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Biomedical subjects

R M Caprioli

Publications and source records attributed to R M Caprioli.

At least 37 records · Page 2Linked to original sources
PubMed1997

Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) has been used to generate ion images of samples in one or more mass-to-charge (m/z) values, providing the capability of mapping specific molecules to two-dimensional coordinates of the original sample. The high sensitivity of the technique (low-femtomole to attomole levels for proteins and peptides) allows the study of organized biochemical processes occurring in, for example, mammalian tissue sections. The mass spectrometer is used to determine the molecular weights of the molecular in the surface layers of the tissue. Molecules desorbed from the sample typically are singly protonated, giving an ion at (M + H)+, where M is the molecular mass. The procedure involves coating the tissue section, or a blotted imprint of the section, with a thin layer of energy-absorbing matrix and then analyzing the sample to produce an ordered array of mass spectra, each containing nominal m/z values typically covering a range of over 50,000 Da. Images can be displayed in individual m/z values as a selected ion image, which would localize individual compounds in the tissue, or as summed ion images. MALDI ion images of tissue sections can be obtained directly from tissue slices following preparative steps, and this is demonstrated for the mapping of insulin contained in an islet in a section of rat pancreas, hormone peptides in a small area of a section of rat pituitary, and a small protein bound to the membrane of human mucosa cells. Alternatively, imprints of the tissue can be analyzed by blotting the tissue sections on specially prepared targets containing an adsorbent material, e.g., C-18 coated resin beads. Peptides and small proteins bind to the C-18 and create a positive imprint of the tissue which can then be imaged by the mass spectrometer. This is demonstrated for the MALDI ion image analysis of regions of rat splenic pancreas and for an area of rat pituitary traversing the anterior, intermediate, and posterior regions where localized peptides were mapped. In a single spectrum from the anterior/intermediate lobe of a rat pituitary print, over 50 ions corresponding to the peptides present in this tissue were observed as well as precursors, isoforms, and metabolic fragments.

Animals↗
PubMed1996

In vitro metabolism of LVV-hemorphin-7 in human plasma studied by reversed-phase high-performance liquid chromatography and micro-electrospray mass spectrometry.

In these experiments we have studied the in vitro metabolism of LVV-hemorphin-7 in human plasma by using reversed-phase high-performance liquid chromatography (RP-HPLC) in combination with micro-electrospray mass spectrometry (micro-ES-MS). Tandem mass spectrometry (MS-MS) was performed in order to verify the structure of the peptide fragments found. Incubations were performed with and without different protease inhibitors. Results showed that LVV-hemorphin-7 was metabolized from the N-terminal end of the peptide, probably by an amastatin-sensitive exopeptidase.

Amino Acid Sequence↗
PubMed1996

Reversed-phase high-performance liquid chromatography combined with tandem mass spectrometry in studies of a substance P-converting enzyme from human cerebrospinal fluid.

The application of reversed-phase HPLC in combination with micro-electrospray mass spectrometry to study a substance P (SP)-hydrolysing endoprotease in human cerebrospinal fluid (hCSF) is reported. The enzyme was partially purified from the hCSF specimens by ion-exchange chromatography and molecular sieving. During the purification procedure the enzyme activity was monitored by measuring the formation of the SP-fragment 1-7 from SP by radioimmunoassay. Regarding its behaviour upon molecular sieve chromatography, the enzyme was suggested to be associated with an apparent molecular mass of around 100 x 10(3). In subsequent experiments using the partially purified endopeptidase, the hydrolysis of SP was demonstrated by HPLC. The reaction product mixture was resolved in several components including the N-terminal fragments 1-8, 1-7 and 1-6 and the C-terminal fragment 8-11. The identity of these fragments were confirmed by tandem mass spectrometry. It was concluded that the present SP-degrading enzyme is different from those previously identified and purified from hCSF. The applied techniques were proven to be highly efficient for the recovery and identification of the released peptide products.

Animals↗
PubMed1996

Capillary electrophoresis combined with matrix-assisted laser desorption/ionization mass spectrometry; continuous sample deposition on a matrix-precoated membrane target.

Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) were combined in an off-line arrangement to provide separation and mass analysis of peptide and protein mixtures in the attomole range. A membrane target, precoated with MALDI matrix, was used for the continuous deposition of effluent exiting from a CE device. A sample track was produced by linear movement of the target during the electrophoretic separation and this track was subsequently analyzed by MALDI/MS. The technique is effective for peptides and proteins, having limits of detection (signal-to-noise >3) of about 50 amol for neurotensin (1673 Da) and 250 amol for cytochrome c (12361 Da) and apomyoglobin (16951 Da). The electrophoretic separation achieved from the membrane target, as measured by theoretical plate numbers from the mass spectrometric data, can be as high as 80-90% of that achieved by on-line UV detection under optimal conditions, although band broadening occurs and with some loss of separation efficiency. Non-volatile buffers such as 10-50 mM phosphate can also be used in the electrophoresis process and directly deposited on the membrane. The use of post-source decay techniques is shown for peptides in the CE sample track in order to obtain sequence verification. The effectiveness of this method of integration of CE and MALDI/MS is demonstrated with both peptide and protein mixtures and with the analysis of a tryptic digest of a protein.

Apoproteins↗
PubMed1995

Isolation and structural characterization of the Leishmania donovani kinetoplastid membrane protein-11, a major immunoreactive membrane glycoprotein.

A novel membrane molecule, previously observed to be co-isolated with lipophosphoglycan and called lipophosphoglycan-associated protein, has been detected in Leishmania donovani promastigotes and amastigotes. This kinetoplastid membrane protein (KMP-11) has been purified by preparative SDS/PAGE after organic solvent extraction of promastigote membranes. Isoelectric-focusing experiments indicated that this was an acidic protein with an isoelectric point of 4.8. Immunoblot analysis of subcellular fractions, together with 125I-labelling experiments, showed this molecule to be associated with the promastigote cell surface membrane. KMP-11 was expressed at a copy number similar to that of lipophosphoglycan (1 x 10(6)-2 x 10(6) molecules per cell), making this glycoprotein one of the major features on the parasite cell surface. The primary structure, less a blocked N-terminal region, was determined by automated Edman degradation of peptides derived from CNBr or enzymic fragmentation. Several post-translational modifications were also found during these studies, including an O-linked oligosaccharide and an NG-monomethylarginine functionality which was verified by m.s. Finally, a set of sequential synthetic peptides was made based on the established partial sequence allowing structural determination of two distinct antibody-binding sites for the monoclonal antibodies L98 and L157.

Amino Acid Sequence↗
PubMed1995

Specific molecular mass detection of endogenously released neuropeptides using in vivo microdialysis/mass spectrometry.

The specific molecular detection of the endogenous neuropeptides methionine ([Met]5) enkephalin and neurotensin released in vivo in rat brain has been accomplished using microdialysis and mass spectrometry. Microdialysis probes were implanted in specific brain regions and were used to collect samples from brain extracellular fluids in unanesthetized, freely moving animals. Microelectrospray/tandem mass spectrometry was used to achieve molecular-specific identification of the neuropeptides with a sensitivity in the amol/microliters range. Measurements of the amounts of neuropeptides in the dialysates obtained from studies of KCl-stimulated release showed that [Met]5-enkephalin from the globus pallidus/ventral pallidum region was present at a level of approximately 4-6 fmol/10 microliters of dialysate and neurotensin from the hypothalamus of approximately 500 amol in 10 microliters of dialysate. In this manuscript, we present the first data of a mass- and molecular-specific detection and quantitation of individual neuropeptides released in response to either intracerebrally or systemically administered compounds.

Animals↗
PubMed1995

In vivo microdialysis and gas chromatography/mass spectrometry for studies on release of N-acetylaspartlyglutamate and N-acetylaspartate in rat brain hypothalamus.

Microdialysis and gas chromatography/mass spectrometry was used for the measurement of extracellular N-acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) in rat hypothalamus. The sensitivity of the method for each of these compounds was approximately 5 pmol/30 microliters of dialysate. Baseline NAA concentrations in dialysate were estimated to be approximately 25 pmol/36 microliters, while that for NAAG was at or below the detection limit of 5 pmol/ 36 microliters. In vivo and in vitro calibrations of microdialysis probes showed that the recovery for NAA was approximately 10 percent. For NAAG, the in vitro recovery was 6.3%, and in vivo recovery, 11%. Depolarization stimulation using 100 mM KCl in the microdialysis perfusate was employed to measure extracellular NAA and NAAG concentrations. Extracellular NAA was elevated to approximately 70 pmol/36 microliters dialysate following depolarization. No significant elevation of NAAG was observed. By infusing known amounts of stable isotopically labeled NAAG-d3 via the microdialysis probe and measuring the isotopically labeled catabolic product, NAA-d3, in collected microdialysate, we were able to confirm the existence of one or more hydrolytic enzymes active towards NAAG in the hypothalamus. This finding suggest the possible involvement of active metabolic processes in the relationship between NAAG and NAA releases.

Animals↗
PubMed1991

Recent advances in liquid chromatography-mass spectrometry and capillary zone electrophoresis-mass spectrometry for protein analysis.

The utility of the combination of separations techniques, such as liquid chromatography and capillary zone electrophoresis, with mass spectrometry in applications involving protein analysis is discussed. The use of continuous-flow fast atom bombardment and electrospray ionization mass spectrometry is compared for the analysis of tryptic digests. For liquid chromatography, both microbore and slurry-packed capillary bore columns were used to separate peptides from proteolytic digests.

Chromatography, Liquid↗
PubMed1991

Identification of nearest-neighbor peptides in protease digests by mass spectrometry for construction of sequence-ordered tryptic maps.

Continuous-flow fast atom bombardment mass spectrometry was used for the identification of the intermediates and end products of the tryptic digest of polypeptides throughout the time-course of the reactions. Precursor/product relationships for these peptides were determined with the aid of a simple personal computer program. The C-terminal tryptic peptides were identified by performing a tryptic digest in 50% 18O-enriched buffer which resulted in labeling of all non-C-terminal peptides with 18O. This information, along with the precursor/product correlations, was used to create a sequence-ordered tryptic map of the original polypeptide. Ion intensities of intermediate hydrolysis products are compared for enzyme to substrate ratios of 1:100 and 1:1000 (w/w) over the course of the reaction. Intermediates were found to have significantly longer lifetimes when lower levels of trypsin were present.

Amino Acid Sequence↗
PubMed1991

Assessing the multimeric states of proteins: studies using laser desorption mass spectrometry.

We have developed a technique which utilizes matrix-assisted laser desorption mass spectrometry to study the subunit association of proteins. Aqueous protein samples are treated with a dilute solution of glutaraldehyde, a cross-linking agent which reacts with free amino groups on proteins. This agent effectively traps the multimeric form, preventing it from dissociating in the sample preparation and desorption process. Proteins measured include lysozyme, carbonic anhydrase, apomyoglobin, glucose 6-phosphate dehydrogenase, ovine lutropin, yeast alcohol dehydrogenase, avidin and pyruvate kinase. Dimeric and tetrameric complexes up to 250,000 Da have been measured in this manner.

Alcohol Dehydrogenase↗
PubMed1990

On-line analysis of penicillin blood levels in the live rat by combined microdialysis/fast-atom bombardment mass spectrometry.

The combination of microdialysis and fast-atom bombardment mass spectrometry has been used to follow the pharmacokinetics of penicillin G directly in the blood-stream of a live rat. After the intramuscular injection of the antibiotic, the blood dialysate was allowed to flow into the mass spectrometer via the continuous-flow/fast-atom bombardment interface. Tandem mass spectrometry provided the means for isolating and recording the ion fragments produced from the drug as the dialysate was exposed to the ionization process. The pharmacokinetic results obtained are compared with those previously reported in the literature, and this on-line method is discussed in terms of its particular advantages.

Animals↗
PubMed1989

Coupling capillary zone electrophoresis and continuous-flow fast atom bombardment mass spectrometry for the analysis of peptide mixtures.

Combined capillary zone electrophoresis (CZE)-continuous-flow fast atom bombardment (CF-FAB) mass spectrometry is described for the analysis of mixtures of peptides. A 90 cm x 50 microns I.D. fused-silica capillary column was used for electrophoretic separations and was connected to the CF-FAB probe via an interface which allows a total flow into the mass spectrometer of about 5 microliters/min. Solutions of peptides were pneumatically loaded onto the CZE capillary, providing sample amounts of 0.1-20 pmol. The magnetic mass spectrometer was scanned over the desired mass range, usually between m/z 500 and 2500. Results are shown for separation and analysis of mixtures of synthetic peptides and also for protease digests of recombinant human growth hormone and horse heart cytochrome c.

Electrophoresis↗
PubMed1989

Direct analysis of drugs by continuous-flow fast-atom bombardment and tandem mass spectrometry.

The techniques of continuous-flow fast-atom bombardment (CF-FAB) and tandem mass spectrometry (MS/MS) are combined and applied to the analysis of small molecular mass drugs (mol.wt less than 500 Da). The approach involves the interfacing of a CF-FAB inlet with a triple-stage quadrupole mass spectrometer, enabling the acquisition of collision-activated decomposition mass spectra of the drugs after FAB ionization. The relationship between a stable sample surface on the CF-FAB probe tip and the quality of the mass spectrum is discussed, as are practical methods for obtaining and maintaining surface stability. CF-FAB MS/MS spectra for several drugs are presented, including penicillin G, phentolamine, cocaine and benzoylecgonine. Minimum detection limits range from 50-500 pg injected, depending on the compound. The reproducibility of the integrated areas of peaks from repetitive injections is approximately five per cent. Data are also presented for the direct CF-FAB MS/MS analysis of cocaine and benzoylecgonine in spiked urine samples.

Cocaine↗
PubMed1989

Synthesis of phosphopeptides containing O-phosphoserine or O-phosphothreonine.

Peptides containing phosphoserine or phosphothreonine were synthesized by solid phase methods. Phosphoserine and phosphothreonine were incorporated into peptides using Boc-diphenylphosphono esters of serine and threonine and standard DCC/HOBt coupling. The phenylphosphoesters were not removed when the peptides were cleaved from the resin by HF or by trifluoromethane sulfonic acid, but were subsequently removed by catalytic hydrogenation. Phosphopeptides were purified by HPLC and by Fe+3-Chelex chromatography and their identity verified by mass spectrometry. Two peptides, Leu-Arg-Arg-Ala-Ser(P)-Leu-Gly and Leu-Arg-Arg-Ala-Thr(P)-Leu-Gly, were prepared by both enzymatic and chemical methods and had identical properties.

Amino Acids↗