Fast Separation Of Proteins Research Articles

Dry-compression packing of hydroxyapatite nanoparticles within a flat cuboid chromatography device and its use for fast protein separation

We describe and discuss a simple dry-compression technique for preparing a flat cuboid chromatography device containing a shallow packed-bed of crystalline hydroxyapatite nanoparticles. We then discuss the use of this device for fast protein separation in the bind-and-elute mode. Such separation could be carried out at quite low pressures, making it possible to use inexpensive low pressure chromatography systems. In the flow rate range examined in this study, the pressure-drop across the device increased linearly with flow rate, indicating negligible media compaction during use. Using this device, binary protein mixtures could be separated in about a minute. Contrary to that observed in most packed-bed chromatographic separations, the width of the flow through and eluted peaks decreased with increase in flow rate. Therefore, both productivity and purity could be simultaneously increased by increasing flow rate. The suitability of this device for preparative protein separations was demonstrated by carrying out purification of a monoclonal antibody (Trastuzumab) from mammalian cell culture supernatant. This study opens up the possibility of developing dry-compression based flat cuboid packed-bed chromatography devices for fast preparative protein separation.

Preparation of Core-Shell Silica Microspheres with Controllable Shell Thickness for Fast High Performance Liquid Chromatography Separation of Alkyl Benzenes and Intact Proteins.

As it is difficult to prevent secondary nucleation and agglomeration during the preparation of core-shell silica microspheres, these issues have been successfully resolved in this study using template-dissolution-induced redeposition. The non-porous particles are transformed into core-shell silica microspheres (CSSMs) in the presence of cetyltrimethylammonium bromide and octyltrimethylammonium bromide under basic conditions. The shell thickness and pore sizes of the CSSMs are controlled by adjusting the etching time and molar ratio of the template, respectively. The CSSMs are modified using octadecyltrimethylammonium chloride to separate the mixture of alkyl benzenes, and a high column separation efficiency is achieved within two minutes. The CSSMs are used for the separation and analysis of proteins and the digests of bovine serum albumin. The chromatographic column packed with core-shell particles affords a significantly higher separation efficiency than the commercial column. Therefore, as a chromatographic stationary phase, these core-shell particles can potentially be used for the fast separation of proteins, small solutes, and complex samples.

A micro-solid phase extraction device to prepare a molecularly imprinted porous monolith in a facile mode for fast protein separation.

A molecularly imprinted polymeric monolith was synthesized in an aqueous environment in 15min via UV-irradiation. The imprinted monolith was composed of hydroxyethyl methacrylate as monomer, dimethyl amino ethyl methacrylate as functional monomer, methylene bisacrylamide and piperazine diacrylamide as crosslinkers and human serum albumin as template molecule. The synthesis took place in a PDMS-based device (2.5cm long) yielding a micro-solid phase extraction column (3×5mm) with two built-in fingertight connectors for an infusion pump and fraction collector. The imprinted monolith displayed the characteristic features of a porous polymeric monolith, had dimethyl amino ethyl methacrylate and human serum albumin as functional groups within the monolith and showed high permeability (0.51×10-13m2). 85% of the imprinted cavities were readily available for rebinding of human serum albumin with an imprinting factor of 1.3. In comparison to a non-imprinted monolith, molecular imprinting increased human serum albumin adsorption by > 30%. Imprinted monolith displayed selectivity for human serum albumin over other competing proteins (human transferrin, ovalbumin and carbonic anhydrase) with similar or different isoelectric points and size. Human serum albumin was adsorbed (in dynamic mode) with > 98% selectivity from diluted human plasma using the imprinted monolith device. Device to device reproducibility and reusability of the device for 5 cycles showcase the imprinted monolith micro-device efficiency.

Synthesis and characterization of (R)-phenylephrine bonded non-porous polystyrene-divinylbenzene for fast separation of proteins by mixed-mode liquid chromatography

Synthesis and characterization of (R)-phenylephrine bonded non-porous polystyrene-divinylbenzene for fast separation of proteins by mixed-mode liquid chromatography

Overload Effects in Reversed Phase Protein Separations using Capillary-Channeled Polymer Fiber Columns.

In preparative chromatography, overloading effects are a fact of life. However, peak distortion is problematic in analytical scale chromatography, especially in the case of bio-macromolecule separations. Capillary-channeled polymer fibers have been employed for fast protein separations in reversed phase, ion exchange, and hydrophobic interaction liquid chromatography. Although the primary advantage of the phase is operation at high linear velocities (~100 mm s-1 ) without van Deemter C-term limitations, the limited specific surface area (<5 m2 g-1 ) suggests that a thorough understanding of overloading effects is needed. We evaluate important factors (injected mass and volume) affecting overload in terms of peak height, width and shape (asymmetry). Overload conditions readily apparent as the peak shape changes from a Gaussian distribution to a left-triangle with slight tailing; more-or-less classical overload behavior. Three methods were used to compute column efficiency in terms of plate counts (N), including the area height, half-height, and the Dorsey-Foley approaches. The half-height method is best-suited for describing column overload, accounting for peak distortion and achieving high levels of consistency. The limiting plate count (N0 ) and the column sample loading capacity (ω0.5 ) were key metrics to characterize overall column performance. Peak capacity (P) was used to assess gradient elution separation performance. Increased flow rates and column length result in enhanced loading capacity. pH mismatch between the sample matrix and the mobile phase, a common cause of protein overload with other phases, was not responsible for distorted peak shapes unless the solvent pH is close to the pI of protein samples.

Synthesis of penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) microsphere and its HPLC application in protein separation

In the present study, the narrow-dispersed penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EDMA)) microspheres were successfully synthesized based on the sacrificial support method. The poly(MAA-co-EDMA) microspheres mirrored the porous structure of the sacrificial support, i.e. penetrable silica, characteristic of copious mesopores and throughpores. In addition, they possessed large surface area, adjustable hydrophobicity and the cation-exchange ability. Owing to their multi functionalities, they were applied as chromatographic stationary phase to separate proteins in different separation modes, including reversed phase, hydrophobic interaction and weak cation exchange. Moreover, thanks to their throughpores, fast separation at low column backpressure could be achieved in these three modes. Both protein recovery and column stability were satisfactory. The penetrable poly(MAA-co-EDMA) microspheres were potential stationary phase matrix for fast protein separation.

On-line Fast Purification of Three Kinds of Active Proteins from Egg White with Two-dimensional Liquid Chromatography Using a Single Column

With the development of biotechnology and life science, both recombinant protein drug productions and proteomics research depend largely on fast and efficient protein separation technology. Analysis of complex samples has put forward higher and higher requirements to separation science. Therefore, developing new separation material, separation mode and more sensitive detection method should be one of the effective ways to solve these problems. Based on a novel strong cation exchange/hydrophobic interaction chromatography (SCX/HIC) dual-function column, which displays HIC character in a high salt concentration, and IEC character in a low salt concentration in mobile phase employed and can be employed to separate proteins with SCX and HIC modes, respectively, a new on-line two-dimensional liquid chromatography technology with a single column (2DLC-1C) was developed. An equipment with automatic control system called as "ChromatoExpert" for pro- cessing fast protein separation online 2DLC-1C was constructed, which has multiple functions, such as on-line fraction col- lection from the first separation mode and quantitatively sample re-injection into the second separation mode, on-line buffer exchange, etc. Eight kinds of protein standards were separated using SCX/HIC 2DLC-1C. The unretained and uncompletely separated proteins in SCX mode were collected online and could be separated completely with each other by online re-injection and second dimensional separation of HIC mode. As a result, these eight kinds of proteins can be separated with- in 2 h. It also was applied to purify three kinds of active intact proteins from egg white successfully. The results indicated that lysozyme, ovotransferrin and ovalbumin from egg white could be purified just within 70 min by online 2DLC-1C. The purity of these proteins obtained was 95%, 93% and 97%, respectively. The new online 2DLC-1C technology can simplify the puri- fication process and lower the separation cost. It is easy to realize automation and scale-up to preparative scale. Therefore, this new technology has a broad application prospect in the fast separation and preparation of active protein. Keywords online 2DLC-1C; protein; purification; egg white

Polyethylenimine modified poly(ethylene terephthalate) capillary channeled-polymer fibers for anion exchange chromatography of proteins

Native poly(ethylene terephthalate) (PET) capillary-channeled polymer (C-CP) fibers have been previously studied as stationary phases for reversed phase and affinity protein separations. In this study, surface modified PET C-CP fibers were evaluated for the anion exchange separation of proteins. The native PET C-CP fibers were aminated using polyethylenimine (PEI) followed by a 1,4-butanediol diglycidyl ether (BUDGE) cross-linking step. Subsequent PEI/BUDGE treatments can be employed to further develop the polyamine layer on the fiber surfaces. The PEI densities of the modified fibers were quantified through the ninhydrin reaction, yielding values of 0.43–0.89μmolg−1. The surface modification impact on column permeability was found to be 0.66×10−11 to 1.33×10−11m2, depending on the modification time and conditions. The dynamic binding capacities of the modified fiber media were determined to be 1.99–8.54mgmL−1 bed volume, at linear velocities of 88–438cmmin−1 using bovine serum albumin as the model protein. It was found that increasing the mobile phase linear velocity (up to 438cmmin−1) had no effect on the separation quality for a synthetic protein mixture, reflecting the lack of van Deemter C-term effects for the C-CP fiber phase. The low-cost, easy modification method and the capability of fast protein separation illustrate great potential in the use of PEI/BUDGE-modified PET C-CP fibers for high-throughput protein separation and downstream processing.

Determination of pore size distributions in capillary-channeled polymer fiber stationary phases by inverse size-exclusion chromatography and implications for fast protein separations

Capillary-channeled polymer (C-CP) fibers have been utilized as liquid chromatography stationary phases, primarily for biomacromolecule separations on the analytical and preparative scales. The collinear packing of the eight-channeled C-CP fibers provides for very efficient flow, allowing operation at high linear velocity (u>100mm s−1) and low backpressure (<2000psi) in analytical-scale separations. To take advantage of these fluid transport properties, there must not be mass transfer limitations as would be imposed by having an appreciably porous phase, wherein solute diffusion limits the overall mass transport rates. To better understand the physical nano-/micro- structure of C-CP fibers, inverse size exclusion chromatography (iSEC) has been employed to determine the pore size distribution (PSD) within C-CP fibers. A diversity of test species (from metal ions to large proteins) was used as probes under non-retaining conditions to obtain a response curve reflecting the apparent partition coefficient (Kd) versus hydrodynamic radii (rm). A mean pore radius (rp) of 4.2nm with standard deviation (sp) of ±1.1nm was calculated by fitting the Kd versus rm data to model equations with a Gaussian pore size distribution, and a pore radius of 4.0±0.1nm was calculated based on a log-normal distribution. The derived mean pore radius is much smaller than traditional support materials, with the standard deviation showing a relatively uniform pore distribution. van Deemter plots were analyzed to provide practical confirmation of the structural implications. Large molecules (e.g., proteins) that are fully excluded from pores have no significant C-terms in the van Deemter plots whereas small molecules that can access the pore volumes display appreciable C-terms, as expected. Fitting of retention data to the Knox equation suggests that the columns operate with a characteristic particle diameter (dp) of ∼53μm.

Rapid fractionation of Al in human serum by the use of HiTrap desalting size exclusion column with ICP-MS detection

To understand the pathways of Al transport in human body it is necessary to identify and quantify Al species present in human serum. It has been demonstrated that about 90% of Al in human serum corresponds to high molecular mass species (HMM-Al) and that Al is bound exclusively to transferrin (Tf). The remaining Al exists in low molecular mass species (LMM-Al) bound predominantly to Al-citrate. In studies of the efficiencies of the chelation therapies and to perform kinetic studies of Al binding to Tf, rapid and reliable analytical procedures are required. Therefore, a new analytical procedure for the efficient, reliable and fast separation of proteins from LMM compounds in serum was developed by the use of a HiTrap desalting size exclusion column. Unspiked and spiked human serum that was left for six hours to reach approximately thermodynamic equilibrium were studied. The separation of HMM-Al from LMM-Al species was accomplished with tris-HCl buffer (pH 7.4) in 10 min. Al elution profile was followed by ICP-MS detection. In the first 5 min HMM-Al compounds were eluted, followed by the elution of LMM-Al species from 5 to 10 min. Results demonstrated that about 94% of Al was present in the HMM (protein) fraction. To prove the reliability of the fractionation procedure developed, the protein peak and LMM separated species were collected in 5 mL fractions, followed by the previously developed speciation procedures. Speciation of the protein peak by CIM® DEAE-ICP-MS verified that about 98% of Al was bound to Tf. In the LMM serum fraction Al-citrate was separated on the FPLC column coupled to ICP-MS. Speciation data confirmed the reliability of the developed fast fractionation procedure. It represents a promising tool for investigations of kinetics of Al binding to Tf and may also be applied in studies of the efficiencies of the chelation therapies in patients overloaded with Al.

Preparation and characterization of porous poly(vinyl ester) resin monoliths as separation media

AbstractPorous poly(vinyl ester) resin monolithic supports were first prepared by radical polymerization of the continuous phase of oil in water high‐internal‐phase emulsions. Vinyl ester (VE) resin was used as the monomer, ethylene glycol dimethacrylate was used as a crosslinker, and poloxamer 127 was used as the emulsifier in the emulsion polymerization. The prepared columns were evaluated by scanning electron microscopy, mercury intrusion porosimetry, and Fourier transform infrared spectroscopy to observe the morphological characteristics and confirm the absorbance based on the VE resin polymer. The obtained monolith showed not only higher column permeability but also lower back pressure and higher column efficiency. To investigate the absorption performance of the monolithic column, a maximum loading capacity experiment was also applied with lysozyme (Lys), and the results show that the maximum adsorption of the poly(vinyl ester) resin monolith was 1.579 mg/g. Moreover, the capabilities of separation on this column in conjunction with high‐performance liquid chromatography were investigated. Immunoglobulin could be separated from human plasma and chicken egg yolk with high resolution within 4 min. Additionally, fast separation of two mode proteins (interleukin‐18 and Lys) was achieved on the monolith within 2 min at the rate of 1445 cm/h, which demonstrated the potential of the poly(vinyl ester) resin monolith for the fast separation of proteins. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011

Rapid Separation of Proteins by Capillary HPLC on a Short Polymethacrylate‐based Strong Cation‐exchange Monolithic Column

AbstractA polymethacrylate‐based strong cation‐exchange capillary monolithic column was prepared by in‐situ copolymerization for the fast separation of proteins. Glycidyl methacrylate (GMA) was used as monomer, ethylenedimethacrylate (EDMA) as cross link agent and the mixture of 1‐propanol, 1,4‐butanediol and water as porogen solvent. The monolith was sulfonated using 1 mol/L Na2SO3 based on a ring opening of epoxides. The influences of the contents of the porogen solvent and GMA and the various concentration ratios of 1‐propanol to 1,4‐butanediol in the polymerization mixture on the morphology, porosity, globule size, stability and column efficiency were investigated. The morphology and pore size distribution of the monolith were characterized by SEM and mercury intrusion porosimetry, respectively. Using only 1.5 cm length of this monolithic capillary column, four kinds of proteins, trypsin, cytochrome C, lysozyme (egg white) and egg albumin, were successfully separated from each other in 5 min at a high flow rate of 110 mm/s.

Novel Porous Monolithic Column Using Poly(high internal phase emulsion) Methacrylate as Materials for Immunoglobulin Separation Performance on HPLC

AbstractA novel porous polymeric monolithic column based on poly(high internal phase emulsion) methacrylate monolith was prepared and applied to fast separation of proteins. The block copolymer chemistry of high internal phase emulsions was used in the experiment. These unique properties, together with high porosity, good mechanical property, chemical modification of the surface make themselves superior in monolithic medium applications. Morphology of the monolithic material was studied by scanning electron microscopy. The stability of the emulsion and the load of hydroxyl groups–the active group of the monolithic column were investigated. Additionally, the capabilities of separation of this column in conjunction with high performance liquid chromatography (HPLC) were investigated. Immunoglobulin was separated from human plasma and chicken egg yolk with high resolution on the hydrophobic interaction chromatographic column in a short time. The effects of pH and concentration of mobile phase (buffer) on the elution of immunoglobulin were investigated. Moreover, fast separation of a two mode protein mixture (α‐amylase and lysozyme) on the monolith was achieved within 1.5 min at a velocity of 1445 cm·h−1. As a result, good separation was achieved, and stable low back pressure drop was ensured at high throughput elution with an even longer column.

Surfactant-bound monolithic columns for separation of proteins in capillary high performance liquid chromatography

A surfactant-bound monolithic stationary phase based on the co-polymerization of 11-acrylamino-undecanoic acid (AAUA) is designed for capillary high performance liquid chromatography (HPLC). Using D-optimal design, the effect of the polymerization mixture (concentrations of monomer, crosslinker and porogens) on the chromatographic performance (resolution and analysis time) of the AAUA–EDMA monolithic column was evaluated. The polymerization mixture was optimized using three proteins as model test solutes. The D-optimal design indicates a strong dependence of chromatographic parameters on the concentration of porogens (1,4-butanediol and water) in the polymerization mixture. Optimized solutions for fast separation and high resolution separation, respectively, were obtained using the proposed multivariate optimization. Differences less than 6.8% between the predicted and the experimental values in terms of resolution and retention time indeed confirmed that the proposed approach is practical. Using the optimized column, fast separation of proteins could be obtained in 2.5 min, and a tryptic digest of myoglobin was successfully separated on the high resolution column. The physical properties ( i.e., morphology, porosity and permeability) of the optimized monolithic column were thoroughly investigated. It appears that this surfactant-bound monolith may have a great potential as a new generation of capillary HPLC stationary phase.

Polymeric strong cation‐exchange monolithic column for capillary liquid chromatography of peptides and proteins

A strong cation-exchange (SCX) monolithic stationary phase was prepared in 75 microm id capillaries by direct in situ polymerization of sulfopropyl methacrylate and polyethylene glycol diacrylate in a ternary porogen system consisting of methanol, cyclohexanol, and water. The resulting monolith exhibited good dynamic binding capacity, fast kinetic adsorption of proteins, and high permeability. The monolith had a dynamic binding capacity of approximately 52 mg/mL of column volume for lysozyme and cytochrome C. The monolith was evaluated for SCX capillary LC of synthetic peptides, natural peptides, and protein standards. Fast separation of proteins was achieved in less than 4 min. The average peak capacity for peptides was 28 using a relatively steep gradient when hydrophobic interactions were suppressed with 40% acetonitrile.

Schrock Catalyst Triggered, Ring-Opening Metathesis Polymerization Based Synthesis of Functional Monolithic Materials

The synthesis of monolithic materials via the Schrock catalyst triggered ring-opening metathesis polymerization (ROMP) is described. Mo(N-2,6-(2-Pr)2-C6H3)(CHCMe2Ph)(OCMe3)2 (1) was used as an initiator. Using various ratios of norborn-2-ene (NBE) and 1,4,4a,5,8,8a-hexahydro-1,4,5,8-exo,endo-dimethanonaphthalene (DMN-H6) in different mixtures of micro- with macroporogens, i.e., 1,2-dichloroethane, toluene and THF with hexane or pentane, monolithic polymeric materials with continuous, interconnected pores in the micrometer range as well as with micro- and mesopores in the 1.5−300 nm range could be synthesized within the confines of 3 × 100 mm glass columns. The resulting monoliths were characterized via inverse size exclusion chromatography (ISEC) in terms of the total pore volume (Vp), the volume fraction of the pore porosity (ϵp), the volume fraction of the interstitial porosity (ϵz) and the pore size distribution. Finally, the novel monoliths were successfully used for the fast separation of proteins as well as of 9-fluorenylmethoxycarbonyl (FMOC) protected amino acids.

Synthesis of zwitterionic stationary phase based on hydrophilic non‐porous poly(glycidymethacrylate‐co‐ethylenedimethacrylate) beads and their application for fast separation of proteins

AbstractA new hydrophilic strong/strong type zwitterionic stationary phase for high performance liquid chromatography (HPLC) was synthesized by chemical modification of 3.0 μm non‐porous monodisperse poly(glycidylmethacrylate‐co‐ethylenedimethacrylate)(PGMA/EDMA) beads in the following steps. First, the beads were reacted with hydrochloride to obtain chlorizated beads; second, chlorizated beads were reacted with dimethylamine to obtain ammoniated beads; third, ammoniated beads were reacted with 1,3‐propanesultone to obtain non‐porous hydrophilic zwitterionic stationary phase. The stationary phase was evaluated in detail to determine its ion‐exchange properties, separability, reproducibility, hydrophilicity, and the effect of column loading and pH on the separation and retention of proteins. The highest dynamic protein loading capacity of the synthesized zwitterionic packing for bovin serum albumin and Lys were 18.3 and 27.4 mg g−1, respectively. The zwitterionic stationary phase was capable of separating two acidic and three basic proteins simultaneously in less than 2.5 min by the flow‐rates of 3.0 mL min−1. The zwitterionic resin was also used for rapid separation and purification of recombinant human interferon‐r (rhIFN‐r) and human granulocyte colony‐stimulation factor (hG‐CSF) from the crude extract solution. The satisfactory results were obtained. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009

Proteomic identification of technologically modified proteins in malt by combination of protein fractionation using convective interaction media and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.

Monolithic poly[(trimethylsilyl-4-methylstyrene)- co- bis(4-vinylbenzyl)dimethylsilane] stationary phases for the fast separation of proteins and oligonucleotides

In this paper the synthesis, optimisation and application of a silane based monolithic copolymer for the rapid separation of proteins and oligonucleotides is described. The monolith was prepared by thermal initiated in situ copolymerisation of trimethylsilyl-4-methylstyrene (TMSiMS) and bis(4-vinylbenzyl)dimethylsilane (BVBDMSi) in a silanised 200 μm I.D. fused silica column. Different ratios of monomer and crosslinker, as well as different ratios of micro- (toluene) and macro-porogen (2-propanol) were used for optimising the physical properties of the stationary phase regarding separation efficiency. The prepared monolithic stationary phases were characterised by measurement of permeability with different solvents, determination of pore size distribution by mercury intrusion porosimetry (MIP). Morphology was studied by scanning electron microscopy (SEM). Applying optimised conditions, a mixture comprised of five standard proteins ribunuclease A, cytochrome c, α-lactalbumine, myoglobine and ovalbumine was separated within 1 min by ion-pair reversed-phase liquid chromatography (IP-RPLC) obtaining half-height peak widths between 1.8 and 2.4 s. Baseline separation of oligonucleotides d(pT) 12–18 was achieved within 1.8 min obtaining half-height peak widths between 3.6 and 5.4 s. The results demonstrate the high potential of this stationary phase for fast separation of high-molecular weight biomolecules such as oligonucleotides and proteins.

Capillary electrophoresis–mass spectrometry of proteins at medium pH using bilayer-coated capillaries

The feasibility of using noncovalently bilayer-coated capillaries for capillary electrophoresis-mass spectrometry (CE-MS) of acidic proteins was investigated using background electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000-300,000 plates) when the ionic strength was sufficiently high. Migration-time reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments showed satisfactory MS responses for studied proteins dissolved in ammonium formate (pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously suppress the MS protein signals. Evaluation of the CE-MS system was performed by analyzing a mixture of intact proteins yielding efficient separations and good-quality mass spectra. CE-MS analysis of a reconstituted formulation of the biopharmaceutical recombinant human growth hormone (rhGH) which was stored for a prolonged time, revealed one degradation product which was provisionally identified as desamido rhGH. Based on the MS responses the amount of degradation was estimated to be ca. 25%.