Charles Mitchell

Relationship Between Cardiac Output and Peripheral Resistance in Borderline Hypertension

Eighty-eight observations on 77 patients with borderline hypertension and 82 single observations in healthy control subjects are reported. Hemodynamic effects of assumption of the sitting position, mild exercise, infusion of dextran, blockade with propranolol and with a combination of propranolol and atropine are evaluated. In the recumbent position, patients with borderline hypertension have increased cardiac output and “normal” peripheral resistance. Under all other experimental conditions, the peripheral resistance in patients with borderline hypertension was elevated. Increased resistance was accompanied by a decrease of the cardiac output. After administration of the atropine and propranolol combination, cardiac output in patients with borderline hypertension was significantly below the normal. Nevertheless, whether cardiac output was high or low and resistance normal or elevated, patients with borderline hypertension maintained mild elevations of the blood pressure. Consequently, borderline hypertension is not caused solely by elevations of cardiac output.

Mass spectrometry of proteins directly from polyacrylamide gels

The direct combination of thin-layer gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry has been demonstrated with good sensitivity and mass accuracy, offering potential advantages in speed and reduced complexity. Mass spectra have been obtained from isoelectric focusing, sodium dodecyl sulfate, and native gels with as little as 660 fmol of α- and β-chain bovine hemoglobin and 1 pmol of horse heart myoglobin loaded. CNBr digests were performed in situ, and the products were probed in-gel. Noncovalent complexes such as multimeric protein systems, enzyme inhibitor complexes, and protein-ligand complexes can also be characterized when gel electrophoresis is run under nondenaturing conditions. This approach shows promise for simplifying the interface between gel electrophoresis and mass spectrometry.

High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins

The molecular weight measurement of intact Escherichia coli proteins separated by isoelectric focusing‐immobilized pH gradient (IEF‐IPG) gels and analyzed by mass spectrometry is presented. Two methods are discussed: (i) electrospray ionization (ESI) mass spectrometry (MS) of extracted proteins, and (ii) matrix‐assisted laser desorption/ionization (MALDI)‐MS analysis directly from IEF‐IPG gels. Both ESI and MALDI methods yield sub‐picomole sensitivity and good mass measurement accuracy. The use of an array detector for ESI‐MS was essential to discriminate against contaminating background ions and to selectively detect high mass protein ions. MALDI‐MS offers high‐throughput analysis of one‐ and potentially two‐dimensional (2‐D) gels. The “virtual 2‐D” gel method with first‐dimensional IEF separation and the second dimension as molecular mass determination by MS, is a particularly promising method for protein analysis due to its ultra high sensitivity and correspondence to classical 2‐D gels. Further sensitivity enhancements for the MALDI‐MS method are provided by post acceleration detection optimized for high mass time‐of‐flight analysis.

Diffusive transfer to membranes as an effective interface between gel electrophoresis and mass spectrometry

Abstract Diffusive transfer was examined as a blotting method to transfer proteins from polyacrylamide gels to membranes for ultraviolet matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The method is well-suited for transfers from isoelectric focusing (IEF) gels. Spectra have been obtained for 11 pmol of 66 kDa albumin loaded onto an IEF gel and subsequently blotted to polyethylene. Similarly, masses of intact carbonic anhydrase and hemoglobin were obtained from 14 and 20 pmol loadings. This methodology is also compatible with blotting high molecular weight proteins, as seen for 6 pmol of the 150 kDa monoclonal antibody anti-β-galactosidase transferred to Goretex. Polypropylene, Teflon, Nafion and polyvinylidene difluoride (PVDF) also produced good spectra following diffusive transfer. Only analysis from PVDF required that the membrane be kept wet prior to application of matrix. Considerations in mass accuracy for analysis from large-area membranes with continuous extraction and delayed extraction were explored, as were remedies for surface charging. Vapor phase CNBr cleavage was applied to membrane-bound samples for peptide mapping.

A Novel Sample Preparation for Shotgun Proteomics Characterization of HCPs in Antibodies

Residual host cell proteins (HCPs) in biopharmaceuticals derived from recombinant DNA technology can present potential safety risks to patients or compromise product stability. Thus, the downstream purification process is designed to demonstrate robust removal of these impurities. ELISA using polyclonal anti-HCP antibodies as reagents for capture, detection, and quantitation purposes is most commonly used to monitor HCP removal during process development, but this technique has limitations. More recently, LC-MS for residual HCP characterization has emerged as a powerful tool to support purification process development. However, mass spectrometry needs to overcome the enormous dynamic range to detect low ppm levels of residual HCPs in biopharmaceutical samples. We describe a simple and powerful methodology to characterize residual HCPs in (monoclonal) antibodies by combining a novel sample preparation procedure using trypsin digestion and a shotgun proteomics approach. Differing from the traditional methodology, the sample preparation approach maintains nearly intact antibody while HCPs are digested. Thus, the dynamic range for HCP detection by MS is 1 to 2 orders of magnitude less than the traditional trypsin digestion sample preparation procedure. HCP spiking experiments demonstrated that our method could detect 0.5 ppm of HCP with molecular weight >60 kDa, such as rPLBL2. Application of our method to analyze a high-purity NIST monoclonal antibody standard RM 8670 derived from a murine cell line expression system resulted in detection of 60 mouse HCPs; twice as many as previously reported with 2D-UPLC/IM/MSE method. A control monoclonal antibody used for 70 analyses over 450 days demonstrated that our method is robust.