Biomedical subjects

D M Lubman

Publications and source records attributed to D M Lubman.

At least 37 records · Page 2Linked to original sources

PubMed1998

On-probe immunoaffinity extraction by matrix-assisted laser desorption/ionization mass spectrometry.

A simple and effective method has been proposed in this work for combination of immunoaffinity extraction with MALDI MS. In this method, an antibody is attached to the surface of a MALDI probe tip via a thin nitrocellulose film. This allows the corresponding antigen to be selectively captured and concentrated on the probe tip from complex plasma solution for MALDI MS analysis. The whole procedure can be completed within 1 h. This combination offers several excellent performance features in the analysis of SNX-111, a therapeutic peptide. It combines the high specificity of affinity chromatography with the high sensitivity of mass spectrometry in a rapid analysis. Direct mass detection provides unambiguous determination by the observation of signals at characteristic m/z values. This method has been used successfully to determine the therapeutic peptide at relevant doses.

Amino Acid Sequence↗

PubMed1998

On-line analysis by capillary separations interfaced to an ion trap storage/reflectron time-of-flight mass spectrometer.

The interface of high-resolution capillary separation methods to time-of-flight mass spectrometry (TOF-MS) has generated considerable interest since TOF can provide the rapid and sensitive detection required by high resolution separations. In recent years, our laboratory has developed a variety of high-resolution capillary separation methods interfaced to TOF-MS via an ion trap storage/reflectron time-of-flight (IT/reTOF) instrument. Using this hybrid configuration, detection of fast separations at very low detection levels has been successfully performed for on-line separations of peptides and protein digests using electrospray ionization. In this report, we review the current status in our laboratory of interfacing high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography to an IT/reTOF-MS instrument and various applications that have been developed involving this technology.

Chromatography, High Pressure Liquid↗

PubMed1998

Genotyping of apolipoprotein E by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Apolipoproteins E↗

PubMed1998

Protein profiles and identification of high performance liquid chromatography isolated proteins of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been used to rapidly profile the protein content of human cell lysates from MCF-10 cell and variant lines. The method was used to study the protein profiles of these cells as they progressed from normal breast epithelium to fully malignant cells. Distinct differences in the protein profiles were observed with progression, and specific proteins associated with carcinogenesis (p53, c-myc, and c-erbB-2) were heavily expressed in these cells as detected by MALDI-TOFMS. These proteins were also isolated using non-porous reversed-phase high performance liquid chromatography (NP-RP-HPLC) and mass analyzed by MALDI-TOFMS to provide molecular weight information without interference from other proteins in the whole cell lysates, and to avoid suppression effects in mixtures of proteins detected by MALDI-TOFMS. In order to confirm the identity of these oncoproteins, the cell lysates were subjected to one-dimensional (1-D) gel separation and subsequently electroblotted onto a poly(vinylidene difluoride) (PVDF) membrane for further analysis. Trypsin and cyanogen bromide digestions were performed on these proteins eluted from excised PVDF bands which were then analyzed by MALDI-TOFMS. The identity of these proteins was confirmed by database matching procedures.

Cell Line, Transformed↗

PubMed1998

Rapid identification and screening of proteins from whole cell lysates of human erythroleukemia cells in the liquid phase, using non-porous reversed phase high-performance liquid chromatography separations of proteins followed by matrix-assisted [correction of multi-assisted] laser desorption/ionization mass spectrometry analysis and sequence database searching.

Non-porous reversed phase (NPRP) high-performance liquid chromatography (HPLC) has been used as a rapid method to separate proteins from whole cell lysates of human erythroleukemia (HEL) cells. Using phosphate-buffered saline (PBS) as a lysis buffer to extract proteins from HEL cells, more than 100 proteins of molecular weight up to 30 kDa were separated by the NPRP HPLC method, using a programmed acetonitrile:H2O gradient. The separated proteins were collected as liquid fractions as they eluted, and were further separated on the NPRP column with a different gradient to separate coeluting peaks. The isolated protein fractions were analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to determine the molecular weight of the protein. The proteins were cleaved by chemical or enzymatic digestion to produce peptide maps, which were analyzed by pulsed delayed extraction MALDI-MS. The peptide maps were matched against a database search to determine the protein identity. In some cases, several enzymes were used in order to find exactly one match against the database. This methodology is demonstrated for several proteins isolated from HEL cells and identified via database matching.

Calmodulin↗

PubMed1998

Development of a three-dimensional topographic map display for capillary electrophoresis/mass spectrometry with an ion trap/reflectron time-of-flight mass spectrometer detector: applications to tryptic digests of isoforms of myelin basic protein.

A three-dimensional (3-D) contour map format has been developed to display the large amount of data continuously collected throughout an on-line capillary separation using an ion trap storage/reflectron time-of-flight detector (IT/reTOF). The resulting data are displayed on a single computer screen with a mass-to-charge ratio value-elution time-intensity representation. The intensity of various components is represented by 16 different colors so that the mass-to-charge ratio value, the elution time, and the intensity can be conveniently determined for each component. In addition, the mass spectrum and total ion chromatogram or total ion electropherogram (TIE) are shown on the same screen as the 3-D map that enables the correlation of a single spot in the 3-D map to the peaks in the TIE and the corresponding mass spectrum. The 3-D map has been used to identify various posttranslational modification sites of bovine myelin basic protein charge isomers, where the datafiles of tryptic digests of proteins analyzed by capillary electrophoresis/mass spectrometry were processed by this software and a comparison could be performed among the isoforms. The feature of in-screen integration over both the separation domain and the mass domain makes the acquisition of the selected ion chromatogram very convenient and greatly improves the ability to detect modified components present in low amounts.

Amino Acid Sequence↗

PubMed1997

Protein digest analysis by pressurized capillary electrochromatography using an ion trap storage/reflectron time-of-flight mass detector.

Pressurized capillary electrochromatography (pCEC) has been coupled to an ion trap storage/reflectron time-of-flight mass spectrometer for the analysis of peptide mixtures and protein digests. Taking advantage of the electroosmotic flow, high separation efficiency has been achieved in pCEC due to a relatively flat flow profile and the use of smaller packing materials. A supplementary pressure was used in these experiments which suppressed bubble formation and also allowed the tuning of the elution of peptides using the electrical field. In this work, a fast separation of a six-peptide mixture has been successfully performed. Using columns only 6 cm long, a tryptic digest of bovine cytochrome c was fully separated in around 14 min by properly tuning the applied voltage and the supplementary pressure. In addition, relatively complex protein digests, such as a tryptic digest of chicken ovalbumin, were analyzed using this pCEC/MS system, and more than 20 peaks were resolved in the total ion current chromatogram within 17 min. The use of an ion trap storage/reflectron time-of-flight mass spectrometer as an on-line detector further increased the resolving power of the pCEC by unambiguously identifying coeluting components. The nonscanning property of the time-of flight mass analyzer and the ion signal integration capability of the ion trap were successfully combined to provide rapid and sensitive full-mass range detection in these experiments.

Amino Acid Sequence↗

PubMed1997

Use of a polybrene capillary coating in capillary electrophoresis for rapid analysis of hemoglobin variants with on-line detection via an ion trap storage/reflectron time-of-flight mass spectrometer.

A polybrene capillary coating in capillary electrophoresis (CE) has been used for rapid analysis of hemoglobin variant digests. The use of the polybrene capillary coating has allowed sufficient separation to resolve the large number of digest products formed upon tryptic digestion of the whole protein, so that prior separation of the hemoglobin alpha and beta chains is not required. The resolution of the digest peaks obtained by CE is sufficient so that even single amino acid substitutions can easily be detected using UV absorption detection. The digest is further analyzed by capillary electrophoresis separation with on-line detection using electrospray ionization interfaced to the ion trap storage/reflectron time of flight device (CE/ESI-IT/reTOF), where a comparison of the total ion electropherograms and mass spectra of the mutant and normal hemoglobins can detect the presence of a mutation site. The CE separation and mass analysis can be accomplished in typically 10-15 min. The unique capability of the CE/ESI-IT/reTOF system for detection of fast separations with narrow peaks that may be under 1 s fwhm is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.

Adult↗

PubMed1997

Open-tubular capillary electrochromatography with an on-line ion trap storage/reflectron time-of-flight mass detector for ultrafast peptide mixture analysis.

In this work, a novel open-tubular column (OTC) capillary electrochromatography (CEC) system has been coupled to an on-line ion trap storage/reflectron time-of-flight mass spectrometer for ultrafast peptide mixture analysis. Reversed-phase OTCs prepared by the sol-gel process were coated with an amine, which greatly enhanced the electroosmotic flow in an acidic buffer solution and considerably reduced nonspecific adsorption between the peptides and the column wall. A six-peptide mixture could be separated to baseline within 3 min on this system. A full mass range detection speed of 8 Hz was used in all these experiments, which was sufficiently rapid to maintain the high efficiency of ultrafast separations. Because of the high duty cycle of the mass spectrometer and the column path length-independent concentration-sensitive feature of the electrospray ionization process, high-quality total ion chromatograms could be obtained with injections of only 1-2 fmol of peptide samples. A concentration limit of detection of 1 x 10(-6) M was also achieved due to the preconcentration capability of CEC. In addition, a novel gradient CEC device was demonstrated which did not result in a pressure-driven flow. A tryptic digest of horse heart myoglobin was successfully separated on the gradient CEC system within 6 min. The use of the mass spectrometer increased the resolving power of this system by clearly identifying coeluting components.

Amino Acid Sequence↗

PubMed1997

The use of on-line capillary electrophoresis/electrospray ionization with detection via an ion trap storage/reflectron time-of-flight mass spectrometer for rapid mutation-site analysis of hemoglobin variants.

Capillary electrophoresis/electrospray ionization using an ion trap storage/reflectron time-of-flight mass spectrometer detector (CE/ESI-IT/reTOF) is used to provide a rapid and sensitive method for analyzing structural variants in the hemoglobin (Hb) beta-chain. The Hb alpha- and beta-chains are separated and the beta-chain is digested by trypsin. The digest is analyzed by CE/ESI-IT/reTOF where a comparison of the total ion electrophorograms and mass spectra of the mutant and normal hemoglobins (Hbs) can detect the presence of a mutation site. In addition, collision-induced dissociation in the vacuum interface-skimmer region can be used to pinpoint the identity of such a site. The unique capability of the CE/ESI-IT/reTOF system for accurately detecting fast separations with narrow peaks that may be under 1 s full width at half maximum is demonstrated. The speed of this system is essential for resolution of the large number of peaks that are separated in a short time duration using CE separations.

Amino Acid Sequence↗

PubMed1997

Improved resolution in the detection of oligonucleotides up to 60-mers in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using pulsed-delayed extraction with a simple high voltage transistor switch.

Pulsed-delayed extraction using a simple high voltage transistor switch together with various sample purification approaches were used to enhance the resolution in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of oligonucleotides up to 60 bases long. This switch can provide a 0-3 kV voltage pulse with a 75 ns fall time. A resolution of 500-900 was typically observed for samples from 5-mers to 60-mers using pulsed-delayed extraction (PDE) with the switch described herein. The resolution deteriorated to < 100 for oligonucleotides of > or = 65-mers. With the TOF acceleration region configuration used in this work, the resolution was found not to vary significantly over a delay range of 2-5 mus. As the DNA size increased to over 35-mer, HPLC purification was required to retain the enhancement in resolution provided by PDE MALDI-MS.

Chromatography, High Pressure Liquid↗

PubMed1997

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as a rapid screening method to detect mutations causing Tay-Sachs disease.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used as a rapid method for the detection of human genetic polymorphisms. In particular, the mutations in the human HEXA gene that cause the infantile Tay-Sachs disease have been studied using MALDI-MS to demonstrate the feasibility of this technique for use in clinical and diagnostic analysis. The protocols involved in this approach include, polymerase chain reaction for the amplification of the mutation site from buccal cell DNA, followed by restriction enzyme digestion of the amplified regions of the template cells. The products of amplification and digestion were studied using MALDI-MS. MALDI-MS experiments are shown to provide essentially the same information as obtained from gel electrophoresis but orders of magnitude faster.

Base Sequence↗

PubMed1997

Detection of oligonucleotides by external injection into an ion trap storage/reflection time-of-flight device.

The detection of oligonucleotides has been studied using external injection of Matrix-assisted laser desorption/ionization (MALDI) produced ions into a quadrupole ion trap storage device. The ions stored in the trap are then detected by pulsed DC injection into a reflection time-of-flight mass spectrometer. It is shown that by using a low acceleration voltage from the probe tip and external injection into the trap that 3-mers to 9-mers can be stored for 20-50 ms and detected as intact ions. In comparison, direct MALDI inside the trap resulted in extensive fragmentation as a function of storage time. Using the external injection with a He buffer gas in the trap yields a resolution of 250-400 in the ion trap storage/reflectron time-of-flight mass spectrometer. The resolution can be further enhanced to 400-800 using a bipolar extraction method. In addition, the trapping efficiency for the injection of externally produced ions into the trap can be enhanced using a dynamic trapping method. The MALDI mass spectra produced by this methodology result in excellent signal-to-noise ratio due to the capabilities of the trap for eliminating low mass matrix background from the spectra.

Electronic Data Processing↗

PubMed1997

Simulation of external ion injection, cooling and extraction processes with SIMION 6.0 for the ion trap/reflectron time-of-flight mass spectrometer.

In this work we have developed a PC-based simulation to study ion injection, cooling and extraction processes for multiple ions in an trap/reflectron time-of-flight (IT/reTOF) system. This simulation is based upon SIMION 6.0 with user written programs in which a 3D collision model is used to describe ion--buffer gas molecule interactions. The results of various simulations describing the relation between the trapping efficiency for external injection of ions into the trap and the RF phase, and the effects of initial kinetic energy and ramp-up rate on dynamic trapping of externally produced ions are discussed. Further, single-pulsing and bipolar-pulsing schemes for ejecting ions from the trap are examined. The simulations show that bipolar pulsing can markedly improve the resolution. In the bipolar ejection mode the relation between resolution and the extraction voltages and RF ramp-off rate are studied.

Buffers↗

PubMed1997

Development of a capillary high-performance liquid chromatography tandem mass spectrometry system using SWIFT technology in an ion trap/reflectron time-of-flight mass spectrometer.

In this work a capillary high-performance liquid chromatography/tandem mass spectrometry system has been developed in a quadrupole ion trap/reflectron time-of-flight mass spectrometer. The broadband excitation for isolating a precursor ion is achieved by generating the stored waveform inverse Fourier transform waveform in real time. Following the isolation and cooling periods, an auxiliary AC voltage (tickle) is applied to the entrance end cap of the ion trap to generate collision induced dissociation. The amplitude of the tickle voltage is optimized on-line using a feedback method which involves having two computers communicating with each other. This system has been tested with a 4-peptide mixture and tandem mass spectra were obtained for each peptide.

Chromatography, High Pressure Liquid↗

PubMed1997

Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used to rapidly detect and profile large proteins from Escherichia coli whole cell lysates in the mass range 25-500 kDa. The bacterial samples were treated with guanidine hydrochloride and Triton X-100 to disrupt and solubilize the large inner membrane proteins. A sample preparation involving a nitrocellulose polymer film, and alpha-cyano-4-hydroxycinnamic acid, sinapinic acid or caffeic acid as matrix was utilized to rapidly monitor the presence of induced and repressed protein synthesis in response to L-arabinose catabolism in E. coli cells. The results were compared to those of 1-D or 2-D gel electrophoresis.

Bacterial Proteins↗

PubMed1996

Use of an ion trap storage/reflectron time-of-flight mass spectrometer as a rapid and sensitive detector for capillary electrophoresis in protein digest analysis.

An ion trap storage (IT)/reflectron time-of-flight mass spectrometer (reTOFMS) has been coupled to capillary electrophoresis (CE) via a sheathless microelectrospray ionization method. This hybrid mass spectrometer has proved to be a rapid and sensitive detector for CE, where mass spectra could be acquired at a speed sufficient to maintain the high-resolution capabilities of CE separations. The nonscanning property of the time-of-flight mass analyzer can provide a full mass range spectral acquisition speed of up to 25 spectra/s with a data system developed in our laboratory. For the work reported herein, a spectral acquisition speed of 4 spectra/s was found to be optimal for maintaining the quality of the separation while achieving high sensitivity. Tryptic digests of bovine cytochrome c and beta-lactoglobulin A were analyzed using the CE/IT/reTOFMS combination, resulting in total ion electropherograms similar to those obtained using UV absorption detection. Taking advantage of the ion storage capability of the ion trap, a detection limit in the lowfemtomole range was routinely obtained for these digests using the total ion electrophoretic mode and CE capillaries of typical dimensions (41 microns i.d.). This high sensitivity was achieved while maintaining a resolution of approximately 1500 for mass identification using the capabilities of the IT/reTOF device. Due to the high acquisition speed and the mass discrimination capabilities of the mass detector, all the peaks in the total ion electropherograms, including some totally or partially unresolved peaks, could be unambiguously identified.

Amino Acid Sequence↗

PubMed1996

Characterization of SDS--PAGE-separated proteins by matrix-assisted laser desorption/ionization mass spectrometry.

A new strategy to characterize SDS--PAGE-separated proteins with MALDI MS is described. The proteins, electroblotted onto nitrocellulose after SDS--PAGE separation and stained with reversible Ponceau S dye, are readily recovered by dissolving the membrane in matrix solutions prepared with acetone. The resulting mixtures are amenable to direct MALDI MS analysis, which provides a rapid and accurate means of measuring the molecular weights of SDS--PAGE-separated proteins and of peptides that result from CNBr digestion of proteins on the nitrocellulose membrane. Compared with the traditional elution method, this procedure provides more efficient detection of proteins and peptides, especially the higher molecular weight proteins from the membrane. As little as 3.5 pmol of lysozyme and 15 pmol of bovine albumin loaded onto a gel can be detected using this method. The detection sensitivity is higher than or comparable to that of the traditional Coomassie Brilliant Blue staining procedure.

Electrophoresis, Polyacrylamide Gel↗