Biomedical subjects

L Pasa-Tolić

Publications and source records attributed to L Pasa-Tolić.

At least 19 recordsLinked to original sources

PubMed2003

Nanoscale proteomics.

Efforts to develop a liquid chromatography (LC)/mass spectrometry (MS) technology for ultra-sensitive proteomics studies (i.e., nanoscale proteomics) are described. The approach combines high-efficiency nanoscale LC (separation peak capacity of approximately 10(3); 15-microm-i.d. packed capillaries with flow rates of 20 nL min(-1), the optimal separation linear velocity) with advanced MS, including high-sensitivity and high-resolution Fourier transform ion cyclotron resonance MS, to perform both single-stage MS and tandem MS (MS/MS) proteomic analyses. The technology enables broad protein identification from nanogram-size proteomics samples and allows the characterization of more abundant proteins from sub-picogram-size samples. Protein identification in such studies using MS is demonstrated from <75 zeptomole of a protein. The average proteome measurement throughput is approximately 50 proteins h(-1) using MS/MS during separations, presently requiring approximately 3 h sample(-1). Greater throughput (approximately 300 proteins h(-1)) and improved detection limits providing more comprehensive proteome coverage can be obtained by using the "accurate mass and time" tag approach developed in our laboratory. This approach provides a dynamic range of at least 10(6) for protein relative abundances and an improved basis for quantitation. These capabilities lay the foundation for studies from single or limited numbers of cells.

Amino Acid Sequence↗

PubMed2001

Basicity of the amino groups of the aminoglycoside amikacin using capillary electrophoresis and coupled CE-MS-MS techniques.

This paper describes the use of capillary electrophoresis (CE), and coupled CE and mass spectrometric techniques, to measure the values of the pKa of the amino groups of the aminoglycoside antibiotic amikacin and of its acetylated derivatives. These values of pKa (8.4, 6.7, 9.7, 8.4) were determined by measuring the electrophoretic mobilities of the molecules as a function of pH; they are within 0.7 unit of certain values reported in the literature (by 13C and 15N NMR spectroscopies) but resolved ambiguities left by these earlier studies. The range of values of pKa of amino groups also indicates the complex dependence of the acidity of a functional group (and thus the extent of ionization at a specified value of pH) on the molecular environment of that group.

Acetylation↗

PubMed2001

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography.

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Amino Acid Sequence↗

PubMed2001

High-throughput proteomics using high-efficiency multiple-capillary liquid chromatography with on-line high-performance ESI FTICR mass spectrometry.

We report on the design and application of a high-efficiency multiple-capillary liquid chromatography (LC) system for high-throughput proteome analysis. The multiple-capillary LC system using commercial LC pumps was operated at a pressure of 10,000 psi to deliver mobile phases through a novel passive feedback valve arrangement that permitted mobile-phase flow path switching and efficient sample introduction. The multiple-capillary LC system uses several serially connected dual-capillary column devices. The dual-capillary column approach eliminates the time delays for column regeneration (or equilibration) since one capillary column was used for a separation while the other was being washed. Several serially connected dual-capillary columns and electrospray ionization (ESI) sources were operated independently and can be used either for "backup" operation or for parallel operation with other mass spectrometers. This high-efficiency multiple-capillary LC system utilizes switching valves for all operations, enabling automated operation. The separation efficiency of the dual-capillary column arrangement, optimal capillary dimensions (column length and packed particle size), capillary regeneration conditions, and mobile-phase compositions and their compatibility with electrospray ionization were investigated. A high magnetic field (11.4 T) Fourier transform ion cyclotron resonance (FTICR) mass spectrometer was coupled on-line with this high-efficiency multiple-capillary LC system using an ESI interface. The capillary LC provided a peak capacity of approximately 650, and the 2-D capillary LC-FTICR analysis provided a combined resolving power of > 6 x 10(7) components. For yeast cytosolic tryptic digests > 100,000 polypeptides were detected, and approximately 1,000 proteins could be characterized from a single capillary LC-FTICR analysis using the high mass measurement accuracy (approximately 1 ppm) of FTICR, and likely more if LC retention time information were also exploited for peptide identification.

Chromatography, Liquid↗

PubMed2001

Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.

We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.

Animals↗

PubMed2001

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics.

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.

Chromatography, Liquid↗

PubMed2001

Rapid quantitative measurements of proteomes by Fourier transform ion cyclotron resonance mass spectrometry.

The patterns of gene expression, post-translational modifications, protein/biomolecular interactions, and how these may be affected by changes in the environment, cannot be accurately predicted from DNA sequences. Approaches for proteome characterization are generally based upon mass spectrometric analysis of in-gel digested two dimensional polyacrylamide gel electrophoresis (2-D PAGE) separated proteins, allowing relatively rapid protein identification compared to conventional approaches. This technique, however, is constrained by the speed of the 2-D PAGE separations, the sensitivity limits intrinsic to staining necessary for protein visualization, the speed and sensitivity of subsequent mass spectrometric analyses for identification, and the limited ability for accurate quantitative measurements based on differences in spot intensity. We are presently developing alternative approaches for proteomics based upon the combination of fast capillary electrophoresis, or other suitable chromatographic separations, and the high mass accuracy and sensitivity obtainable with unique Fourier transform ion cyclotron resonance (FTICR) mass spectrometers available at our laboratory. Several approaches are presently being pursued; one based upon the analysis of intact proteins and the second upon approaches for global protein digestion and accurate peptide mass analysis. Quantitation of protein/peptide levels are based on using two or more stable-isotope labeled versions of proteomes which are combined to obtain precise quantitation of relative protein abundances. We describe the status of our efforts towards the development of a high-throughput proteomics capability and present initial results for application to several microorganisms and discuss our efforts for extending the developed capability to mammalian proteomes.

Bacterial Proteins↗

PubMed2000

Separation and detection of intact noncovalent protein complexes from mixtures by on-line capillary isoelectric focusing-mass spectrometry.

Separation and mass spectrometric analysis of intact noncovalent protein-protein complexes from mixtures is described. Protein complexes were separated using isoelectric focusing in a capillary under native conditions. During the mobilization, molecular masses of the intact complexes were measured on-line (as they emerged from the capillary) using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. An FTICR "in-trap" ion cleanup procedure was necessary for some complexes to reduce high levels of adduction and to obtain accurate molecular mass measurements. Optimization of the conditions for analysis of different intact complexes is discussed. We have shown that either the intact noncovalent complexes or their constituent protein subunits can be detected by variation of sheath liquid (i.e., NH4OAc vs HOAc) added at the electrospray-mass spectrometer interface. Thus, two successive experiments permit a fast and efficient characterization of intact complex stoichiometry, the individual complex subunits and the possible presence of metal or other adducted species.

Animals↗

PubMed2000

Utility of accurate mass tags for proteome-wide protein identification.

An enabling capability for proteomics would be the ability to study protein expression on a global scale. While several different separation and analysis options are being investigated to advance the practice of proteomics, mass spectrometry (MS) is rapidly becoming the core instrumental technology used to characterize the large number of proteins that constitute a proteome. To be most effective, proteomic measurements must be high-throughput, ideally allowing thousands of proteins to be identified on a time scale of hours. Most strategies of identification by MS rely on the analysis of enzymatically produced peptides originating from an isolated protein followed by either peptide mapping or tandem MS (MS/MS) to obtain sequence information for a single peptide. In the case of peptide mapping, several peptide masses are needed to unambiguously identify a protein with the typically achieved mass measurement accuracies (MMA). The ability to identify proteins based on the mass of a single peptide (i.e., an accurate mass tag; AMT) is proposed and is largely dependent on the MMA that can be achieved. To determine the MMA necessary to enable the use of AMTs for proteome-wide protein identification, we analyzed the predicted proteins and their tryptic fragments from Saccharomyces cerevisiae and Caenorhabditis elegans. The results show that low ppm (i.e., approximately 1 ppm) level measurements have practical utility for analysis of small proteomes. Additionally, up to 85% of the peptides predicted from these organisms can function as AMTs at sub-ppm MMA levels attainable using Fourier transform ion cyclotron resonance MS. Additional information, such as sequence constraints, should enable even more complex proteomes to be studied at more modest mass measurement accuracies. Once AMTs are established, subsequent high-throughput measurements of proteomes (e.g., after perturbations) will be greatly facilitated.

Cyclotrons↗

PubMed2000

Stepwise mobilization of focused proteins in capillary isoelectric focusing mass spectrometry.

A stepwise mobilization strategy has been developed for the elution of complex protein mixtures, separated by capillary isoelectric focusing (CIEF) for detection using on-line electrospray ionization mass spectrometry (ESI-MS). Carrier polyampholytes are used to establish a pH gradient as well as to control the electroosmotic flow arising from the use of uncoated fused-silica capillaries. Elution of focused protein zones is achieved by controlling the mobilization pressure and voltage, leaving the remaining protein zones focused inside the capillary. Protein zones are stepwise eluted from the capillary by changing the mobilization conditions. Stepwise mobilization improves separation resolution and simplifies coupling with multistage MS (i.e., MSn) analysis since it allows more effective temporal control of protein elution from the CIEF capillary. We also describe a modified configuration for coupling CIEF with ESI-MS using a coaxial sheath flow interface that facilitate the automation of on-line CIEF-ESI-MS analyses. The stepwise mobilization strategy is demonstrated for the analysis of standard protein mixtures and soluble E. coli lysate proteins using CIEF-ESI-MS. These results indicate that inlet pressure or voltage programming to control the elution of the protein zones from the capillary (i.e., gradient mobilization) may allow for the optimization of the mobilization conditions and provide higher resolution for CIEF separation of complex mixtures with on-line MS.

Electrophoresis, Capillary↗

PubMed2000

Mass spectrometric detection for capillary isoelectric focusing separations of complex protein mixtures.

Capillary isoelectric focusing (CIEF) can provide high-resolution separations of complex protein mixtures, but until recently it has primarily been used with conventional UV detection. This technique would be greatly enhanced by much more information-rich detection methods that can aid in protein characterization. We describe progress in the development of the combination of CIEF with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry and its application to proteome characterization. Studies have revealed 400-1000 putative proteins in the mass range of 2-100 kDa from total injections of approximately 300 ng protein in single CIEF-FTICR analyses of cell lysates for both Escherichia coli (E. coli) and Deinococcus radiodurans (D. radiodurans). We also demonstrate the use of isotope labeling of the cell growth media to improve mass measurement accuracy and provide a means for quantitative proteome-wide measurements of protein expression. The ability to make such comprehensive and precise measurements of differences in protein expression in response to cellular perturbations should provide new insights into complex cellular processes.

Bacterial Proteins↗

PubMed2000

Proteome analysis using selective incorporation of isotopically labeled amino acids.

A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.

Amino Acids↗

PubMed2000

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor beta and derivatives.

The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor beta (rhM-CSF beta) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (approximately 25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.

Amino Acid Sequence↗

PubMed1999

Electrospray ionization-mass spectrometry study of the interaction of cisplatin-adducted oligonucleotides with human XPA minimal binding domain protein.

Nucleotide excision repair (NER) is the process responsible for eliminating most ultraviolet (UV) radiation damage from DNA, as well as base alterations caused by a variety of mutagens. The xeroderma pigmentosum group A complementing protein (XPA) is believed to be involved in the early step of NER by recognizing and binding damaged DNA. Recent work has suggested that electrospray ionization-mass spectrometry (ESI-MS) can be an effective tool for the study of protein-DNA complexes. We have used ESI-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to examine the cisplatin-adducted oligonucleotide and its interaction with the human XPA minimal binding domain (XPA-MBD). High-resolution FTICR experiments of the binding products showed that both double-stranded damaged 20-mer and double-stranded undamaged 20-mer formed 1:1 noncovalent complexes with XPA-MBD. A 2:1 binding stoichiometry complex was also observed between XPA-MBD and double-stranded damaged 20-mer. Competitive binding experiments indicated only slightly preferential binding of XPA-MBD with the double-stranded damaged 20-mer compared to the undamaged 20-mer. The results demonstrate that ESI-FTICR mass spectrometry provides a fast and efficient approach for characterizing weak protein-DNA interactions such as the binding between XPA-MBD and a 20-mer oligonucleotide system.

Base Sequence↗

PubMed1999

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.

Bacterial Proteins↗

PubMed1997

A dual-trap design and its applications in electrospray ionization FTICR mass spectrometry.

A new arrangement consisting of two separate Fourier transform ion cyclotron resonance (FTICR) ion traps was used to develop methods for the manipulation of the ions produced by an electrospray ionization source (ESI). A first, "accumulation" trap, is generally maintained at a higher pressure than the second, high-performance "analyzer" trap. The manipulations developed and demonstrated include the following: (1) mass-selective ion transfers between the traps; (2) mass-selective step-wise accumulation of low-abundance ions of different mass-to-charge ratios transferred from the first trap to the analyzer trap; (3) simultaneous detection of ions in the analyzer trap and ion accumulation in the source trap; (4) simultaneous ion detection in the accumulation trap and ion storage in the analyzer trap; (5) sequential multiple transfers of the ions into the analyzer trap from the same ion population stored in the accumulation trap; (6) collision-induced dissociation of ions stored in the accumulation trap followed by mass-selective transfer of the product ions into the analyzer trap; (7) sequential transfer of the ions of different mass-to-charge ratios into the analyzer trap from the same ion population stored in the accumulation trap followed by the collision-induced dissociation of transferred ions in the analyzer trap. These ion manipulations benefit multistage studies and are projected to be useful in many biochemical applications of ESI-FTICR, including structural determination of biopolymers and study of noncovalent complexes.

Cyclotrons↗

PubMed1996

High-resolution multistage MS, MS2, and MS3 matrix-assisted laser desorption/ionization FT-ICR mass spectra of peptides from a single laser shot.

By combined and repeated use of sustained off-resonance irradiation (SORI) for ion dissociation, stored waveform inverse Fourier transform (SWIFT) waveforms for ion isolation, and ion axialization and remeasurements techniques, we obtain for the first time MS, MS2, and MS3 FT-ICR mass spectra from peptide ions (enzymatic digest products of horse cytochrome c) produced from a single laser shot. The successive fragmentation of gas-phase ions detected from the same initial batch of ions increases the sensitivity of analysis of trace amounts of biological samples in structural mass spectrometry, and fragment identification is facilitated by resolution of carbon-13 isotopic distributions. The method is illustrated by analyses of subfemtomole amounts of crudely purified samples of tryptic digest solutions of horse cytochrome c and bovine cytochrome c. The high-resolution primary ion mass spectrum, along with the collision-induced dissociation (CID) and MSn capabilities of FT-ICR, help to determine the primary amino acid sequence of the fragment ions beyond what is obtained from enzymatic digestion alone, without prior chromatographic separation and purification.

Animals↗

PubMed1996

Characterization of PCR products from bacilli using electrospray ionization FTICR mass spectrometry.

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetronitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Bacillus↗