George M. Janini

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.

Analysis of the human serum proteome

Changes in serum proteins that signal histopathological states, such as cancer, are useful diagnostic and prognostic biomarkers. Unfortunately, the large dynamic concentration range of proteins in serum makes it a challenging proteome to effectively characterize. Typically, methods to deplete highly abundant proteins to decrease this dynamic protein concentration range are employed, yet such depletion results in removal of important low abundant proteins.A multi-dimensional peptide separation strategy utilizing conventional separation techniques combined with tandem mass spectrometry (MS/MS) was employed for a proteome analysis of human serum. Serum proteins were digested with trypsin and resolved into 20 fractions by ampholyte-free liquid phase isoelectric focusing. These 20 peptide fractions were further fractionated by strong cation-exchange chromatography, each of which was analyzed by microcapillary reversed-phase liquid chromatography coupled online with MS/MS analysis.This investigation resulted in the identification of 1444 unique proteins in serum. Proteins from all functional classes, cellular localization, and abundance levels were identified.This study illustrates that a majority of lower abundance proteins identified in serum are present as secreted or shed species by cells as a result of signalling, necrosis, apoptosis, and hemolysis. These findings show that the protein content of serum is quite reflective of the overall profile of the human organism and a conventional multidimensional fractionation strategy combined with MS/MS is entirely capable of characterizing a significant fraction of the serum proteome. We have constructed a publicly available human serum proteomic database (http://bpp.nci.nih.gov) to provide a reference resource to facilitate future investigations of the vast archive of pathophysiological content in serum.

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 °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.

Micellar Electrokinetic Capillary Chromatography: Basic Considerations and Current Trends

Abstract A brief review of micellar electrokinetic capillary chromatography is presented. Basic theory of MECC, a discussion of types of micelles in MECC and its application to different classes of compounds is presented. Selected examples, which illustrate the advantages of MECC over capillary zone electrophoresis are also given.

Laser-induced fluorescence detection of 9-fluorenylmethyl chloroformate derivatized amino acids in capillary electrophoresis.

Laser-induced fluorescence (LIF) was applied to the detection of 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatized amino acids separated by capillary electrophoresis. Fluorescence excitation was provided by a pulsed, KrF laser operating at 248 nm. A limit of detection of 5 x 10(-10) M was obtained for FMOC-alanine (S/N = 2). Separation of FMOC-derivatized proline, hydroxyproline, and sarcosine was achieved with a 20 mM borate buffer (pH 9.2), and the separation of FMOC-derivatized amino acid standard mixture was obtained using a 20 mM borate buffer (pH 9.2) containing 25 mM sodium dodecyl sulfate.

Capillary electrophoresis separation of small peptides : effect of pH, buffer additives, and temperature

Abstract The separation of dipeptides and small peptides by various modes of capillary electrophoresis was investigated in order to identify the best separation conditions and to compare the different charge-to-size parameters used in correlating peptide migration. For a series of equally charged polyalanines the best linear correlation was obtained when the electrophoretic mobility was plotted against q/(MW)2/3. Deviations from linearity with other peptides are due to an imprecise charge calculation procedure. The best separations were achieved at low pH (∼2.5) when a large metal ion such as Zn++ was added to the buffer. Under these conditions, peptides are positively charged and differences in charge are-maximized. The separation of peptides at pH 2.5 improved as temperature was decreased. A set of five 9-residue peptides with no significant difference in charge-to-size ratio were separated at pH 7.0 with a buffer composed of 50 mM Tris + 50 mM DTAB.

Peptide mobility and peptide mapping in capillary zone electrophoresis. Experimental determination and theoretical simulation.

The electrophoretic mobilities of 58 peptides that varied in size from 2 to 39 amino acids and varied in charge from 0.65 to 7.82 are presented. The measurements were conducted at 22 degrees C using a 10% linear polyacrylamide-coated column and a 50 mM phosphate buffer at pH 2.5. Excellent separation of peptides and highly reliable peptide maps of protein digests are routinely obtained using these experimental conditions. The electrophoretic data were used to test existing theoretical models that correlate electrophoretic mobility with physical parameters. The results indicate that the Offord model that correlates electrophoretic mobility with the charge-to-size parameter q/M2/3 offers the best fit of our reliable experimental data. Furthermore, we also obtained the capillary zone electrophoretic profile of the endoproteinase Lys-C digests of a peptide sequencing standard, melittin, and horse myoglobin under the same experimental conditions as described above. The resulting peptide maps were compared with corresponding theoretical simulation.

Pulsed UV Laser-Induced Fluorescence Detection of Native Peptides and Proteins in Capillary Electrophoresis

Abstract A pulsed UV laser operating at 248 nm was used for the laser-induced fluorescence (LIF) detection of native tryptophan-containing compounds in capillary electrophoresis. The limit of detection (LOD) of tryptophan was found to be 3.3×10−9 M (S/N=2). The LODs of the model proteins conalbumin and bovine serum albumin were found to be 1.3×10−9 and 4×10−9 M (S/N=2), respectively. These results were at least two orders of magnitude more sensitive when compared to UV absorption at 214 nm.

A simple two-dimensional high performance liquid chromatography/high performance capillary electrophoresis set-up for the separation of complex mixtures.

A two‐dimensional high performance liquid chromatography/capillary electrophoresis (HPLC/CE) instrumental set‐up was assembled from commercially available equipment. Fractions of the effluent from the HPLC system are collected into microtiter plates with a microfraction collector. The fractions are then dried under vacuum at room temperature, reconstituted, and analyzed by capillary zone electrophoresis (CZE). This method allows the collection of samples by time, drops, or external signal (peaks). Any size or type of HPLC or CE column can be used with no limitation on the amount of sample injected into the HPLC. Any CE detection, laser‐induced fluorescence (LIF), mass spectrometry (MS), ultraviolet (UV) or other, can be used. This set‐up is practical, simple, robust and allows the separation of complex mixtures. Preliminary results show the utility of this system for the analysis of protein digest.

Separation of pyridinecarboxylic acid isomers and related compounds by capillary zone electrophoresis: Effect of cetyltrimethylammonium bromide on electroosmotic flow and resolution

The effect of the addition of cetyltrimethylammonium bromide (CTAB) to the buffer system in capillary electrophoresis on electroosmotic flow (EOF) is examined. At a CTAB concentration of 2.5 x 10(-4) M, EOF is anodal (flow towards the positive detector column end). With bare silica columns, anodal EOF first increases with increasing pH, up to a maximum in the pH range 4-6 depending on CTAB concentration, then decreases as pH is further increased. Optimum resolution of pyridinecarboxylic acid isomers is obtained at pH 2.7 with a 10 mM phosphate buffer and 30 mM CTAB. Using the same buffer system, optimum resolution for hydroxy-substituted pyridinecarboxylic acid isomers is obtained at pH 7.5. The use of CTAB results in a dramatic improvement in peak shape. Preliminary results, using an excimer laser operated at 248 nm, show that the fluorescence intensity of isonicotinic acid is substantially enhanced with the addition of 0.3% hydrogen peroxide to the phosphate buffer system.