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PubMed2003

Time-of-flight secondary ion mass spectrometry: techniques and applications for the characterization of biomaterial surfaces.

The surface of a biomaterial plays a critical role in the success of an implant. Much effort is currently being focused on controlling the chemistry at biomaterial surfaces to ensure favorable results in vivo. The successful tailoring of the surface chemistry will require a detailed surface characterization to verify that the desired changes have been made. This will include the ability to determine the composition, structure, orientation, and spatial distribution, of the molecules and chemical structures on the surface. TOF-SIMS is a powerful surface characterization technique that is able to address these requirements through both spectral analysis and direct chemical state imaging. The flexibility of the TOF-SIMS technique, and the wealth of data produced have generated much interest in its use for biomaterial characterization. This review discusses the strengths, weaknesses, and challenges of static TOF-SIMS for biomaterial surface characterization. First the basic principles of TOF-SIMS are introduced, giving an overview of the technique. Next, sample type, and other sample considerations are discussed. Then data interpretation is overviewed using examples from both spectral and imaging data. Finally, quantitative SIMS analysis is discussed and an outlook for TOF-SIMS analysis of biomaterials will be given.

Biocompatible Materials↗

PubMed1998

Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded.

The interaction of Salmonella and Yersinia with macrophages is critical to the pathogenesis of these organisms. After internalization into macrophages, these bacteria reside in membrane-enclosed vacuoles. In this report, we present an approach to isolate and characterize bacteria-containing vacuoles (BCVs) to study intracellular trafficking of pathogenic bacteria within the membrane system of host cells. Using the mouse monocyte-macrophage cell line J774A.1, we found that Salmonella typhimurium replicated intracellularly to approximately 5 times its original numbers over a 9 hour infection course, while Yersinia pseudotuberculosis and Escherichia coli did not replicate inside these cells. Analysis of isolated latex bead-containing vacuoles confirmed that they trafficked normally from endosomes to lysosomes within the endocytic pathway of J774A.1 cells. We isolated BCVs free of contaminating endosomes and lysosomes using sucrose step gradients, and used quantitative immunoblotting to characterize the contents of these vacuoles at different time points after internalization. We found that the isolated BCVs contained endosomal and lysosomal marker proteins including lamp-1, mannose 6-phosphate receptor (M 6-PR), cathepsin D and cathepsin L. Further, we report on differential processing of lysosomal hydrolases (such as cathepsin D and cathepsin L) associated with the isolated BCVs. Although there was some contamination of the S. typhimurium-containing vacuoles with endoplasmic reticulum (ER) marker protein calnexin, the Y. pseudotuberculosis-containing vacuoles were predominately free of ER contamination. The Y. pseudotuberculosis-containing vacuoles displayed properties of lysosomes, containing the M 6-PR-dependent lysosomal hydrolases cathepsin D and cathepsin L, which were shown to be processed to their mature forms incrementally over time. These results, coupled with intracellular growth and microscopic examination of infected cells over time, indicated that Y. pseudotuberculosis traffics to lysosomes where they are degraded. The described method for isolation and characterization of BCVs proved to be a valuable tool to characterize the vacuolar compartment occupied by Y. pseudotuberculosis, and has potential to be applied to other vacuole resident pathogens whose trafficking is thought to play a role in pathogenesis.

Animals↗

PubMed2001

Structural characterization of erythroid and megakaryocytic differentiation in Friend erythroleukemia cells.

OBJECTIVE: The aim of this study was to examine the structural characterization of erythroid and megakaryocytic cell differentiation in Friend erythroleukemic cells using spectral imaging and electron microscopy. MATERIALS AND METHODS: Two variants of Friend erythroleukemia cells were treated with hexamethylene bisacetamide (HMBA) to induce differentiation: 1) MEL, which exhibit the normal phenotype and are susceptible to differentiation; and 2) the resistant R1 cells. The cells were analyzed by spectral imaging along with transmission and scanning electron microscopy. The expression of cell cycle regulatory proteins was analyzed by Western blotting. RESULTS: Spectral imaging of HMBA-treated MEL and R1 cells stained by May-Grünwald-Giemsa and subjected to spectral similarity mapping revealed five morphologic cell types: proerythroblast-like cells, normoblast-like cells, reticulocyte-like cells, megakaryocytes, and apoptotic cells. In MEL cells, both megakaryocytic differentiation characterized by nuclear lobes and erythroid differentiation characterized by accumulation of hemoglobin were detected; R1 cells were not committed to terminal differentiation. HMBA-induced cell cycle arrest at G(1) affected the expression of regulatory proteins in a similar manner in both types of cells. Expression of cyclin-dependent kinase 4 decreased and expression of p21(WAF1) increased. The level of the underphosphorylated form of phosphorylated retinoblastoma protein increased, inducing a decrease in the level of c-myc. In addition, we detected a decrease in the expression of the anti-apoptotic regulator, Bcl-2, and an increased expression of the pro-apoptotic regulator, Bax. CONCLUSIONS: Spectral imaging provides new insight for the morphologic characterization of erythroid and megakaryocytic cell differentiation as well as apoptosis. Image analysis was well correlated to cell cycle arrest and the expression of regulatory proteins.

Acetamides↗

PubMed2003

CBED and LACBED: characterization of antiphase boundaries.

Convergent-beam electron diffraction (CBED) and large-angle convergent-beam electron diffraction (LACBED) techniques are well adapted to the characterization of several types of crystal defects. In fact, dislocations, grain boundaries and stacking faults have already been successfully characterized with these methods. In the present paper, we describe the CBED and LACBED characterization of another type of crystal defect showing a special interest in materials science: antiphase boundaries (APBs). The first part of the paper is devoted to the determination of the effects of antiphase boundaries on CBED and LACBED patterns that could be expected from a theoretical point of view. It indicates that the superlattice excess lines present on these patterns are split into two lines with equal intensity when the incident beam is located on an APB. In the second part, we experimentally test these theoretical predictions on a specimen showing two different known types of antiphase boundaries. In a third part we indicate how these methods could be used to identify unknown APBs in a specimen. Finally, the advantages and disadvantages of both methods for the characterization of antiphase boundaries are discussed.

Journal Article↗

PubMed1999

Molecular characterization and mapping of canine cGMP-phosphodiesterase delta subunit (PDE6D).

cGMP-phosphodiesterase (PDE) is composed of two catalytic (alpha and beta) and two identical inhibitory (gamma) subunits. The human gene (PDE6D) encoding a new subunit (delta) has been characterized and mapped to the long arm of chromosome 2 (HSA2q35-q36) where a new autosomal recessive retinitis pigmentosa (arRP) locus (RP26) has been localized. Characterization of the canine PDE6D shows the gene is about 4.2kb containing four exons interrupted by three introns; the size of the cDNA is 1059bp with an open reading frame (ORF) of 453bp. A single transcript of identical size (1.43kb) was detected in all tissues examined (liver, lung, spleen, kidney, heart, brain and retina), with the highest abundance in the retina. Canine PDE6D has been localized to canine radiation hybrid group 14-a, which extends conserved synteny between the dog, human chromosome 2q and mouse chromosome 1. The characterization of the canine PDE6D gene and its mapping provide important information for testing causal association of the gene with canine retinal degenerations, in particular rod-cone dysplasia 2 (rcd2) in collie dogs. This disease is characterized by abnormal retinal cGMP metabolism due to a deficiency in cGMP-PDE activity, yet the alpha, beta and gamma subunits of PDE have been excluded as candidate gene loci.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2002

Comparison of heterogeneous and homogeneous radioactivity flow detectors for simultaneous profiling and LC-MS/MS characterization of metabolites.

Methods for simultaneous liquid chromatography-radioactivity monitor (LC-RAM) metabolite profiling and LC-tandem mass spectrometry (MS/MS) characterization of metabolites are described. Profiling and characterization of metabolites from three drug candidates from different therapeutic areas were compared using in-line heterogeneous LC-RAM-MS/MS and homogeneous LC-RAM-MS/MS methods. Although comparison shows that simultaneous metabolite profiling and characterization can be achieved using either heterogeneous or homogeneous-LC-RAM-MS/MS systems, a homogeneous system has the advantage in the following aspects, (1) sensitivity; (2) ease of method transfer; (3) less peak broadening problems due to the drug or metabolites adhering to the RAM cell; (4) accuracy in quantitation of the metabolites; and (5) the ability to load larger volumes of unprocessed biological fluids. Furthermore, the study shows that some of the possible metabolites that do not ionize well with electrospray ionization (ESI) and eluded detection by heterogeneous-LC-RAM detection could be very easily detected and characterized using a homogeneous-LC-RAM-MS/MS system.

Amidines↗

PubMed1984

Applicability of ultrasonic tissue characterization for longitudinal assessment and differentiation of calcification and fibrosis in cardiomyopathy.

Progress in tissue characterization of myocardium with ultrasound suggests that quantitative recognition of ischemic or scarred tissue will be achieved. Despite the increasing recognition and importance of cardiomyopathy, its diagnosis generally requires invasive procedures such as cardiac catheterization and biopsy. To investigate methods that permit the characterization of longitudinal cardiomyopathic changes that might ultimately be extended for noninvasive studies in patients, quantitative ultrasonic methods were utilized for in vitro tissue characterization of hearts from Syrian hamsters of selected age of either 2 to 3 or 5 to 7 months. Normal hamsters were used as controls. Myocardial sites (n = 600) from the young Syrian hamsters exhibited values (+/- standard error) of integrated ultrasonic backscatter averaging -53.87 +/- 0.26 dB, which were significantly different from values (n = 500) in age-matched control hamsters (-58.07 +/- 0.08 dB; p less than 0.001). Cardiomyopathic hearts from older animals exhibited backscatter values (n = 500 sites) averaging -50.87 +/- 0.22 dB, again significantly different from values (n = 300 sites) in age-matched control hamsters (-55.91 +/- 0.11 dB; p less than 0.001). In addition, ultrasonic attenuation was significantly different for hearts from the control and cardiomyopathic hamsters of both age ranges. The results correlated with sequential calcification and fibrosis characteristics assessed histopathologically. This study indicates that quantitative characterization of myocardium with ultrasound may permit longitudinal assessment of cardiomyopathic changes in diverse disease entities and their response to therapy.

Age Factors↗

PubMedDate not supplied

Dobutamine stress ultrasonic myocardial tissue characterization in patients with dilated cardiomyopathy.

Although acoustic properties of the myocardium are different between patients with cardiomyopathy and normal subjects, the frequency of the abnormal properties in patients with cardiomyopathy is unknown. We assessed the frequency of abnormal acoustic properties of the myocardium detectable with integrated backscatter in patients with cardiomyopathy and attempted more sensitive ultrasonic tissue characterization by combining dobutamine stress testing in patients with cardiomyopathy with apparently normal acoustic properties of the myocardium at rest. The magnitude of cyclic variation of integrated backscatter and calibrated myocardial integrated backscatter at end diastole were measured in 36 normal subjects and 40 patients with dilated cardiomyopathy. Either one of the integrated backscatter parameters was abnormal in 30 of 40 patients with cardiomyopathy. Dobutamine stress ultrasonic tissue characterization was performed in 10 patients with cardiomyopathy with normal values of both integrated backscatter parameters and 10 normal subjects. Calibrated myocardial integrated backscatter did not change during dobutamine infusion in any subject. The magnitude of cyclic variation in integrated backscatter increased in normal subjects but did not change in patients with cardiomyopathy despite a comparative associated increase in the systolic wall thickening during dobutamine infusion. Abnormal acoustic properties are detectable at rest with myocardial integrated backscatter about in three quarters of patients with cardiomyopathy. A combination of dobutamine stress testing would provide more sensitive ultrasonic myocardial tissue characterization and may make it possible to detect subtle changes in the acoustic properties of the myocardium in patients with dilated cardiomyopathy. Therefore dobutamine stress ultrasonic tissue characterization may detect mild dilated cardiomyopathy.

Adult↗

PubMed1997

Characterization of avian Chlamydia psittaci strains using omp1 restriction mapping and serovar-specific monoclonal antibodies.

In the present study, 60 avian Chlamydia psittaci isolates were characterized using restriction fragment length polymorphism as well as serovar-specific monoclonal antibodies, enabling a comparison between the two characterization methods. Sixty avian C. psittaci isolates were characterized by Alul restriction mapping of the major outer membrane protein gene omp1 obtained after amplification by the polymerase chain reaction. The 60 avian C. psittaci strains were also characterized using serovar-specific monoclonal antibodies in a microimmunofluorescence test. Digestion of 60 avian C. psittaci omp1 amplicons by Alul generated 5 of the 6 known distinct restriction patterns (A, B, D, E and F). Restriction pattern C was not observed. Serotyping revealed 4 avian C. psittaci serovars (A, B, C and D). None of the 60 isolates was typed as serovar E. AluI restriction patterns A, B, D and E corresponded in 98% of the cases to serovars A, B, C and D, respectively. One isolate, classified as serovar A, generated restriction pattern F instead of A. Genotyping enabled a more precise differentiation of avian C. psittaci serovar A strains. Serovar A strains were divided into two groups according to their Alul restriction pattern (A or F). For epidemiological studies, genotyping can thus be a highly valuable alternative to serotyping, especially when applied directly to the clinical samples.

Animals↗

PubMed2000

A new theoretical model to characterize the densification behavior of tableting materials.

The purpose of the study was to develop a new three-dimensional model using force, time and displacement to characterize the densification behavior of tableting materials. Normalized time (x), displacement converted to ln(1/1 - D(rel)) according to Heckel (y) and force presented as pressure (z) were used to plot a graph. A twisted plane was fitted to this three-dimensional plot. This plane was characterized by three parameters d, the slope over time called 'time plasticity', e, the slope over pressure called 'pressure plasticity' and omega, the angle of rotation called 'fast elastic decompression'. These parameters were used to characterize the densification behavior of the well-known materials microcrystalline cellulose, dicalcium phosphate dihydrate, theophylline monohydrate, cellulose acetate and hydroxypropyl methylcellulose at different rho(rel, max). It could be shown that brittle, elastic and plastic compression properties could be very well distinguished and differentiated. Further on, it could be shown whether these properties were due to pressure or time. Thus this model has the prevailing advantage to characterize tableting materials in one step according to time and pressure and it is a useful tool to develop tablet formulations or new excipients.

Algorithms↗

PubMed2003

Characterizing volume and surface deformations in an atlas framework: theory, applications, and implementation.

Given deformations for mapping images or surfaces into an atlas configuration, methods are described for characterizing the mean deformation and deviations from this mean. Jacobian matrices are used to characterize the deformations locally, and the method can be applied to any image warping method for which Jacobian matrices can be computed. The method makes use of the fact that each matrix descriptor of the local deformation required to match an image to the atlas corresponds to a point on a semi-Riemannian manifold. By assuring that the mean matrix lies within this manifold, fundamental geometric properties common to all of the images can be preserved. Local deviations from the mean can be characterized in a euclidean space tangent to the semi-Riemannian manifold at the mean and can be accumulated globally across multiple sampling locations within the atlas to generate a global multivariate characterization of how each image deviates from the mean.

Algorithms↗

PubMed2003

US characterization of focal hepatic lesions with intermittent high-acoustic-power mode and contrast material.

RATIONALE AND OBJECTIVES: This study was performed to determine whether ultrasound (US) performed with SonoVue, a contrast agent that contains microbubbles filled with sulfur hexafluoride vapor, depicts differential patterns of contrast enhancement in focal hepatic lesions. MATERIALS AND METHODS: Forty focal hepatic lesions (15 hepatocellular carcinomas [HCCs], 10 metastases, 11 hemangiomas, and four focal nodular hyperplasias) in 39 patients were evaluated by means of US, color Doppler US, and contrast-enhanced US performed by using intermittent high-acoustic-power mode. Contrast-enhanced helical computed tomography (11 patients) and US-guided fine needle aspiration (28 patients) were used as reference procedures. Contrast enhancement patterns were defined by means of both subjective and objective analysis, and baseline and contrast-enhanced US scans were reviewed offline. RESULTS: Thirteen of 15 HCCs, eight of 10 metastases, and all four hemangiomas with an atypical pattern at baseline US were correctly characterized after SonoVue injection. Two of 15 HCCs and two of 10 metastases remained indeterminate, with no characteristic baseline or contrast-enhanced patterns identified. Baseline US was essential in characterizing all hemangiomas with a typical pattern (n = 7), and color Doppler US with spectral analysis of tumoral vessels was essential in characterizing focal nodular hyperplasia. The percentage of diagnostic agreement with reference procedures was significantly increased (P < .001) for contrast-enhanced US compared with baseline US. CONCLUSION: Characteristic patterns of US contrast enhancement with SonoVue help in characterizing and differentiating focal hepatic lesions.

Carcinoma, Hepatocellular↗

PubMed2002

Positron emission tomography and drug discovery: contributions to the understanding of pharmacokinetics, mechanism of action and disease state characterization.

As an imaging modality, positron emission tomography (PET) provides unique quantitative in vivo information of value to drug discovery studies. These non-invasive studies span the pharmacokinetic/pharmacodynamic evaluation of potential drug candidates, receptor occupancy as an important determinant of efficacy, the pharmacological characterization of potential mechanisms of action, and the biological characterization of disease with well-characterized PET ligands. PET techniques are also being applied to the assessment of gene-level activities and the longitudinal evaluation of disease progression and therapeutic intervention. As the availability of PET scanners, cyclotrons, and specific PET ligands grows, the techniques highlighted in this review will become central to target validation, drug candidate selection, pharmacokinetic characterization, and clinical evaluation.

Journal Article↗

PubMedDate not supplied

Maturation of somatostatin immunoreactivity in the pigeon retina: morphological characterization and quantitative analysis.

In addition to a modulatory function, somatostatin (SS) is likely to exert a morphogenetic and/or trophic role in the developing nervous system. In this study, a mouse monoclonal antibody directed to SS was used to investigate the posthatching development of SS-immunoreactivity (SS-ir) in the pigeon retina to provide a basis for a better understanding of the role of this peptide in retinal maturation. In the adult, SS-ir was observed in amacrine cells located in the inner nuclear layer (INL) of the entire retina. Two cell types were recognized according to their morphology. They showed a differential density distribution. Cell type indicated as "adult 1" (AD1) was characterized by pear-shaped cell bodies with single primary processes directed to the inner plexiform layer (IPL) and was mostly present in the red field. In contrast, cell type indicated as "adult 2" (AD2) was characterized by round-shaped somata with 1-3 primary processes and was highly represented in the fovea and the dorsal periphery. Posthatching maturation of the pigeon retina was characterized by drastic changes in the pattern of SS-ir. Over the first days posthatching, SS-ir was observed in sparsely distributed somata mostly located in the ganglion cell layer (GCL). This cell type indicated as "hatch" (H) was characterized by dense granular staining and became extremely rare at 7 days. Over the same period, growing SS-positive axons displaying enlarged growth cones were found in the optic tract (TrO). These observations suggest the possibility that ganglion cells transiently expressing SS are present at early stages of posthatching development. Of the two types of SS-containing cells observed in the adult, the first to be recognized morphologically was cell type AD1 which appeared at 2 days after hatching in the INL. These cells were virtually adult-like in morphology by 7 days. In contrast, cell type AD2 was not apparent until 7 days posthatching. The density (defined as number of cells/mm2 of retinal tissue) and the total number of SS-containing cells changed during posthatching maturation. In particular, the adult number of cell type AD1 was reached at about 10 days, while the number of cell type AD2 was reached at about 3 weeks posthatching. At this stage, both cell types also displayed their mature density distribution. The present findings suggest a temporal relationship between the maturation of SS-ir and developmental events which include the onset of light-driven activity and the maturation of retinal acuity.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗

PubMed2004

Advanced electron microscopy characterization of nanostructured heterogeneous catalysts.

Heterogeneous catalysis is one of the oldest nanosciences. Although model catalysts can be designed, synthesized, and, to a certain degree, characterized, industrial heterogeneous catalysts are often chemically and physically complex systems that have been developed through many years of catalytic art, technology, and science. The preparation of commercial catalysts is generally not well controlled and is often based on accumulated experiences. Catalyst characterization is thus critical to developing new catalysts with better activity, selectivity, and/or stability. Advanced electron microscopy, among many characterization techniques, can provide useful information for the fundamental understanding of heterogeneous catalysis and for guiding the development of industrial catalysts. In this article, we discuss the recent developments in applying advanced electron microscopy techniques to characterizing model and industrial heterogeneous catalysts. The importance of understanding the catalyst nanostructure and the challenges and opportunities of advanced electron microscopy in developing nanostructured catalysts are also discussed.

Catalysis↗

PubMed2001

Automated tandem mass spectrometry by orthogonal acceleration TOF data acquisition and simultaneous magnet scanning for the characterization of petroleum mixtures.

The automated acquisition of the product ion spectra of all precursor ions in a selected mass range by using a magnetic sector/orthogonal acceleration time-of-flight (oa-TOF) tandem mass spectrometer for the characterization of complex petroleum mixtures is reported. Product ion spectra are obtained by rapid oa-TOF data acquisition and simultaneous scanning of the magnet. An analog signal generator is used for the scanning of the magnet. Slow magnet scanning rates permit the accurate profiling of precursor ion peaks and the acquisition of product ion spectra for all isobaric ion species. The ability of the instrument to perform both high- and low-energy collisional activation experiments provides access to a large number of dissociation pathways useful for the characterization of precursor ions. Examples are given that illustrate the capability of the method for the characterization of representative petroleum mixtures. The structural information obtained by the automated MS/MS experiment is used in combination with high-resolution accurate mass measurement results to characterize unknown components in a polar extract of a refinery product. The exhaustive mapping of all precursor ions in representative naphtha and middle-distillate fractions is presented. Sets of isobaric ion species are separated and their structures are identified by interpretation from first principles or by comparison with standard 70-eV EI libraries of spectra. The utility of the method increases with the complexity of the samples.

Journal Article↗

PubMedDate not supplied

Dendritic iodinated contrast agents with PEG-cores for CT imaging: synthesis and preliminary characterization.

The purpose of this study was to design, synthesize, and initially characterize a representative set of novel constructs for large-molecular radiographic/computed tomography (CT) contrast agents, intended for a primarily intravascular distribution. A new assembly of well-known and biocompatible components consists of paired, symmetrical dendritic polylysines initiated from both ends of a poly(ethylene glycol) (PEG) core, yielding an array of multiple free amino groups to which were conjugated highly soluble and stable triiodophthalamide ("triiodo") moieties. An array of six dendritic contrast agents was synthesized originally, using three different PEG cores (3, 6, 12 kDa) with t-Boc lysine-generated dendrimer "amplifiers" (from three to five generations) containing 16 to 64 amino groups for conjugation with reactive triiodo moieties. A clinically used, nonionic, small molecular CT contrast agent, iobitridol, was derivatized via a hydroxyl protection/deprotection strategy, introducing a new carboxyl group available for conjugation to the lysine amino groups of dendrimers. Final products were purified by size exclusion chromatography and characterized by NMR, UV, HPLC, and elemental analysis. Preliminary evaluations were conducted for physicochemical characterization and in vivo CT contrast enhancement in a rat model. All six iodinated PEG-core dendrimer conjugates were synthesized in good yields, with a high degree of size monodispersity, large apparent molecular weight, favored physicochemical properties. A representative compound, PEG12000-carbamate-Gen4-IOB conjugate, 27% (w%) rich in iodine, demonstrated a desirable strong and persistent intravascular enhancement with a monoexponential blood half-life of approximately 35 min assayed by dynamic CT imaging and also showed high water solubility (>550 mg/mL at 25 degrees C), large apparent molecular size (comparable to a 143-kDa protein), high hydrophilicity (butanol-water partition coefficient 0.015), and stability to autoclaving conditions. This study showed the synthetic feasibility, desired basic characteristics, and potential utility for CT contrast enhancement achieved with a new type of iodinated, large-molecular PEG-core dendritic construct. Further development of this class of macromolecular contrast agents will be required to define the optimal formulation, pharmacology, safety profile, and the full range of diagnostic applications including tumor microvascular quantitative characterization by CT imaging.

Chromatography, High Pressure Liquid↗

PubMedDate not supplied

Rapid characterization of chemically-modified proteins by electrospray mass spectrometry.

Electrospray mass spectrometry (ESI-MS) has been used to examine monoclonal antibodies (MAbs), antibody fragments (Fab and Fc), modified fragments, and a range of other chemically-modified proteins as part of a study aimed at establishing ESI-MS as a method for the characterization of radioimmunoconjugates. This has been approached from two angles. Firstly, ESI-MS of complexes formed between chelators and other small molecules conjugated to hen egg white lysozyme (HEL) (14 kDa) demonstrate the considerable advantages of this powerful new technique compared with existing methods for the characterization of chemically-conjugated proteins. Molecular weights can be determined rapidly to within 0.01-0.05% and with good sensitivity (10-50 pmol total), thus providing specific structural information and opening the way for ESI-MS to be applied widely for the structural characterization of radioimmunoconjugates. Secondly, the conditions for ESI-MS of intact antibodies and antibody fragments have been examined in detail, and we have shown that the addition of up to 10 biotin molecules to the 50 kDa Fab fragment can be easily detected in ESI mass spectra, thus demonstrating the potential for the characterization of modified MAb fragments and metabolites. Finally, the strengths and limitations of ESI-MS of intact antibodies are discussed, and these results indicate that it may only be possible to detect average shifts in the mass of intact antibodies following modification.

Animals↗