Biomedical subjects

C Masselon

Publications and source records attributed to C Masselon.

5 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

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

Accurate mass multiplexed tandem mass spectrometry for high-throughput polypeptide identification from mixtures.

We report a new tandem mass spectrometric approach for the improved identification of polypeptides from mixtures (e.g., using genomic databases). The approach involves the dissociation of several species simultaneously in a single experiment and provides both increased speed and sensitivity. The data analysis makes use of the known fragmentation pathways for polypeptides and highly accurate mass measurements for both the set of parent polypeptides and their fragments. The accurate mass information makes it possible to attribute most fragments to a specific parent species. We provide an initial demonstration of this multiplexed tandem MS approach using an FTICR mass spectrometer with a mixture of seven polypeptides dissociated using infrared irradiation from a CO2 laser. The peptides were added to, and then successfully identified from, the largest genomic database yet available (C. elegans), which is equivalent in complexity to that for a specific differentiated mammalian cell type. Additionally, since only a few enzymatic fragments are necessary to unambiguously identify a protein from an appropriate database, it is anticipated that the multiplexed MS/MS method will allow the more rapid identification of complex protein mixtures with on-line separation of their enzymatically produced polypeptides.

Amino Acid Sequence↗

PubMed2000

Characterization of human alcohol dehydrogenase isoenzymes by capillary isoelectric focusing-mass spectrometry.

The human liver alcohol dehydrogenase (ADH) isoenzymes are currently believed to play a major role in ethanol metabolism, accounting for most of the ethanol oxidized in the liver. They have similar molecular masses and similar isoelectric point (pI) values (the 13 possible isoenzymes having pIs in the range of 8.26-8.87), making their characterization a significant analytical challenge. Capillary isoelectric focusing (CIEF) coupled on-line with electrospray ionization - Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry was applied to separate and characterize mixtures of alphaalpha, beta1beta1 and beta3beta3 ADH isoenzymes. Seven different species were resolved by the separation in the pI 8.26-8.67 range. ESI-FTICR analysis of native ADHs revealed that each noncovalent ADH complex contains two monomeric protein units and four zinc atoms. The combination of CIEF separations with mass spectrometry appears well-suited for detailed characterization of ADH isozymes, and the attomole level sensitivity of FTICR should allow very small samples to be addressed.

Alcohol Dehydrogenase↗

PubMed1998

Matrix-assisted laser desorption/ionization mass spectrometry of noncovalent protein-transition metal ion complexes.

Transition metal ion complexes with proteins and peptides are important in many areas of analytical and biological chemistry. We used positive and negative ion MALDI-MS to detect complexes with Cu and Ni ions, and show that the specific and non-specific transition metal ion-peptide complexes can be distinguished by the use of different analytical protocols. The pH dependent stability of these complexes is also reflected in the MALDI data. We further show that triple complexes of peptides or protein with chelated metal ions can be detected efficiently and rapidly by MALDI mass spectrometry. Such triple complexes play an important role in metal chelate affinity chromatography, where histidine containing biopolymers in particular are thought to bind metal-ligand complexes, depending on the oxidation state of the metal and the number of unoccupied coordination sites of the ligand.

Amino Acid Sequence↗