Multi-dimensional HPLC/MS of the nucleolar proteome using HPLC-chip/MS.

The proteome of the human nucleolus was investigated in a single analysis using off-line strong cation exchange chromatography and microfraction collection combined with HPLC-chip/MS. The analysis was conducted either as a 1-D workflow with HPLC-chip alone or as a 2-D workflow. Two hundred and six unique proteins were identified in the International Protein Index human database corresponding to 2024 unique tryptic peptides identified in the 2-D analysis. In contrast, only 34 proteins and 151 corresponding tryptic peptides were found by applying a 1-D separation strategy. This clearly indicated that the complexity of the samples required the combination of more than one orthogonal separation technique. Stringent database search criteria, including reversal of sequences and therefore better exclusion of false-positive identifications, were applied for reliable protein identification.

Nanoliquid chromatography-mass spectrometry of oligosaccharides employing graphitized carbon chromatography on microchip with a high-accuracy mass analyzer.

The nanoLC separations of oligosaccharides using microchip-based columns are described. Mixtures of alditols from mucins and human milk are separated on graphitized carbon. The nanoLC-MS device showed high mass accuracy for the oligosaccharides ranging between 1 and 6 ppm on routine analyses. The high mass accuracy readily allowed identification of oligosaccharide peaks and the determination of their compositions. High retention time reproducibility was exhibited by the microchip LC. Little variation was observed for standard sample either alone or in a complex heterogeneous mixture. The nanoLC-MS exhibits excellent capabilities in profiling mixtures of oligosaccharides.

Purification and characterization of a flavonol 3-O-beta-heterodisaccharidase from the dried herb of Fagopyrum esculentum Moench.

A flavonol-3-O-beta-heterodisaccharide glycosidase (FHG I) was isolated from dried aerial tissues of Fagopyrum esculentum Moench (Fagopyri herba). It has a specific enzyme activity of ca. 3.5 nkat mg(-1) protein in buffered extracts when rutin (quercetin-3-O-rutinoside) was used as substrate and an optimal enzyme activity was seen at around pH 4.8 and 30 degrees C. FHG I was purified about 156-fold to apparent homogeneity by hydrophobic interaction, anion exchange and size exclusion chromatographic steps. The apparent molecular mass of FHG I was 74.5+/-2 kDa as determined by SDS-PAGE and it is a monomeric glycoprotein with a carbohydrate content of 23%. The isoelectric point as determined by isoelectric focusing was 5.7 and the energy of activation was 32 kJ mol(-1). FHG I exhibits a high substrate specificity, preferring flavonol 3-O-glycosides comprising the disaccharide rutinose. The K(m) and V(max) values for the natural substrate rutin were calculated to be 0.561 microM and 745 nkat mg (-1) protein, respectively. Two oligopeptide fragments obtained after enzymatic digestion of FHG I were sequenced and showed similarities to sequences of beta-glucohydrolases from other plant species. Polyclonal antibodies were raised and their specificities determined. Another flavonol 3-O-beta-heterodisaccharide glycosidase (FHG II) could also be detected in buckwheat herb, having a molecular mass of 85.3+/-2 kDa and an isoelectric point between pH 6.0 and 6.5.

Phosphorylation of maize and Arabidopsis HMGB proteins by protein kinase CK2alpha.

In plants, a variety of chromatin-associated high mobility group (HMG) proteins belonging to the HMGB family have been identified. We have examined the phosphorylation of the HMGB proteins from the monocotyledonous plant maize and the dicotyledonous plant Arabidopsis by protein kinase CK2alpha. Maize CK2alpha phosphorylates the maize HMGB1 and HMGB2/3 proteins and the Arabidopsis HMGB1, HMGB2/3, and HMGB4 proteins. Maize HMGB4 and HMGB5 and Arabidopsis HMGB5 are not phosphorylated by CK2alpha. Depending on the HMGB protein up to five amino acid residues are phosphorylated in the course of the phosphorylation reaction. The HMGB1 proteins from both plants are markedly more slowly phosphorylated by CK2alpha than the other HMGB substrate proteins, indicating that certain HMGB proteins are clearly preferred substrates for CK2alpha. The rate of the phosphorylation reaction appears to be related to the ease of interaction between CK2alpha and the HMGB proteins, as indicated by chemical cross-linking experiments. MALDI/TOF mass spectrometry analyses demonstrate that the HMGB1 and HMGB2/3 proteins occur in various phosphorylation states in immature maize kernels. Thus, HMGB1 exists as monophosphorylated, double-phosphorylated, triple-phosphorylated, and tetraphosphorylated protein in kernel tissue, and the tetraphosphorylated form is the most abundant version. The observed in vivo phosphorylation states indicate that protein kinase(s) other than CK2alpha contribute(s) to the modification of the plant HMGB proteins. The fact that the HMGB proteins are phosphorylated to various extents reveals that the existence of differentially modified forms increases the number of distinct HMGB protein variants in plant chromatin that may be adapted to certain functions.

Protein kinase CK2 phosphorylates the high mobility group domain protein SSRP1, inducing the recognition of UV-damaged DNA.

The structure-specific recognition protein SSRP1 plays a role in transcription and replication in the chromatin context. Mediated by its C-terminal high mobility group (HMG) box domain, SSRP1 binds DNA non-sequence specifically but recognizes certain DNA structures. Using acetic acid urea polyacrylamide gel electrophoresis and mass spectrometry, we have examined the phosphorylation of maize SSRP1 by protein kinase CK2 alpha. The kinase phosphorylated several amino acid residues in the C-terminal part of the SSRP1 protein. Two phosphorylation sites were mapped in the very C-terminal region next to the HMG box domain, and about seven sites are localized within the acidic domain. Circular dichroism showed that the phosphorylation of the two C-terminal sites by CK2 alpha resulted in a structural change in the region of HMG box domain, because the negative peak of the CD spectrum at 222 nm was decreased by approximately 10%. In parallel, the phosphorylation induced the recognition of UV-damaged DNA, whereas the non-phosphorylated protein does not discriminate between UV-damaged DNA and control DNA. The affinity of CK2 alpha-phosphorylated SSRP1 for the DNA correlates with the degree of UV-induced DNA damage. Moreover, maize SSRP1 can restore the increased UV-sensitivity of a yeast strain lacking the NHP6A/B HMG domain proteins to levels of the control strain. Collectively, these findings indicate a role for SSRP1 in the UV response of eukaryotic cells.