Enrichment and analysis of nonenzymatically glycated peptides: Boronate affinity chromatography coupled with electron-transfer dissociation mass spectrometry

Nonenzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. However, no effective high-throughput methods exist for identifying proteins containing this low-abundance posttranslational modification in bottom-up proteomic studies. In this report, phenylboronate affinity chromatography was used in a two-step enrichment scheme to selectively isolate first glycated proteins and then glycated, tryptic peptides from human serum glycated in vitro. Enriched peptides were subsequently analyzed by alternating electron-transfer dissociation (ETD) and collision induced dissociation ( CID) tandem mass spectrometry. ETD fragmentation mode permitted identification of a significantly higher number of glycated peptides (87.6% of all identified peptides) versus CID mode (17.0% of all identified peptides), when utilizing enrichment on first the protein and then the peptide level. This study illustrates that phenylboronate affinity chromatography coupled with LC-MS/MS and using ETD as the fragmentation mode is an efficient approach for analysis of glycated proteins and may have broad application in studies of diabetes mellitus.

Mass spectrometric analysis of glucose-modified ribonuclease

RNase A (1 mM) was incubated with glucose (0.4 M) at 37degreesC for up to 14 days in phosphate buffer (0.2 M, pH 7.4), digested with trypsin and analysed by LC-MS. The major sites of fructoselysine formation were Lys(1), Lys(7), Lys(37) and Lys(41). Three of these sites (Lys(7), Lys(37) and Lys(41)) were also the major sites of N-epsilon-(carboxymethyl)lysine formation.

A Brown Dwarf Mass Donor in an Accreting Binary

A long-standing and unverified prediction of binary star evolution theory is the existence of a population of white dwarfs accreting from substellar donor stars. Such systems ought to be common, but the difficulty of finding them, combined with the challenge of detecting the donor against the light from accretion, means that no donor star to date has a measured mass below the hydrogen burning limit. We applied a technique that allowed us to reliably measure the mass of the unseen donor star in eclipsing systems. We were able to identify a brown dwarf donor star, with a mass of 0.052 ± 0.002 solar mass. The relatively high mass of the donor star for its orbital period suggests that current evolutionary models may underestimate the radii of brown dwarfs.

The rotational broadening of V395 Carinae. Implications on the compact object's mass

Context: The masses previously obtained for the X-ray binary 2S 0921-630 inferred a compact object that was either a high-mass neutron star or low-mass black-hole, but used a previously published value for the rotational broadening (v sin i) with large uncertainties. Aims: We aim to determine an accurate mass for the compact object through an improved measurement of the secondary star's projected equatorial rotational velocity. Methods: We have used UVES echelle spectroscopy to determine the v sin i of the secondary star (V395 Car) in the low-mass X-ray binary 2S 0921-630 by comparison to an artificially broadened spectral-type template star. In addition, we have also measured v sin i from a single high signal-to-noise ratio absorption line profile calculated using the method of Least-Squares Deconvolution (LSD). Results: We determine v sin i to lie between 31.3±0.5 km s-1 to 34.7±0.5 km s-1 (assuming zero and continuum limb darkening, respectively) in disagreement with previous results based on intermediate resolution spectroscopy obtained with the 3.6 m NTT. Using our revised v sin i value in combination with the secondary star's radial velocity gives a binary mass ratio of 0.281±0.034. Furthermore, assuming a binary inclination angle of 75° gives a compact object mass of 1.37±0.13 M_?. Conclusions: We find that using relatively low-resolution spectroscopy can result in systemic uncertainties in the measured v sin i values obtained using standard methods. We suggest the use of LSD as a secondary, reliable check of the results as LSD allows one to directly discern the shape of the absorption line profile. In the light of the new v sin i measurement, we have revised down the compact object's mass, such that it is now compatible with a canonical neutron star mass.

The massive neutron star or low-mass black hole in 2S 0921-630

We report on the optical spectroscopy of the eclipsing halo low-mass X-ray binary 2S 0921-630, which reveals the absorption-line radial velocity curve of the K0 III secondary star with a semiamplitude K-2=92.89+/-3.84 km s(-1), a systemic velocity gamma=34.9+/-3.3 km s(-1), and an orbital period P-orb of 9.0035+/-0.0029 days (1 sigma). Given the quality of the data, we find no evidence for the effects of X-ray irradiation. Using the previously determined rotational broadening of the mass donor and applying conservative limits on the orbital inclination, we constrain the compact object mass to be 2.0-4.3 M-circle dot (1 sigma), ruling out a canonical neutron star at the 99% level. Since the nature of the compact object is unclear, this mass range implies that the compact object is either a low-mass black hole with a mass slightly higher than the maximum possible neutron star mass (2.9 M-circle dot) or a massive neutron star. If the compact object is a black hole, it confirms the prediction of the existence of low-mass black holes, while if the object is a massive neutron star, its high mass severely constrains the equation of state of nuclear matter.