Plasma protein map: an update by microsequencing.

The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.     

Anthracene-BODIPY cassettes: syntheses and energy transfer

Compounds based on the 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) framework are excellent fluorescent markers. When BODIPY dyes of this type are conjugated to functionalities that absorb at relatively short wavelengths, those functionalities can, in some molecules, transmit the absorbed energy to the BODIPY which then fluoresces. In such cases the BODIPY fragment acts as an acceptor while the other group serves as a donor. Energy transfer efficiencies in such donor-acceptor cassette systems must vary with the relative orientation of the two components, and with the structure of the linkers that attach them. This study was designed to probe these issues for the special case in which the linkers between the donor and acceptor fragments are conjugated. To do this, the cassettes 3-10 were prepared. Electrochemical studies were performed to provide insight into the degree of donor-acceptor conjugation in these systems. X-ray Crystallographic studies on single crystals of compounds 7 and 9 revealed the favored conformations of the donor and acceptor fragments in the solid state. Absorption, fluorescence, and time-resolved fluorescence spectra of the compounds were recorded, and quantum yields for the cassettes excited at the donor lambda(max) were measured. Fluorescence steady-state anisotropy data were determined for cassettes 3 and 9 to provide information about the mutual direction of the transition dipole moments.     

Motions of single molecules and proteins in trehalose glass

The fluorescence intensity-time records of individual metal-free porphyrin cytochrome-c and Zn porphyrin cytochrome-c molecules whose translational motions are restricted by encapsulation in trehalose are examined by single-molecule spectroscopy by means of a two-channel confocal microscope that records transient fluorescence signals in two orthogonal polarization directions. Large angular motions often occur on time scales ranging to many seconds. Measurements of the photobleaching time distributions indicate that the trehalose glass restricts the accessibility of the fluorescent molecules to oxygen.     

Two-dimensional electrophoresis resources available from ExPASy

This paper describes the set of two-dimensional electrophoresis (2-DE) resources currently available from the ExPASy proteomics Web server. These resources include the SWISS-2DPAGE database, 2-DE software packages, 2-DE technical and educational services, as well as indexes and search engines for 2-DE related sites over the Internet.     

Modified expression of plasma glutathione peroxidase and manganese superoxide dismutase in human renal cell carcinoma

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is a powerful tool to separate thousands of polypeptides and to highlight the modification of protein expression in malignant diseases. By applying 2-D PAGE to ten normal human kidney and ten homologous renal cell carcinoma (RCC) tissues, we found two peptides in all ten normal tissues but not in RCCs and, conversely, two peptides were detected in all RCCs but not in normal tissues. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and internal sequence analysis, the two first peptides were identified as two isoforms of plasma glutathione peroxidase (GPxP). The two other peptides isolated in all RCCs but not in normal tissues were identified by N-terminal sequence analysis as multimeric forms of manganese superoxide dismutase (Mn-SOD). No multimeric Mn-SODs and only two monomeric forms were detected in normal tissues. GPxP and Mn-SOD are metallo-enzymes encoded on chromosome 5q32 and on chromosome 6p25, respectively. Their regions are within the locus 5q21-->qter and 6q21-6q27 on which deletions and translocations are described in some cytogenetic studies of RCC transformation. Therefore, our results might suggest a correlation between the modified expression of GPxP and Mn-SOD in tumor tissues and chromosomal modifications, and that the two proteins may be putative markers for diagnosis of RCC.     

Imaging mass spectrometry using peptide isoelectric focusing

Imaging Mass Spectrometry (IMS) has emerged as a powerful technique in the field of proteomics. The use of Immobilized pH Gradient-IsoElectric Focusing (IPG-IEF) is also a new trend, as the first dimension of separation, in shotgun proteomics. We report a combination of these two outstanding technologies. This approach is based on the separation of shotgun-produced peptides by IPG-IEF. The peptides are then transferred by capillarity to a capture membrane, which is then scanned by the mass spectrometer to generate MS images. This high-throughput methodology allows a preview of the sample to be obtained in a single day. We report the application of this new pipeline for differential comparison of the membrane proteome of two different strains of Staphylococcus aureus bacteria in a proof-of-principle experiment.     

Stark effects and electric dipole coupling of nπ* and ππ* states

The coupling of nπ* states amongst themselves or with ππ* states can be induced by static electric fields. The resulting linear or non-linear Stark effects expose hitherto unknown aspects of the properties of the n-orbitals. The matrix elements of the dipole operator are calculated for most of the important two-electron configurations that are expected to describe the low energy electronic states of azines. The case of s-triazine is treated in detail and results are given for pyridine, pyrazine, pyrimidine, pyridazine and s-tetrazine.

On the basis of these calculations a number of predictions of measurable effects are made including estimates of the intensities of new infra-red triplet-triplet (and singlet-singlet) absorption bands of azines, and lifetimes of some electronic transitions in the infra-red that may be important in electronic relaxation in isolated molecules.     

The 2D IR responses of amide and carbonyl modes in water cannot be described by Gaussian frequency fluctuations

The two-dimensional (2D) IR spectral shapes seen for aqueous amide-I' or carbonyls having apparently single bands are not those predicted by Gaussian frequency fluctuations. Their population evolution exposes discrete distributions undergoing picosecond time scale exchange. The energy transfer to other modes provides a clear view of this underlying structure, which is largely attributed to exchanging water configurations. The results suggest new approaches to examine protein-bound water at the residue level.     

Correlation of the vibrations of the aqueous azide ion with the O-H modes of bound water molecules

Dual frequency two-dimensional infrared spectroscopy (2D-IR) has been used to investigate the dynamics of the azide-water solvation shell. The memory of the azide transition frequencies is detected in the echo emitted by the OH stretching mode of the ion-bound water molecules. There is a significant positive correlation of the two frequency distributions that decays on a 140 fs time scale. The result confirms that the O-H bond of water molecules in the solvent shell have frequency fluctuations that are considerably slowed from those that are known in bulk water. The positive correlation is attributed to cooperative interactions of coordinated water molecules with an azide ion.