spectrometry

HPLC fractionation combined with mass spectrometry can become a powerful tool for analyzing the proteome in the mass range below 15 kDa where efficient protein separation by gel electrophoresis can be difficult. For sensitive and high-resolution separation of the low-mass proteome, the use of analytical rather than preparative HPLC columns is preferred. However, individual fractions collected by a conventional HPLC separation usually contain a small amount of proteins whose concentrations may not be sufficiently high for subsequent enzyme digestion and protein identification by mass spectrometry. In this work, we present a high sensitivity nanoliter sample handling technique to analyze proteins fractionated by HPLC. In this technique, an individual HPLC fraction in hundreds of microliter volume is pre-concentrated to several microliters. About 700 pl of the pre-concentrated fraction is then drawn into a 20-μm I.D. capillary and dried in a small region near the capillary’s entrance. This process can be repeated many times to concentrate a sufficient amount of protein to the small region of the capillary. After protein concentration, protein digestion is achieved by drawing 1 nl of chemical or enzymatic reagent into the capillary and placing it in the same region where the dried protein sits. The resulting peptides are then deposited onto a microspot in a MALDI probe for mass analysis. The performance of this technique is demonstrated with the use of a standard protein solution. This technique is applied to the identification of low-mass proteins separated by HPLC from a complex mixture of an E. coli extract.

Two-dimensional protein analysis by isoelectric focusing in a multi-cell plate system.

A practical and low cost system for isoelectric protein focusing (IEF) was developed. The system uses a multi-cell glass plate compatible with a common vertical one-dimensional electrophoresis chamber, dispensing specific IEF apparatus. After focusing, the IEF gels are easily recovered. The resulting two-dimensional (2-D) gels have provided efficient protein separation for different concentrations and extracts.

Alkylation kinetics of proteins in preparation for two-dimensional maps: a matrix assisted laser desorption/ionization-mass spectrometry investigation.

All existing protocols for protein separation by two-dimensional (2-D) gel electrophoresis require the full reduction, denaturation, and alkylation as a precondition for an efficient and meaningful separation of such proteins. Existing literature provides a strong evidence to suggest that full reduction and denaturation can be achieved in a relatively short time; the same thing, however, can not be said for the alkylation process, which the present study shows that more than 6 h are required for a complete alkylation. We have used matrix assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) to monitor protein alkylation by iodoacetamide over the period 0-24 h at pH 9. The present, fast and specific MS method provided clear indication on the extent and speed of alkylation which reached approximately 70% in the first 2 min, yet the remaining 30% resisted complete alkylation up to 6 h. The use of sodium dodecyl sulfate (SDS) during the alkylation step resulted in a strong quenching of this reaction, whereas 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) exerted a much reduced inhibition. The implications of the present measurements on 2-D gel analysis in particular and proteomics in general are discussed.

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Evaluation of volatile eluents and electrolytes for high-performance liquid chromatography–electrospray ionization mass spectrometry and capillary electrophoresis–electrospray ionization mass spectrometry of proteins: II. Capillary electrophoresis

Peptides and proteins were separated by capillary electrophoresis (CE) in fused-silica capillaries coated with an irreversibly adsorbed monolayer of derivatized polystyrene nanoparticles. Whereas phosphate buffer, pH 3.10, enabled the highly efficient separation of basic proteins with plate counts up to 1 400 000 m −1, volatile buffer components such as formic acid or acetic acid titrated with ammonia to the desired pH had to be used for the direct coupling of CE with electrospray ionization mass spectrometry (ESI-MS). Compared to 40 m M phosphoric acid–sodium hydroxide, pH 3.10, a background electrolyte containing 125 m M formic acid–ammonia, pH 4.00, was shown to yield equivalent separation efficiency. Investigation of the influence of buffered electrolytes on the ESI-MS signal of lysozyme at pH 2.70–4.00 showed that the charge state distribution shifted to lower charge states at higher pH with a concomitant five-fold decrease in signal intensity of the most abundant signal. The presence of trifluoroacetic acid in the background electrolyte greatly increased the level of baseline noise and completely inhibited the observation of any mass signals related to proteins. Full scan spectra could be acquired from 50–500 fmol amounts of proteins during coupled CE–ESI-MS utilizing 100–125 m M formic acid–ammonia, pH 3.10. However, compared to UV detection, considerable band broadening is observed with ESI-MS detection which is mainly attributed to column overloading, band spreading in the interface, and scanning data acquisition. Finally, the major whey proteins β-lactoglobulin A, β-lactoglobulin B, and α-lactalbumin were identified in a whey drink by comparison of molecular masses determined by CE–ESI-MS to molecular masses calculated from the amino acid sequence.

Reference map of the low molecular mass proteins of Haemophilus influenzae.

Analysis of the proteome of Haemophilus influenzae by two-dimensional polyacrylamide gel electrophoresis on conventional Tris-glycine gels does not usually result in efficient separation of the proteins in the 5-20 kDa range, which are mainly accumulated in the lower acidic and basic regions. In order to improve the separation of the low molecular mass proteins, we used homogeneous Tricine gels of two urea concentrations in the second-dimensional separation. The Tricine gel systems allowed the efficient and reproducible separation of the proteins of the microorganism with masses between 5 and 20 kDa, however, no proteins with masses below 5 kDa could be visualized. Approximately 80 proteins migrating in the 5-25 kDa region were identified by matrix assisted laser desorption/ionization - mass spectrometry, of which 40 identified for the first time. The digestion of the low mass proteins often produced only few peptides, which were insufficient for confident identification by mass spectrometry. Therefore, the identification was occasionally achieved by a sequential digestion with two proteases, trypsin or endoproteinase Lys-C as first and carboxypeptidase P as second enzyme. The gel system described may be useful for the efficient separation of low molecular mass proteins from other organisms to construct standard maps.

Application of micropellicular poly-styrene/divinylbenzene stationary phases for high-performance reversed-phase liquid chromatography electrospray-mass spectrometry of proteins and peptides

Short columns packed with highly crosslinked 2.3 μm poly-styrene/divinylbenzene (PS/DVB) particles were used for rapid and efficient separation of proteins and peptides by reversed-phase high-performance liquid chromatography at elevated temperatures. Enhancement of the diffusivities of the sample components at elevated temperatures together with the short diffusion pathlength with the micropellicular polymeric stationary phases were responsible for high efficiency, high speed of analysis, and short column regeneration times. Underivatized PS/DVB beads as well as PS/DVB microspheres which have been modified with polyvinylalcohol or octadecyl chains on the surface were synthesized, employed, and compared to HY-TACH-C18, a commercially available micropellicular octadecyl-silica stationary phase, for the separation of proteins, octapeptides and tryptic protein digests. Highest performance was obtained with the silica- and PS/DVB-based octadecyl stationary phases, which exhibited similar column efficiencies but different selectivities for proteins and peptides. The minimum detectability at 214 nm and the maximum loading capacity for ribonuclease A using analytical 30×4.6 mm I.D. columns were 10 ng (0.6 pmol) and 1 μg, respectively. Finally, reversed-phase HPLC with a 60×2 mm I.D. narrow-bore column packed with micropellicular octadecyl PS/DVB was coupled successfully to electrospray mass spectrometry at a flow-rate of 0.15 mL min−1 and on-line full-scan mass spectra for molecular mass determination and identification of proteins in the lower picomol range were obtained.

Highly efficient protein separations in high-performance capillary electrophoresis, using hydrophilic coatings on polysiloxane-bonded columns

Three methods for preparing hydrophilic coatings on polysiloxane bonded CElect H-type capillary electrophoresis columns have been shown. The polyalkylsiloxane-bonded phase is the first coating layer on the capillary surface, and nonionic surfactant, hydrophilic polymer, or polymer surfactant, adsorbed onto this first layer through hydrophobic interactions, forms the second coating layer. The resultant capillary surfaces are inert, hydrophilic, and suitable for highly efficient protein separations. The effectiveness and applicability of these capillary surface modification methods were tested for the separations of a variety of proteins over a wide range of buffer pH values under different capillary electrophoretic operation modes.

N,N,N',N'-tetramethyl-1,3-butanediamine as effective running electrolyte additive for efficient electrophoretic separation of basic proteins in bare fused-silica capillaries.

The effect of N,N,N',N'-tetramethyl-1,3-butanediamine (TMBD) in the running electrolyte on the electroosmotic flow and the migration behavior of four standard basic proteins in bare fused-silica capillaries was examined at pH 4.0, 5.5, and 6.5. Depending on the electrolyte pH and additive concentration the electroosmotic flow was either cathodic or anodic. A similar Langmuirian-type dependence of the electroosmotic flow on the concentration of TMBD in the running electrolyte was found at the three experimented pH values, which may be indicative of the specific adsorption of the additive in the immobilized region of the electric double layer at the interface between the capillary wall and the electrolyte solution. Electrophoretic separations of the four standard basic proteins performed at the three above pH values, showed well-resolved, efficient and symmetric peaks, demonstrating the utility of this additive for protein electrophoresis in bare fused-silica capillaries. The variations in separation efficiency, peak capacity, resolution and reproducibility of migration times as a function of the additive concentration at pH 6.5 were also examined.

High resolution of multiple forms of rabbit reticulocyte hexokinase type I by hydrophobic interaction chromatography

Hydrophobic interaction chromatography (HIC) has been employed extensively in the separation of proteins by elution using a descending salt gradient, with and without the use of detergents or denaturing agents. In this study, a new hydrophobic interaction chromatographic support, Toyopearl Phenyl 650 S, was investigated in order to examine the distribution of multiple forms of rabbit reticulocyte hexokinase type I. These distinct forms of the enzyme, designated hexokinase Ia, Ia* and Ib, show similar kinetic and physical properties, similar molecular masses (ca. 100 000) and a different intracellular distribution. The results obtained using Toyopearl Phenyl 650 S of 20–50-μm particle diameter show that this HIC support allows very high resolution, comparable to that obtainable with HIC-HPLC columns but with the advantage of charging a higher amount of starting material even with a high protein concentration. These characteristics render Toyopearl Phenyl 650 S suitable for analytical and preparative purposes. Further, in the separation of multiple forms of rabbit reticulocyte hexokinase, the HIC method was shown to be superior to RP-HPLC, making possible the efficient separation of proteins with high molecular mass and their recovery in active forms. The Toyopearl Phenyl 650 S column was also shown to be more efficient than the ion-exchange chromatographic media previously used, allowing a quicker analysis of the multiple forms of rabbit reticulocyte hexokinase under different biological conditions.

Size-dependent separation of proteins in the presence of sodium dodecyl sulfate and dextran in capillary electrophoresis: effect of molecular weight of dextran.

Dextran solutions are widely used as sieving medium in protein analysis by capillary electrophoresis in the presence of sodium dodecyl sulfate. We studied the effect of dextran molecular weight on the separation efficiency using different dextran preparations with wide and very narrow molecular weight distributions, in the range between 1270 and 2,000,000. Migration times and band broadening of proteins were significantly affected by the molecular weight of dextran. Migration times of proteins decreased as molecular weights of the dextrans decreased. Satisfactory separation of all the proteins was possible with all dextrans except those with molecular weights of 70,000 and 23,800 where bovine serum albumin and phosphorylase b failed to be separated. Unexpectedly rapid separation of all the proteins with enhanced resolution could be observed using two dextrans with a narrow molecular weight distribution, with molecular weights of 1270 and 5220. Clearly the use of dextran with higher molecular weight is not the only way to achieve efficient separation of proteins. The separation mechanism in the presence of the low molecular weight dextrans remains to be made clear in a future study.

Rapid analysis of biopolymers on modified non-porous polystyrene-divinylbenzene particles

The use of short columns packed with alkylated nonporous 2.1 μm polystyrene — divinylbenzene particles has enabled the efficient separation of proteins, oligonucleotides, and DNA fragments in less than 60 seconds. An increase in the flow-rate resulted in only a minor reduction in the resolution of the proteins. Careful temperature control and small gradient dead volume were essential if reproducible quantitative results were to be achieved. Analysis at elevated temperatures not only reduced the viscosity of the eleents and, hence, column back-pressure but also resulted in higher column efficiency. The best resolution of proteins was achieved by performing the separation at 80°C whereas the optimum temperatures for the separation of oligonucleotides and DNA fragments ranged from 40–50°C. High speed analysis was used both to control the purity of oligonucleotides following automated solid-phase synthesis and to evaluate the expression of multidrug resistance genes in patients suffering from chronic lymphatic leukemia.

High-performance capillary electrophoresis for the determination of trypsin and chymotrypsin inhibitors and their association with trypsin, chymotrypsi

High-performance capillary electrophoresis (HPCE) was adapted for the determination of Kunitz soybean trypsin inhibitor, Bowman Birk inhibitor from soybean and protein-type proteinase inhibitors from pea ( Pisum sativum L.). The method was developed for the determination and characterization of the inhibitors, the enzymes trypsin and chymotrypsin and the monoclonal antibodies (mAbs) raised against the inhibitors, and also the inhibitor—enzyme and inhibitor—mAb association complexes. The results from studies involving the use of various types of buffers revealed the advantages of having zwitterions such as trimethylammoniumpropyl sulphonate (AccuPure) or taurine included in the buffer. The use of capillaries dynamically coated with zwitterions efficiently reduced the interactions of the proteins with the silica capillary surface, which was important for the analyses for trypsin, chymotrypsin and mAbs and their association complexes with the inhibitors. The influence of temperature, voltage, pH and buffer type on migration times, resolution, peak areas and number of theoretical plates was investigated for the proteins studied. The proposed HPCE method is very suitable for studies of proteinase inhibitors compared with traditional inhibitor studies, and it gives efficient protein separations with the possibility of 245 000 plates/m.

Use of mixed-mode, high-performance liquid chromatography for the separation of peptide and protein mixtures

The packing material recently introduced for use in a radial compression chamber, with a partially flexible cartridge, has a low C 18−-coating (5% w/w) combined with an absence of secondary capping. This report demonstrates that such a support, which contains significant concentrations of both free silanol and hydrocarbon groups, can allow a mixed-mode separation to occur via adsorption and reversed-phase separation mechanisms. In any given separation, the predominant mechanism depends both on the nature of the sample and the mobile phase. For efficient peptide and protein separations, it was necessary to suppress most silanol group interactions by the use of a mobile phase which contained a high concentration of an amine phosphate, e.g., 0.17 M triethylammonium phosphate, pH 3.2. In addition, it was necessary to deactivate further the silanol groups by an initial column wash of at least 20 column volumes of methanol. Samples which contained strongly basic groups, for example the guanidino group of arginine, can still exhibit poor separation efficiencies on such a support. These problems were largely overcome, however, with the use of isopropanol as an organic modifier. If these precautions were followed, the packing material gave excellent selectivities in the separation of closely related materials, well as allowing increased sample capacities. These observations will be supported by an examination of the chromatographic properties of a range of small peptides, the C-apolipoprotein mixture present in human very-low-density lipoproteins and the purification of an 8-mg sample of a synthetic pentadecapeptide in a single chromatographic run.

蛋白の高速液体クロマトグラフィー(第2報)逆相高速液体クロマトグラフィーによる蛋白の分離

Recently several papers have shown that proteins can be separated by using gradient elution method in reversed phase partition high performance liquid chromatography (HPLC) system, but there has been no report on the use of isocratic elution method. The HPLC method, which has been developed in previous report, was examined whether it can be applied to achieve efficient separation and identification of proteins. Under isocratic elution system by use of Nucleosil CN as stationary phase and sodium octanesulfonate as ion-pair reagent in mobile phase, we studied simultaneous spearation of five kinds of proteins ; insulin, ribonuclease, myoglobin, lysozyme chloride and cytochrome C. Under optimal conditions all of the proteins tested could be satisfactorily chromatographed.

DETERMINATION OF GLUCOSE IN FISH BLOOD: A MODIFICATION OF THE FOLIN–MALMROS MICROPROCEDURE

A modification of the Folin–Malmros micromethod is described for the determination of fish blood glucose. A more efficient separation of blood proteins is obtained by precipitating 0.1 ml blood in 10.0 ml tungstic acid. This is followed by hot alkaline reduction of ferricyanide, and colorimetric measurement of a Prussian blue – sodium lauryl sulphate suspension. The method, which eliminates certain difficulties found in other procedures, has been successfully applied in more than a thousand analyses of blood from two species of fish (Opsanus and Ictalurus) having glucose concentrations between 15 and 250 mg per cent.

DETERMINATION OF GLUCOSE IN FISH BLOOD: A MODIFICATION OF THE FOLIN–MALMROS MICROPROCEDURE

A modification of the Folin–Malmros micromethod is described for the determination of fish blood glucose. A more efficient separation of blood proteins is obtained by precipitating 0.1 ml blood in 10.0 ml tungstic acid. This is followed by hot alkaline reduction of ferricyanide, and colorimetric measurement of a Prussian blue – sodium lauryl sulphate suspension. The method, which eliminates certain difficulties found in other procedures, has been successfully applied in more than a thousand analyses of blood from two species of fish (Opsanus and Ictalurus) having glucose concentrations between 15 and 250 mg per cent.