Comparative proteome analysis of wild-type and klotho-knockout mouse kidneys using a combination of MALDI-IMS and LC-MS/MS.

PURPOSE: Mutation of the klotho gene in mice elicits a syndrome resembling accelerated human aging. However, there is limited evidence for the role of Klotho in the kidney. We conducted a comparative proteome analysis of wild-type (WT) and klotho-knockout (kl-/- ) mouse kidneys to identify proteins involved in Klotho deficiency. EXPERIMENTAL DESIGN: MALDI imaging MS (MALDI-IMS) of frozen kidney sections from 7-wk-old male WT and kl-/- mice was used to determine genotype-specific differences in the MS distribution. Proteins uniquely distributed in kl-/- kidneys were identified by subsequent analysis of adjacent trypsinized sections by MALDI-IMS in combination with LC-MS/MS. Immunohistochemistry and western blotting were adopted in qualitative and quantitation analysis. RESULTS: Ninety-seven and 69 proteins identified by LC-MS/MS were matched to the MALDI-IMS spectra in WT and kl-/- mouse kidneys, respectively. Among protein types matched, nucleic acid binding proteins were most abundant, followed by enzymes. We identified secretogranin-1 (SCG1), which was predominately distributed in the glomeruli and renal tubules of kl-/- mouse kidneys. Immunohistochemistry for SCG1 mirrored images of MALDI-IMS. CONCLUSIONS: SCG1 may be a candidate protein involved in Klotho deficiency. Although further research is needed to investigate the role of SCG1 in the kidney, we show the usefulness of MALDI-IMS combined with LC-MS/MS.

Nucleotide incorporation against 7,8-dihydro-8-oxoguanine is influenced by neighboring base sequences in TLS DNA polymerase reaction.

7,8-Dihydro-8-oxoguanine (8-oxoG) is a well-known oxidative lesion in DNA and is related to carcinogenesis and ageing processes. Misincorporation of dATP opposite to 8-oxoG leads to G --> T transversion mutations. DNA sequence has been proved as an important factor influencing the replication and enzymatic repair of various types of damages. To explore the influence of sequence effect on the properties of translesion synthesis (TLS) polymerase bypass of 8-oxoG, oligonucleotides with an 8-oxoG in different sequence contexts were used. We conclude that the 5'-nearest base next to 8-oxoG has significant effects in the G --> T mutation by hpoleta.

Oligonucleotide transformation for the study of mutagenic specificities of DNA lesions in yeast.

We developed a method for the analyzing mutagenic potential of DNA damage based on the oligonucleotide transformation technique in yeast. Using this assay we have analyzed mutagenic specificities of various DNA lesions. In the present study, we analyzed the mutagenic properties of 2-hydroxyadenine and 5-hydroxycytosine in yeast. Oligonucleotides containing 2-hydroxyadenine or 5-hydroxycytosine were used for the transformation. The oligonucleotides showed transforming activities similar to unmodified oligonucleotides. This indicates that no repair systems were working on them. The sequencing data of the transformants showed that 5-hydroxycytosine and 2-hydroxyadenine are read mainly as cytosine and adenine. We will also discuss the mechanism of oligonucleotide transformation and its application to the mutagenesis study.