Biomedical subjects

T P Conrads

Publications and source records attributed to T P Conrads.

7 recordsLinked to original sources

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

Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses.

A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins. The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine. The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process. The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein. After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry. PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified. The PhIAT labeling method was also applied to a yeast protein extract. The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins.

Affinity Labels↗

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

Design and performance of an ESI interface for selective external ion accumulation coupled to a Fourier transform ion cyclotron mass spectrometer.

The coupling of Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) with electrospray ionization has advanced the analysis of large biopolymers and provided the basis for high-throughput protein characterization (e.g., for rapid "proteome" analyses). In this work, the combination of high-performance capillary liquid chromatography with FTICR mass spectrometry and external ion accumulation has been shown to increase both sensitivity and analysis duty cycle. Instrument versatility is further improved by ion preselection followed by ion accumulation in an external linear quadrupole ion trap. The interface was tested with a 3.5-T FTICR mass spectrometer and evaluated with a number of peptides and proteins whose molecular weights ranged from 500 to 66000. A significant increase in the sensitivity, duty cycle, and dynamic range over that of the previously used accumulated trapping was achieved, exhibiting a detection limit of approximately 10 zmol (approximately 6000 molecules) for smaller proteins such as cytochrome c. Capillary LC external accumulation interface with FTICR was successfully applied for the study of whole-proteome mouse tryptic digests.

Animals↗

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

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↗