Shiaw-Lin Wu

Characterization of human growth hormone by capillary electrophoresis

Production of proteins by recombinant DNA technology for use as pharmaceuticals requires the use of the most powerful tools of analytical protein chemistry in order to confirm purity and identity of the product and reliability of the process. Capillary electrophoresis is an emerging technology that shows high sensitivity and selectivity and may have promise in this application. The technique combines the instrumental control and quantification features of high-performance liquid chromatography with the separating power of electrophoresis, and thereby has attracted broad interest. In this report, human growth hormone expressed in bacteria has been analyzed by both free zone electrophoresis and isoelectric focusing in a coated capillary to demonstrate the separation of the native molecule from its deamidated variant. A capillary zone electrophoretic tryptic map has also been developed and characterized. This map complements the widely employed reversed-phase high-performance liquid chromatography tryptic mapping systems that are important in protein characterization. Certain drawbacks to capillary zone electrophoresis compared to other analytical methods are noted, including relatively poor reproducibility and low sample tolerance. For applications as demonstrated here, however, the speed, separating power and sensitivity of the technique compensate for these shortcomings.

Protein conformational effect in hydrophobic interaction chromatography : Retention characterization and the role of mobile phase additives and stationary phase hydrophobicity

Abstract We have studied the conformational behavior of α-lactalbumin (α-LACT) in hydrophobic interaction chromatography (HIC). Retention characterization in terms of Z (Slope of plot 1n k ′ vs . 1n o B , where k ′ is the capacity factor and O B is the volume fraction of mobile phase B) has been explored, and the relationship of Z to other slopes, such as S (slope of the plot of 1n k ′ vs . o B f) has been derived. The reason for the sensitivity of Z to conformational change are discussed. The enhanced broadening of α-LACT in a temparature transition region of conformational change has been studied by spectral analysis using on-line photodiode array detection. The influence of Ca 2+ and Mg 2+ addition to the mobile phase is further explored. Since α-LACT is a calcium binding protein, addition of this metal leads to stabilization, i.e higher column temperatures are required for conformational change. On the other hand, addition of Mg 2+ appears to destabilize the protein. We have explored the use of a more hydrophobic support, C 2 -(ethyl) ether phase, for the elution of α-LACT. In this case, two widely separated peaks ae observed. By spectral analysis the first peak is shown to be native and the later eluted, broad second peak to ber an unfolded mixture of species. As previously observed in reversed-phase liquid chromatography, the second peak grows at the expense of the first, as the column temperature is raised. The second peak also grows as the contact time of the protein with the surface increases. This behavior can be ascribed to the conformational change of α-LACT in the column, the late eluted species under the second peak binding significantly more strongly to the phase than the native peak. Reinjection of the late eluted fraction reveals the reformation of the native species takes place in solution within 30 min. As before, addition of Ca 2+ reduces the extent of unfolding under any specific condition. These results add further to our understand and ability to control conformational changes in high-performance liquid chromatography.

Mass Spectrometric Determination of Disulfide Linkages in Recombinant Therapeutic Proteins Using Online LC−MS with Electron-Transfer Dissociation

In the biotechnology industry, the generation of incorrectly folded recombinant proteins, either from an E.coli expression system or from an overexpressed CHO cell line (disulfide scrambling), is often a great concern as such incorrectly folded forms may not be completely removed in the final product. Thus, significant efforts have been devoted to map disulfide bonds to ensure drug quality. Similar to ECD, disulfide bond cleavages are preferred over peptide backbone fragmentation in ETD. Thus, an online LC-MS strategy combining collision-induced dissociation (CID-MS(2)), electron-transfer dissociation (ETD-MS(2)), and CID of an isolated product ion derived from ETD (MS(3)) has been used to characterize disulfide-linked peptides. Disulfide-linked peptide ions were identified by CID and ETD fragmentation, and the disulfide-dissociated (or partially dissociated) peptide ions were characterized in the subsequent MS(3) step. The online LC-MS approach is successfully demonstrated in the characterization of disulfide linkages of recombinant human growth hormone (Nutropin), a therapeutic monoclonal antibody, and tissue plasminogen activator (Activase). The characterization of disulfide-dissociated or partially dissociated peptide ions in the MS(3) step is important to assign the disulfide linkages, particularly, for intertwined disulfide bridges and the unexpected disulfide scrambling of tissue plasminogen activator. The disulfide-dissociated peptide ions are shown to be obtained either directly from the ETD fragmentation of the precursors (disulfide-linked peptide ions) or indirectly from the charge-reduced species in the ETD fragmentation of the precursors. The simultaneous observation of disulfide-linked and disulfide-dissociated peptide ions with high abundance not only provided facile interpretation with high confidence but also simplified the conventional approach for determination of disulfide linkages, which often requires two separate experiments (with and without chemical reduction). The online LC-MS with ETD methodology represents a powerful approach to aid in the characterization of the correct folding of therapeutic proteins.

Dynamic Profiling of the Post-translational Modifications and Interaction Partners of Epidermal Growth Factor Receptor Signaling after Stimulation by Epidermal Growth Factor Using Extended Range Proteomic Analysis (ERPA)

In a recent report, we introduced Extended Range Proteomic Analysis (ERPA), an intermediate approach between top-down and bottom-up proteomics, for the comprehensive characterization at the trace level (fmol level) of large and complex proteins. In this study, we extended ERPA to determine quantitatively the temporal changes that occur in the tyrosine kinase receptor, epidermal growth factor receptor (EGFR), upon stimulation. Specifically A 431 cells were stimulated with epidermal growth factor after which EGFR was immunoprecipitated at stimulation times of 0, 0.5, 2, and 10 min as well as 4 h. High sequence coverage was obtained (96%), and methods were developed for label-free quantitation of phosphorylation and glycosylation. A total of 13 phosphorylation sites were identified, and the estimated stoichiometry was determined over the stimulation time points, including Thr(P) and Ser(P) sites in addition to Tyr(P) sites. A total of 10 extracellular domain N-glycan sites were also identified, and major glycoforms at each site were quantitated. No change in the extent of glycosylation with stimulation was observed as expected. Finally potential binding partners to EGFR were identified based on changes in the amount of protein pulled down with EGFR as a function of time of stimulation. Many of the 19 proteins identified are known binding partners of EGFR. This work demonstrates that comprehensive characterization provides a powerful tool to aid in the study of important therapeutic targets. The detailed molecular information will prove useful in future studies in tissue.

Structure of Sad1-UNC84 Homology (SUN) Domain Defines Features of Molecular Bridge in Nuclear Envelope

Background: The SUN domain mediates mechanical linkage across the nuclear envelope. Results: The structure of the SUN2 protein SUN domain was solved. The structure features important for SUN domain function were identified. Conclusion: The SUN domain forms a homotrimer. The SUN-KASH domain interaction is required for nuclear migration. Significance: The study provides insights into how the SUN protein complex functions. The SUN (Sad1-UNC-84 homology) domain is conserved in a number of nuclear envelope proteins involved in nuclear migration, meiotic telomere tethering, and antiviral responses. The LINC (linker of nucleoskeleton and cytoskeleton) complex, formed by the SUN and the nesprin proteins at the nuclear envelope, serves as a mechanical linkage across the nuclear envelope. Here we report the crystal structure of the SUN2 protein SUN domain, which reveals a homotrimer. The SUN domain is sufficient to mediate binding to the KASH (Klarsicht, ANC-1, and Syne homology) domain of nesprin 2, and the regions involved in the interaction have been identified. Binding of the SUN domain to the KASH domain is abolished by deletion of a region important for trimerization or by point mutations associated with nuclear migration failure. We propose a model of the LINC complex, where the SUN and the KASH domains form a higher ordered oligomeric network in the nuclear envelope. These findings provide the structural basis for understanding the function and the regulation of the LINC complex.

Characterization and Comparison of Disulfide Linkages and Scrambling Patterns in Therapeutic Monoclonal Antibodies - Using LC-MS with Electron Transfer Dissociation

The disulfides in three monoclonal antibodies (mAb), the anti-HER2, anti-CD11a, and GLP-1 with IgG4-Fc fusion protein, were completely mapped by LC-MS with the combination of electron-transfer dissociation (ETD) and collision induced dissociation (CID) fragmentation. In addition to mapping the 4 inter- and 12 intrachain disulfides (total 16), the identification of scrambled disulfides in degraded samples (heat-stress) was achieved. The scrambling was likely attributed to an initial breakage between the light (Cys 214) and heavy (Cys 223) chains in anti-HER2, with the same observation found in a similar therapeutic mAb, anti-CD11a. On the other hand, the fusion antibody, with no light chain but containing only two heavy chains, generated much less scrambling under the same heat-stressed conditions. The preferred sites of scrambling were identified, such as the intrachain disulfide for CxxC in the heavy chain, and the C194 of the heavy chain pairing with the terminal Cys residue (C214) in the light chain. The interchain disulfides between the light and heavy chains were weaker than the interchain disulfides between the two heavy chains. The relative high abundance ions observed in ETD provided strong evidence for the linked peptide information, which was particularly useful for the identification of the scrambled disulfides. The use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) helped the separation of these misfolded proteins for the determination of scrambled disulfide linkages. This methodology is useful for comparison of disulfide stability generated from different structural designs and providing a new way to determine the scrambling patterns, which could be applied for those seeking to determine unknown disulfide linkages.

Development of different analysis platforms with LC-MS for pharmacokinetic studies of protein drugs.

Three different analysis platforms using LC-MS were successfully developed for pharmacokinetic (PK) studies of an antibody drug in serum. These analysis platforms can be selectively used for different types of protein drugs, which ranged from a very specific for a particular drug (antibody enrichment) to a less specific for any antibody drugs with an Fc domain (protein A enrichment), and to a very generic method that can be used for any protein drugs (albumin depletion method). In this manner, the three platforms will be applicable to a wide range of antibody therapeutic studies for different species. The analysis using an albumin depletion method (with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) achieved the detection of the drug (to 1 ng) in an aliquot of serum (30 microL) with a 5-order magnitude of linearity. The analysis using protein A enrichment (with SDS-PAGE) achieved the detection of the drug at a 50-fold lower level (to 0.02 ng). Without the use of SDS-PAGE for separation, the use of protein A enrichment achieved the detection to 10 ng and using the antidrug antibody enrichment achieved the detection to 0.1 ng, with a similar linear dynamic range. These three analysis platforms produced good agreement with a mimic PK study of the drug in monkey serum, as compared to the enzyme-linked immunosorbent assay (ELISA) approach. In addition, these analysis platforms can be selectively applied for PK studies of drugs with different requirements of development time and resources. Such as, the antibody enrichment method can be used in a high-throughput manner but limited to a specific protein drug only. On the other hand, the albumin depletion method can be applied to many types of protein drugs but with the laborious sample preparation steps (SDS-PAGE and the subsequent in-gel digestion). When antidrug antibodies are not available for antibody drugs or the sensitivity requirement is not stringent (e.g., >10 ng), using protein A enrichment (without using SDS-PAGE) seems to be a good choice for PK studies which require fast throughput.

The challenges of developing a sound proteomics strategy

This paper will review the challenges of developing a proteomics strategy. A key issue is the integration of the two‐dimensional (2‐D) gel platform with mass spectrometry measurements. The use of both matrix‐assisted laser/desorption ionization (on off‐line coupling) and electrospray (on‐line) ionization are complementary. While the use of one‐dimensional and 2‐D gels are essential to many aspects of proteomics research (sample preparation, preliminary fractionation and quantitation, storage of protein components), the emergence of shotgun sequencing based on high performance liquid chromatography and tandem mass spectrometry offers a powerful new approach. The latter has particular utility in the characterization of low level samples and complex post‐translational modifications. The development of capillary columns, such as 75 to 150 micron, that can be packed in a reproducible manner has been a key step in the development of high sensitivity liquid chromatography/mass spectrometry analysis.

Hydrophilic Interaction 10 μm I.D. Porous Layer Open Tubular Columns for Ultratrace Glycan Analysis by Liquid Chromatography-Mass Spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI-MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 microm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared ( approximately 4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI-MS, low detection limits (0.3fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5mx10 microm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from approximately 50 fmol amounts injected. As an example of the high sensitivity of the column, MS(6) characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC-MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC-MS analysis of glycans at the ultratrace level.

Study of the human plasma proteome of rheumatoid arthritis

In this study, we report a combined proteomic and peptidomic analysis of human plasma from patients with rheumatoid arthritis (RA) and controls. We used molecular weight cut-off filters (MWCO: 10kDa) to enrich low-molecular-weight (LMW) peptides from human plasma. The peptide fraction was analyzed without trypsin digestion by capillary reversed-phase high-performance liquid chromatography (HPLC) coupled to a linear ion trap-FT-MS system, which identified 771 unique peptides in the peptidome study (from 145 protein progenitors). An anti-albumin/anti-IgG column was used to remove albumin and immunoglobulin G (IgG) from the human plasma. After that, the albumin/IgG-depleted sample was fractionated into a bound fraction and an unbound fraction on a multi-lectin affinity column (M-LAC). LC-MS analysis of the corresponding tryptic digests identified 308 proteins using the M-LAC approach. Relative differences in the following protein classifications were observed in the RA human plasma samples compared with controls: structural proteins, immuno-related proteins, protease inhibitors, coagulation proteins, transport proteins and apolipoproteins. While some of these proteins/peptides have been previously reported to be associated with RA disease such as calgranulin A, B, C and C-reactive protein, several others were newly identified (such as thymosin beta4, actin, tubulin, and vimentin), which may further the understanding of the disease pathogenesis. Moreover, we have found that the peptidomic and glycoproteomic approaches were complementary and allow a more complete picture of the human plasma proteome which can be valuable in studies of disease etiology.