Mark A. Strege

Hydrophilic interaction chromatography-electrospray mass spectrometry analysis of polar compounds for natural product drug discovery

For the drug discovery efforts currently taking place within the pharmaceutical industry, natural product extracts have been found to provide a valuable source of molecular diversity which is complementary to that provided by traditional synthetic organic methods or combinatorial chemistry. However, there exists a need for analytical tools that can facilitate the separation and characterization of components from these sources in a rapid manner. Specifically, the evaluation of highly polar compounds (i.e., compounds that cannot be retained on traditional reversed-phase stationary phases) has been challenging, and a hydrophilic interaction chromatography-electrospray ionization mass spectrometry (HILIC-ESI-MS) method was developed to meet this need. In this investigation, amide-, Polyhydroxyethyl Aspartamide-, and cyclodextrin-based packings provided superior performance for the analysis of a set of polar natural product compounds. The properties of the mobile-phase buffers also greatly impacted the separations, and relative to other volatile buffering agents, ammonium acetate at a concentration of approximately 6.5 mM was determined to facilitate optimal HILIC retention, reproducibility, and durability. An optimized HILIC-ESI-MS system was successfully applied for the analysis of complex natural product mixtures. The techniques described in this report should also prove useful for the analysis of polar compounds from synthetic sources of molecular diversity such as combinatorial chemistry.

High-performance liquid chromatographic-electrospray ionization mass spectrometric analyses for the integration of natural products with modern high-throughput screening

Within the pharmaceutical industry, significant resources have been applied to the identification of new drug compound leads through the use of high-throughput screening (HTS). To meet the demand for rapid analytical characterization of biologically active samples identified by HTS, the technique of high-performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) has been utilized, and the application of this technique specifically for the integration of natural product sample mixtures into modern HTS is reviewed. The high resolution provided by reversed-phase HPLC coupled with the gentle and relatively universal ionization facilitated by the electrospray process has had significant impact upon a variety of procedures associated with the HTS of natural products, including extract sample diversity evaluation, dereplication, structure elucidation, preparative isolation, and affinity-based biological activity evaluation.

Capillary electrophoretic protein separations in polyacrylamide-coated silica capillaries and buffers containing ionic surfactants

Abstract Capillary electrophoretic protein separations of high efficiency and resolution were obtained using polyacrylamide-coated silica capillaries and buffers containing ionic surfactants. The presence of micellar concentrations of sodium dodecyl sulfate or cetyltrimethyl- ammonium chloride minimized protein-capillary wall interactions, and facilitated concurrent separations of a mixture of both acidic and basic proteins, while the polyacrylamide coating provided increased resolution and migration time reproducibility via a reduction in electroosmotic flow. Attempts to obtain size-based protein separations via sieving through buffers containing the hydrophilic polymers methylcellulose and polyethylene glycol were unsuccessful.

Studies of migration time reproducibility of capillary electrophoretic protein separations

Abstract Studies of the migration time reproducibility of trypsinogen were performed using a capillary electrophoresis system designed for high resolution protein separations, where analyte adsorpt...

Capillary electrophoresis of proteins and peptides

Surfactant-Based Methods for Prevention of Protein Adsorption in Capillary Electrophoresis Charles A. Lucy, Nicole E. Baryla, and Ken K.-C. Yeung Capillary Coating for Protein Separation Based on Si-O and Si-C Covalent Bond Formation for Capillary Electrophoresis With Laser-Induced Fluorescence Detection Hossein Ahmadzadeh, Norman J. Dovichi, and Sergey Krylov On-Column Labeling Reaction for Analysis of Protein Contents of a Single Cell Using Capillary Electrophoresis With Laser-Induced Fluorescence Detection Hossein Ahmadzadeh and Sergey Krylov Covalent and Noncovalent Labeling Schemes for Near-Infrared Dyes in Capillary Electrophoresis Protein Applications John Sowell, Jozef Salon, Lucjan Strekowski, and Gabor Patonay Capillary Electrophoresis in the Analysis and Monitoring of Biotechnological Processes Vadim Klyushnichenko Capillary Electrophoresis of Proteins in a Quality Control Environment David L. Good, Stacey Cummins-Bitz, Raeann M. Fields, and Brian K. Nunnally Analysis of Neutral N-Linked Oligosaccharides From Antibodies Using Free-Solution Capillary Electrophoresis in Bare Fused-Silica Capillaries Jeffrey S. Patrick, Brenda P. Rener, Gregory S. Clanton, and Avinash L. Lagu Affinity Capillary Electrophoresis to Examine Receptor-Ligand Interactions Maryam Azad, John Kaddis, Valerie Villareal, Lili Hernandez, Catherine Silverio, and Frank A. Gomez Screening Major Binding Sites on Human Serum Albumin by Affinity Capillary Electrophoresis Hee Seung Kim, John Austin, and David S. Hage Using Charge Ladders and Capillary Electrophoresis to Measure the Charge, Size, and Electrostatic Interactions of Proteins Upma Sharma and Jeffrey D. Carbeck Frontal Analysis Continuous Capillary Electrophoresis for Protein-Polyelectrolyte Binding Studies Emek Seyrek, Toshiaki Hattori, and Paul L. Dubin Analysis of Proteins by CE, CIEF, and Microfluidic Devices With Whole-Column-Imaging Detection Jiaqi Wu, Xing-Zheng Wu, Tiemin Huang, and Janusz Pawliszyn Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry of Amino Acids, Peptides, and Proteins Mehdi Moini Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes Suzana Martinovic, Ljiljana Pasa-Tolic, and Richard D. Smith Integrated System for Rapid Proteomics Analyses Using Microfluidic Devices Coupled to Nanoelectrospray Mass Spectrometry Jianjun Li, Tammy-Lynn Tremblay, Jed Harrison, and Pierre Thibault Index

Micellar electrokinetic chromatography of proteins

Abstract Micellar electrokinetic capillary chromatography (MECC) of proteins is a high resolution capillary electrophoretic (CE) analysis method that utilizes the hydrophobic and electrostatic interaction of protein analytes with surfactant micelles present in the buffer medium to facilitate separation. Through the manipulation of the protein-micelle interaction by the adjustment of variables such as surfactant concentration, solution pH, ionic strength, the presence of an organic modifier and the use of coated capillaries, MECC analyses of a wide variety of proteins have been optimized. MECC has been demonstrated to provide resolution of mixtures consisting of proteins with minor structural variations and also has provided the successful quantitative analysis of protein present in complex matrices. The adoption of protein MECC as a routine analytical technique may be dependent upon the successful interface of MECC with detection methodology, such as mass spectrometry, which can provide analyte characterization information.

Capillary electrophoresis as a tool for the analysis of protein folding

Abstract The utility of capillary electrophoresis (CE) was demonstrated for the analysis of a model protein, bovine trypsinogen, as it underwent oxidation from a fully reduced molecule through a distribution of intermediate species until the disulfide bond conformation corresponding to that of the globular native structure was reached. Through the use of CE, completely refolded (native) trypsinogen was resolved from both the reduced protein and intermediate refolded conformations. In addition, the presence of polyethylene glycol in the separation buffer was determined to provide size-based protein separations of significantly higher resolution than those obtained in phosphate buffer alone. Quantitation of native trypsinogen in refolded material revealed an excellent correlation between CE determinations and analyses by established techniques such as biological activity assay and high-performance size-exclusion and cation-exchange chromatography. These results, together with a comparative consistency of CE separations with those obtained via non-denaturing slab gel electrophoresis and isoelectric focusing, suggest that CE can be an effective technique useful for the analysis of protein refolding.

Evaluation of a new macrocyclic antibiotic as a chiral selector for use in capillary electrophoresis

Abstract A new macrocyclic antibiotic, LY307599, has been evaluated as a chiral selector for the separation of the enantiomers of flurbiprofen using capillary electrophoresis (CE). The effect of varying separation buffer parameters such as buffer strength, pH, LY307599 concentration and methanol concentration were assessed. Using the optimized CE conditions, the separation of flurbiprofen enantiomers can be achieved using LY307599 as a chiral selector.

At-Line Quantitative Ion Mobility Spectrometry for Direct Analysis of Swabs for Pharmaceutical Manufacturing Equipment Cleaning Verification

The potential for ion mobility spectrometry (IMS) to provide rapid at-line quantitation of residues on surfaces via direct analysis of swabs is attractive for pharmaceutical manufacturing equipment cleaning verification. In this study, the development of an IMS method to provide acceptable quantitation of active pharmaceutical ingredients and cleaning agents is described. Key modifications to commercially available instrumentation were made to achieve a dynamic range of 5-100 microg per 25 cm2 surface area and acceptable analyte recovery in the presence of ionizable matrix components. The results of this study effectively demonstrate the capability of IMS to serve as an at-line quantitative analytical method.

Analysis of recombinant human growth hormone in Escherichia coli fermentation broth by micellar high-performance liquid chromatography

A method for the reversed-phase high-performance liquid chromatographic (HPLC) determination of recombinant methionylaspartyl-human growth hormone (MD-HGH) in Escherichia coli fermentation broth is described. The technique utilizes mobile phases containing n-propanol and the anionic surfactant sodium dodecyl sulfate (SDS) under micellar conditions at pH 6.4. The methodology is directly applicable to the analysis of samples solubilized via sulfitolysis in the presence of SDS, and offers superior resolution in comparison with chromatography in the absence of the surfactant. Using this method, acceptable precision (day-to-day R.S.D. = 4.9%), accuracy, selectivity, range, linearity and ruggedness were achieved.