Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.

Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.

Multidimensional separation of peptides for effective proteomic analysis.

Current solution based proteomic analysis methods are generally based on enzymatic digestion of a protein mixture followed by separation using multidimensional liquid chromatography and/or electrophoresis where peptide identification is typically accomplished by tandem mass spectrometry (MS/MS). It is generally accepted that no single chromatographic or electrophoretic procedure is capable of resolving the complex mixture of peptides that results from a global proteolytic digest of a proteome. Therefore, combining two or more orthogonal (multimodal) separation procedures dramatically improves the overall resolution and results in a larger number of peptides being identified from complex proteome digests. Separation of a proteome digest is a particularly challenging analytical problem due to the large number of peptides and the wide concentration dynamic range. While it has been demonstrated that increasing the number of dimensions of separation prior to MS analysis increases the number of peptides that may be identified, a balance between the time invested and the overall results obtained must be carefully considered. This manuscript provides a review of two- and three-dimensional peptide separation strategies combined with MS for the analysis of complex peptide mixtures.

Sheathless electrospray ionization interfaces for capillary electrophoresis-mass spectrometric detection advantages and limitations.

A review of sheathless interfaces for capillary electrophoresis (CE)-mass spectrometry (MS) is presented. The review discusses the on-line CE-MS system requirements, advantages and weaknesses of current sheathless interface designs for CE-electrospray ionization MS, and comparison between sheath flow and sheathless interfaces. The advantages and limitations of three sheathless designs are discussed and commented upon, these include single-capillary, two-capillary and three-piece designs.

Two-dimensional liquid chromatography-capillary zone electrophoresis-sheathless electrospray ionization-mass spectrometry: evaluation for peptide analysis and protein identification.

A peptide separation strategy that combines two-dimensional (2-D) liquid chromatography (LC)-capillary zone electrophoresis (CZE) with tandem mass spectrometry (MS/MS) is described for the identification of proteins in complex mixtures. To test the effectiveness of this strategy, a serum sample was depleted of the high-abundance proteins by methanol precipitation, digested with trypsin to generate a complex peptide mixture, and separated into 96 fractions by reversed-phase (RP)-LC. Compared to ion-exchange LC separations, RPLC provides much higher resolution and peak capacity. Fractions were collected off-line from the RPLC separation, and subjected to short 20 min CZE separations. The separated zones were introduced to the mass spectrometer through a sheathless electrospray ionization interface that is integrated on the separation capillary. The ease of fabrication of the interface and its durability allowed for the analysis of all fractions on a single capillary in a relatively short analysis time. A stable electrospray was produced at nanoliter flowrates by augmenting analyte electrophoretic and electroosmotic mobilities with pressure-assisted flow. Unlike first-dimensional ion-exchange LC fractionation, where there is a large degree of overlap, the CZE-MS results show less than 15% overlap between neighboring RPLC fractions.

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome.

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.

A detergent- and cyanogen bromide-free method for integral membrane proteomics: application to Halobacterium purple membranes and the human epidermal membrane proteome.

A simple and rapid method for characterizing hydrophobic integral membrane proteins and its utility for membrane proteomics using microcapillary liquid chromatography coupled on-line with tandem mass spectrometry (microLC-MS/MS) is described. The present technique does not rely on the use of detergents, strong organic acids or cyanogen bromide-mediated proteolysis. A buffered solution of 60% methanol was used to extract, solubilize, and tryptically digest proteins within a preparation of Halobacterium (H.) halobium purple membranes. Analysis of the digested purple membrane proteins by microLC-MS/MS resulted in the identification of all the predicted tryptic peptides of bacteriorhodopsin, including those that are known to be post-translationally modified. In addition, 40 proteins from the purple membrane preparation were also identified, of which 80% are predicted to contain between 1 and 16 transmembrane domains. To evaluate the general applicability of the method, the same extraction, solubilization, and digestion conditions were applied to a plasma membrane fraction prepared from human epidermal sheets. A total of 117 proteins was identified in a single microLC-MS/MS analysis, of which 55% are known to be integral or associated with the plasma membrane. Due to its simplicity, efficiency, and absence of MS interfering compounds, this technique can be used for the characterization of other integral membrane proteins and may be concomitantly applied for the analysis of membrane protein complexes or large-scale proteomic studies of different membrane samples.

On-column sample enrichment for capillary electrophoresis sheathless electrospray ionization mass spectrometry: evaluation for peptide analysis and protein identification.

Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CE-separated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-microm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry.

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.

Development of a two-dimensional protein-peptide separation protocol for comprehensive proteome measurements.

We have developed an effective two-dimensional fractionation protocol of complex proteome mixtures that extends the ability to conduct more comprehensive proteome measurements. A sample containing intact proteins extracted from Saccharomyces cerevisiae was fractionated by liquid phase isoelectric focusing, followed by tryptic digestion and solid-phase extraction (SPE) clean-up and reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS-MS) of the resultant peptides. The clean-up step is designed to desalt the fractions and rid them of urea and ampholytes prior to analysis by LC-MS-MS. Fifty milligrams of protein were separated into 20 fractions by liquid-phase isoelectric focusing, spanning a pH range of 3-10. The effectiveness of the removal of ampholytes was monitored by capillary zone electrophoresis and LC-MS-MS. The ability to analyze all of the 20 fractions without any noticeable decrease in the separation efficiency demonstrates the overall effectiveness of the SPE clean-up step. The results show that the separation strategy is effective for high throughput characterization of proteins from complex proteomic mixtures.

Methods for fractionation, separation and profiling of proteins and peptides.

In the last few years there has been an increased effort to develop technologies capable of identifying and quantifying large numbers of proteins expressed within a cell system (i.e., the proteome). The complexity of the mixtures being analyzed has made the development of effective fractionation and separation methods a critical component of this effort. This review highlights many of the protein and peptide fractionation and separation methods, such as electrophoresis and high-performance liquid chromatography (HPLC), which have experienced significant development over the past forty years. Modern instrumental strategies for the resolution of cell proteins, based on separations employing a single high-resolution or multidimensional approach, and the relative merits of each, will be discussed. The focus of this manuscript will be on the development of multidimensional separations such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), HPLC/HPLC, and HPLC-capillary electrophoresis and their application to the characterization of complex proteome mixtures.