Charge-reduced nano electrospray ionization combined with differential mobility analysis of peptides, proteins, glycoproteins, noncovalent protein complexes and viruses.

This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

Controlling charge states of large ions.

The charge state of ions produced in electrospray ionization (ESI) was reduced in a controlled manner to yield predominantly singly charged species by exposure of the aerosol to a bipolar ionizing gas. Analysis of the resulting ions on an orthogonal time-of-flight mass spectrometer yielded mass spectra greatly simplified compared with conventional ESI spectra. The decreased spectral complexity afforded by the charge reduction facilitates the analysis of mixtures by ESI mass spectrometry.

Analysis of a 3.6-MDa hexagonal bilayer hemoglobin from Lumbricus terrestris using a gas-phase electrophoretic mobility molecular analyzer.

The recent successful use of electrospray gas-phase electrophoretic mobility molecular analysis (GEMMA) to separate globular proteins (mass 6 to 670 kDa) and the excellent correlation found between the electrophoretic mobility diameter (EMD), or Millikan diameter, and the protein mass (S. L. Kaufman et al., 1996, Anal. Chem. 68, 1895-1904; 1996, Anal. Chem. 68, 3703), prompted the examination of a large protein complex, the 3.6-MDa, heteromultimeric, hexagonal bilayer hemoglobin (Hb) and its subunits from the earthworm Lumbricus terrestris. The native Hb had an EMD of 25.7 nm and the products of its dissociation at pH >8 and <5 were resolved into peaks with EMDs of 10.5, 6.3, 5.0, and 4.2 nm, identified as a dodecamer of globin chains ([a+b+c]3d3, 213 kDa), the disulfide-bonded trimer of globin chains ([a+b+c], 52.7 kDa), all the linker chains (L1, 27.5 kDa; L2, 32.1 kDa; L3, 24.9 kDa; L4, 24. 1 kDa), and the monomer subunit (chain d, 17 kDa), respectively. Reassembly of the Hb complex was observed on restoring the pH from >8 to 7. The EMDs and the masses of the Hb and its subunits are in excellent agreement with the correlation found earlier, under the assumption of nearly spherical shape with an effective density around 0.7 g/cm3. GEMMA also provided a profile of the Hb completely dissociated in 0.1% SDS; its deconvolution permitted a quantitative determination of the subunit stoichiometry, providing a globin to linker ratio of 3 to 1.

DNA analysis using an electrospray scanning mobility particle sizer.

A scanning mobility particle sizer (SMPS) allows size separation of gas phase particles according to their electrophoretic mobilities. The addition of an electrospray source (ES) recently allowed extension of SMPS analysis to the macromolecular range. We demonstrate here the application of ES-SMPS to nucleic acids analysis. Single- and double-stranded DNA molecules ranging from 6.1 kDa (single-stranded DNA 20 nucleotides in length) to 300 kDa (500 base-pair double-stranded DNA) were separated and detected by ES-SMPS at the picomole to femtomole levels. The measured electrophoretic mobility diameters were found to correlate with the analytes' molecular weights, while the peak areas could yield quantitative information. No fragmentation of DNA was observed under the conditions employed. Different apparent densities were observed for single-stranded and double-stranded DNAs, showing a different behavior for each type of biomolecule. The total analysis time was about 3 min/spectrum. Further optimization of ES-SMPS is expected to make it a fast and sensitive technique for biopolymer characterization.

Capillary zone electrophoresis with electrospray condensation particle counting detection.

A new sheath flow microelectrospray interface combines capillary electrophoretic separation of protein standards with condensation particle counting detection. Protein separations are performed in both coated and uncoated fused-silica columns. The electrospray needle utilizes a sheath flow rate between 0 and a few microL/min. Typically, the makeup flow is equal to or less than the electroosmotic flow from the separation capillary; thus there is minimal dilution while still providing pH adjustment and solid electrical contact with the separation capillary. While there are some inherent restrictions to a condensation particle counting detector, the microelectrospray needle performs well and has direct application to electrospray mass spectrometry.

Macromolecule analysis based on electrophoretic mobility in air: globular proteins.

Globular proteins ranging in molecular mass from 5.7 to 669 kDa were separated and analyzed using an aerosol technique based on the electrophoretic mobility of singly-charged molecular ions in air. The ions were produced by electrospraying and drying 100-nm-diameter droplets of a liquid suspension of the proteins, using ionized air to remove the droplet charge due to the spray process. The electrophoretic mobility was measured using a modified commercial continuous-flow differential mobility analyzer operated near atmospheric pressure. An unmodified commercial condensation particle counter was used for detection. The concentrations analyzed ranged from 0.02 to 200 µg of protein/mL of buffer, with a liquid sample flow rate of approximately 50 nL/min. Sampling time of 3 min was used for each complete distribution measured. The electrophoretic mobilities measured were determined entirely from air flow rates, apparatus geometry, and applied potentials. Results were expressed as electrophoretic mobility equivalent diameters using a Millikan formula.

Percutaneous palliation of unresectable pancreatic cancer.

Percutaneous biliary drainage is one of several methods of palliating patients with unresectable carcinoma of the pancreas. In most cases, patients with unresectable carcinoma of the pancreas causing biliary obstruction are optimally managed by endoscopic stent placement. Percutaneous biliary drainage may be performed when endoscopic biliary drainage is unsuccessful or with obstruction at or above the hepatic duct bifurcation. Patients with common bile duct obstruction today are optimally managed by placement of expandable metal endoprostheses. In such patients with short life expectancies, symptoms of jaundice can be effectively palliated with a low rate of recurrence and with a complication rate no higher than that associated with percutaneous biliary drainage. Metal endoprostheses are associated with less pain during placement than are plastic endoprostheses and may have a lower rate of recurrence of jaundice than plastic endoprostheses in patients with common bile duct obstruction due to carcinoma of the pancreas. Patients with hilar biliary obstruction are better treated with internal-external biliary drainage catheters.

Transjugular intrahepatic portal systemic shunt for the management of symptomatic cirrhotic hydrothorax.

OBJECTIVE: To investigate the safety and effectiveness of performing transjugular intrahepatic portal systemic shunt (TIPS) for the management of symptomatic cirrhotic hydrothorax in patients with advanced cirrhosis. METHODS: TIPS was performed by standard technique after portal vein patency had been established by ultrasound. Portal-hepatic vein pressure gradient was determined before and after placement of the shunt. A portal-hepatic vein gradient of less than 12 mm Hg was the treatment goal. RESULTS: Five patients underwent TIPS placement over an 11-month period. Despite use of diuretics, the patients had required a median of seven thoracenteses (range 2-11) for control of symptoms preceding placement of the shunt. A TIPS was placed without serious complications in all five patients. In two patients, insertion of the shunt was associated with no further need for thoracentesis. The other three patients had recurrent need for thoracentesis. These three patients were found to have occluded shunts which were rendered patent by angioplasty and/or urokinase. Subsequently, two required no further thoracentesis, whereas, in the other patient, the need for thoracentesis was decreased dramatically. CONCLUSIONS: TIPS appears to be a safe and useful technique for the management of patients with symptomatic cirrhotic hydrothorax that is refractory to medical therapy. Recurrence of the pleural effusion after placement of TIPS may be an indication of shunt occlusion.

Electrospray-condensation particle counter: a molecule-counting LC detector for macromolecules.

A new detector for macromolecular separations is described. The detector counts individual macromolecules (molecular weights greater than about 10,000) and reports counts per second. The chromatographic effluent is electrosprayed, neutralized, and swept to the detector by a stream of air. The detector is a condensation particle counter that detects individual particles by light scattering from droplets condensed on the particles. When used as the detector for a size exclusion separation of proteins, the detector has a linear range of 4 orders of magnitude with detection limits as low as 0.1 microgram/mL. The detector can be directly interfaced (no makeup flow) with effluent flows as low as 10 nL/min. A Monte Carlo model based on size measurements of the electrosprayed droplets correctly predicts the observed detector behavior.

Calculation of actuarial life tables with use of a microcomputer spreadsheet program.

PURPOSE: To simplify the calculation of the life-table method of survival analysis. MATERIALS AND METHODS: A template was developed for use with a popular microcomputer spreadsheet program to perform life-table survival calculations and to perform statistical comparisons between the survivals of groups of patients with use of the log-rank test. RESULTS: The template successfully performed life-table and log-rank calculations when compared with two standard, accepted statistical computing programs. CONCLUSION: The template can be used to quickly perform life-table calculations that can be used in the long-term follow-up of patients undergoing interventional radiologic procedures.

Long-term results of angioplasty in 110 patients with renal artery stenosis.

Percutaneous transluminal renal angioplasty (PTRA) is generally considered of little benefit in the treatment of ostial renal artery stenosis. This report contains long-term follow-up (> 12 months in all patients; mean follow-up, 38 months) for 110 patients who underwent PTRA for treatment of ostial renal artery stenosis. There was no significant difference in patient benefit related to bilaterality or multiplicity of lesions treated or to renal function before angioplasty (P > .1). Although there was no statistically significant difference in benefit among groups of patients who received treatment, certain trends were apparent. The least benefit occurred in patients with insulin-dependent diabetes and those with symptoms or history of vascular disease in another organ system. Treatment of lesions with proportionately larger balloons did not result in greater benefit. Restenoses were redilated in 16 patients for whom initial treatment failed. Eleven of these were ostial restenoses. The ostial stenosis in one patient was redilated a second time. At the end of follow-up, primary, secondary, and tertiary clinical benefits were 48%, 57%, and 58%, respectively. This was not statistically different (P = .14) from a control group of 94 patients with nonostial stenoses who had 68% long-term benefit. The authors conclude that ostial renal artery stenosis is not a contraindication to PTRA, and balloon angioplasty can play an important role in blood pressure control in this patient population.

Peripheral transcatheter embolization with platinum microcoils.

Embolotherapy with platinum microcoils delivered through the Tracker-18 microcatheter (Target Therapeutics, San Jose, Calif) was performed in 16 patients when peripheral superselective catheterization with standard angiographic catheters was not possible. The Tracker-18 catheter could be directed distally into small peripheral vessels for delivery of the microcoils. These microcoils, with attached fiber strands, resulted in vascular occlusion within a few minutes in all cases. Embolotherapy was technically successful in all patients. The procedures were clinically successful in 15 of 16 patients (94%), and no complications were encountered. Embolization with platinum microcoils through the Tracker-18 catheter is useful when standard methods of embolization are not possible.

Arterial injuries associated with complete dislocation of the knee.

To determine the frequency and type of arterial injuries associated with complete dislocation of the knee and to correlate the findings at physical examination with those at arteriography, a computerized search for patients with a diagnosis of dislocation of the knee at discharge from an urban trauma hospital was performed. Nineteen patients with complete dislocation of the knee were found. Arterial injuries were seen in six patients (32%). Four of the 19 patients had no pedal pulse at physical examination. In three of these four patients, occlusion of the popliteal artery was seen on arteriograms. The fourth patient had occlusion of an anomalous anterior tibial artery and a compartmental syndrome. Two of the 15 other patients with pedal pulses (13%) had nonoccluding intimal defects of the popliteal artery; these two patients did well without surgery. It is concluded that abnormal peripheral pulses associated with complete knee dislocation are highly predictive of major arterial injury. If peripheral pulses are normal, a low but definite frequency of arterial damage exists.

Urokinase thrombolysis using a multiple side hole multilumen infusion catheter.

A multilumen, multiple side hole infusion catheter was used for urokinase thrombolysis in 13 patients with thromboembolic occlusions of peripheral arteries and grafts. Balloon angioplasty was performed following urokinase infusion in 6 patients and atherectomy in 1 patient. There was one hemorrhagic complication. The major advantage of the multiple sidehole infusion catheter was the elimination of the need to reposition the catheter during the infusion and the reduction of the time burden on the angiographic facility. The success rate for the thrombolysis (77%) was comparable to results recorded in the literature. The total duration of infusion was not reduced compared to other series.

Young Investigator Award. Raman spectroscopy of human atherosclerotic plaque: implications for laser angioplasty.

Raman spectroscopy is a specialized technique that permits highly specific identification of specimens, in contrast to fluorescence spectroscopy with which analysis of arterial tissues generates spectra that are broad and featureless, with little difference seen between normal artery and atheroma. Various plaque types and the contributions of different arterial fluorophores were studied to determine if Raman spectroscopy could function as a potential guidance modality for laser angioplasty. Arterial specimens obtained at atherectomy and post mortem were studied in air and while immersed in blood. One hundred fifty-six Raman spectra were collected from arterial specimens and chromatographic samples of collagen, elastin, cholesterol, beta-carotene, and L-tryptophan. Analysis showed both fatty and fibrous atherosclerotic plaques to have characteristic spectral peaks at 1,002, 1,154, and 1,516 cm-1, while the Raman spectrum of normal vessel was featureless. Spectral peaks of beta-carotene were nearly identical to those of fatty plaque. The arterial fluorophores collagen, elastin, cholesterol, and L-tryptophan were non-contributory. The Raman spectrum of fatty plaque immersed in a blood field was also detectable, suggesting that this technique may be useful for in vivo plaque recognition.

Angioplasty from the contralateral approach: use of a guiding catheter and coaxial angioplasty balloons.

An 8-F guiding catheter has been developed for use with coaxial angioplasty balloon catheters in angioplasty of the femoral artery from the contralateral approach when antegrade puncture of the ipsilateral femoral artery is not possible. The catheter may be used for angioplasty of the femoral artery bifurcation and the superficial femoral artery and for arterial stenoses in patients with renal transplants.

Angioplasty for long-term treatment of patients with Budd-Chiari syndrome.

The goal of radiologic intervention in patients with Budd-Chiari syndrome is to control portal hypertension and prevent further hepatocellular damage until collateral hepatic venous outflow channels can develop. Percutaneous balloon angioplasty was used to treat six patients with this syndrome who were followed up for an average of 43 months (range, 12-92 months). Standard interventional radiologic techniques were used to dilate the hepatic veins (two patients), inferior vena cava (three patients), and proximal anastomosis of a mesoatrial shunt (one patient). Angioplasty was the only invasive treatment in three patients, whereas the remaining three patients had previous portosystemic shunts. Clinical and hemodynamic improvement occurred after each angioplasty. Multiple dilatations were required in all patients (average, 3.2; range, 2-5) because of restenosis at the angioplasty site and ongoing hepatocyte necrosis shown by biopsy. Long-term benefit occurred in five patients despite ultimate caval occlusion in two patients and restenosis in one patient. One patient who was almost free of symptoms for 36 months developed gastrointestinal bleeding caused by portal hypertension. This experience suggests that balloon angioplasty is a safe and effective treatment for patients with Budd-Chiari syndrome. The therapy is not definitive, but serves to moderate the severity of the disease until collateral venous pathways develop. Multiple angioplasties are required for the long-term care of these patients.