[Using of metal-affinity chromatography for identification of alkylated rat globin].

Rat globin peptides alkylated by sulfur mustard on amino-acid residues C-126, C-93 and E-27 with MH+ 1444.62 Da, 1561.66 Da, 1676.78 Da, respectively, were concentrated using metal-affinity chromatography on Cu2+. The peptides were received by trypic digestion after in vitro incubation of rat globin with 60 microM HD. Aklylated peptide with MH+ 1444.62 Da is the most sensitive biomarker, which can be concentrated from globin trypic digest, incubated with 3 microM sulfur mustard.

[Analysis of nuclear protein complexes comprising alpha-actinin-4 by 2D-electrophoresis and mass-spectrometry].

Actin-binding protein alpha-actinin-4 is a member of spectrin super family. It is located in the cytoplasm and in the nucleus. However, nuclear functions of alpha-actinin-4 are still not clear. In this study, we analyzed composition of nuclear protein complexes associated with alpha-actinin-4 in A431 cells. Using 2D electrophoresis, we have determined that about 50 different proteins may be associated with nuclear alpha-actinin-4. Using mass-spectrometry, we analyzed major proteins of these complexes. beta-Actin, alpha- and beta-tubulins, ribonucleoprotein A2/B1, which regulates splicing and is associated with beta-actin, peroxiredoxin-1, which is involved in oxidative stress, and glycolytic enzyme D-3-phosphoglycerate dehydrogenase were identified by MALDI-TOF. Detection of these proteins in nuclear complexes with alpha-actinin-4 may suggest that alpha-actinin-4 is involved in transcription and splicing. Presence of beta-actin in the investigated complexes was confirmed by tandem mass-spectrometry (MALDI-TOF-TOF). Immunoprecipitation of nuclear proteins with antibodies against alpha-tubulin confirmed association of alpha-actinin-4 with alpha-tubulin in the protein complex. Nuclear alpha-actinin-4 constitutes of 105 KDa fullsize isoform and two truncated isoforms of 65 and 75 kDa, whereas only the truncated isoform have been found in nuclear complexes with alpha-tubulin. These data suggest that alpha-actinin-4 is associated with a number of different nuclear protein complexes which may carry out different functions in the cell nucleus.