We have developed a new mass spectrometry (MS) technology, the Single-probe MS, capable of real-time, in situ metabolomic analysis of individual living cells. The Single-probe is a miniaturized multifunctional sampling and ionization device that is directly coupled to the mass spectrometer. With a sampling tip smaller than individual eukaryotic cells (<10 μm), the Single-probe can be inserted into single cells to sample the intracellular compounds for real-time MS analysis. We have used the Single-probe to detect several cellular metabolites and the anticancer small molecules paclitaxel, doxorubicin, and OSW-1 in individual cervical cancer cells (HeLa). Single cell mass spectrometry (SCMS) is an emerging scientific technology that could reshape the analytical science of many research disciplines, and the Single-probe MS technology is a novel method for SCMS that, through its accessible fabrication protocols, can be broadly applied to different research areas.

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3, and H4 to form nuclosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails, e.g., acetylation and methylation of histones. These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence has been provided that biotinidase and holocarboxylase synthetase catalyze the covalent binding of biotin to histones. Primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation, and to determine whether biotinidase and holocarboxylase synthetase localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histone H2A and H2AX are targets for biotinylation, and that K125, K127, and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines, but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modificationspecific antibodies. Antibodies to biotinidase and holocarboxylase synthetase localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that biotinylation sites include amino acid residues from the Cterminus.

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3′ regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.

Histones H1, H2A, H2B, H3 and H4 are DNA-binding proteins that mediate the folding of DNA into chromatin. Various posttranslational modifications of histones regulate processes such as transcription, replication and repair of DNA. Recently, a novel posttranslational modification has been identified: covalent binding of the vitamin biotin to lysine residues in histones, mediated by biotinidase and holocarboxylase synthetase. Here we describe a novel peptide-based technique, which was used to identify eight distinct biotinylation sites in histones H2A, H3 and H4. Biotinylation site-specific antibodies were generated to investigate biological functions of histone biotinylation. Evidence was provided that biotinylation of histones plays a role in cell proliferation, gene silencing and cellular response to DNA damage.