Monolithic sorbents for microscale separations

Over the last decade, the miniaturization of analytical systems has become an increasingly important and interesting research area. Miniaturized systems offer many advantages, including reduced reagent and sample consumption, shorter analysis times, portability and disposability. This dissertation describes novel approaches in this direction, focusing on two areas: the miniaturization of existing column chromatographic systems and the development of microfluidic systems in which the separation is performed in a channel on a microchip. A new type of methacrylate-based monolithic capillary columns for liquid chromatography and capillary electrochromatography were prepared within the confines of fused-silica tubing using Starburst dendrimers to affect porosity. The polyamidoamine (PAMAM) dendrimers were incorporated into a solution of functionalized monomer, cross-linker, solvents, and polymerization initiator. Thermal polymerization, followed by the removal of solvent and dendrimers, produced a continuous rod of polymer with uniform porosity. Different column porosities were obtained by varying the amount of the dendrimer template. The chromatographic performance of these monolithic columns was evaluated using a peptides mixture obtained by tryptic digestion of chicken egg lysozyme. A distinct advantage of polymer monolithic stationary phases over conventional packed chromatographic beds is the ability to prepare them easily and rapidly via free radical polymerization within the channels of a microfluidic device. In this work, continuous polymeric beds were prepared within a channel of three different microchip substrates: glass, poly(dimethylsiloxane) and polycarbonate. The methacrylate-based monolith was cast in-situ via UV-initiated polymerization. The functionalization of the inner wall of the channel with methacryloyl groups enabled the covalent binding of the monolith to the wall. The morphology of the wall-anchored monolith was studied by SEM of chip sections, and by SEM of an extruded segment of non-anchored monolith from a separate chip

Mass spectrometric analysis of UV-crosslinked protein-nucleic acid complexes

The DNA-binding domains of E. coli uracil-DNA glycosylase (Ung) and human replication protein A (hRPA) were studied using a general protocol developed in our laboratory for probing protein-DNA interactions. The procedure involves purification and mass spectrometric analysis of the nucleopeptide-products of a tryptically digested UV-crosslinked protein-nucleic acid complex. In the case of Ung x dT₂₀ nucleoprotein complex, three nucleopeptide isomers having the same peptide backbone (T₁₈ peptide) but with dinucleotides attached to different aminoacids were separated. The tandem mass spectra from the isomers provided new structural information about Ung binding to DNA. Specifically, His₁₈₇, Ser₁₈₉, and His₁₉₄ from T₁₈ nucleopeptide were putatively identified as sites that photocrosslink to dT₂₀. Photochemical crosslinking of hRPA to oligonucleotide dT₃₀ produced two covalent hRPA70 x dT₃₀ complexes involving one of the protein's subunits (hRPA7O). Three crosslinked tryptic peptides were isolated using the same protocol as used for Ung and MALDI-TOF and nanoLC-ESI-MS/MS analyses revealed the identity of these peptides as T₄₃, T₂₈/₂₉, and a truncated *T₂₄/₂₅ (without the last 5 aminoacids from the C-teminal). Additional experiments showed that at least one amino acid from the sequence 383-VSDF-386 (located in T43), at least one residue from 235-ATAFNE-240 (*T₂₄/₂₅), and at least one residue from F269/T270 (T₂₈/₂₉) is involved in crosslinking. Aromatic residues contained in these peptides (F23 8, F269 and F386), which can form base stacking interactions with the DNA, are the residues most likely to be involved in crosslinking. These observations are in good agreement with previously published data regarding the single stranded-DNA binding site of hRPA obtained from crystal structure and from site-directed mutagenesis experiments

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ: a study of an industrial mAb producing CHO-S cell line

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO-S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs

Using Automated Glycan Assembly (AGA) for the Practical Synthesis of Heparan Sulfate Oligosaccharide Precursors

Herein we report synthesis of complex heparan sulfate oligosaccharide precursors by automated glycan assembly using disaccharide donor building blocks. Rapid access to a hexasaccharide was achieved through iterative solid phase glycosylations on a photolabile resin using Glyconeer™, an automated oligosaccharide synthesiser, followed by photochemical cleavage and glycan purification using simple flash column chromatography

Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization

We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC—with only a single sample injection event—as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans

LC-MS characterization of antibody-based therapeutics: recent highlights and future prospects

Abstract Antibody-based therapeutics constitute a major growth area in medicine today. However, antibodies as drugs present significant analytical challenges as they are large, complex and heterogenous molecules produced in living cells. The key attributes that affect safety, stability and efficacy must be identified and controlled to ensure regulatory compliance. Liquid Chromatography-Mass Spectrometry (LC-MS) is a powerful analytical technology that is well suited to the task of analyzing antibody-based therapeutics. LC-MS is used to characterize antibody features ranging from the relatively simple (e.g. intact molecular weight determination and post-translational modification analysis) to the complex (e.g., higher order structure analysis and epitope identification). Few other analytical technologies are as versatile as LC-MS for monitoring a wide range of attributes or as capable of keeping pace with the innovation happening today in biotherapeutic design. In this chapter we will provide an overview of the LC-MS methods currently used for the characterization of antibody-based therapeutics, with an emphasis on the analysis of post-translational modifications. We will also highlight some recent innovations, new technologies and trends that are likely to significantly impact the manner in which antibody-based therapeutics are analyzed in the future

Age-related increase in 4-hydroxynonenal adduction to rat heart alpha-ketoglutarate dehydrogenase does not cause loss of its catalytic activity

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (p< or =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (p< or =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (p< or =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction