A method for rapid sequencing of intact proteins simultaneously from the N and C termini (1-2 s) with online chromatography is described and applied to the characterization of histone H3.1 posttranslational modifications and the identification of an additional member of the H2A gene family. Proteins are converted to gas-phase multiply charged positive ions by electrospray ionization and then allowed to react with fluoranthene radical anions. Electron transfer to the multiply charged protein promotes random dissociation of the NOC␣ bonds of the protein backbone. Multiply charged fragment ions are then deprotonated in a second ion͞ion reaction with the carboxylate anion of benzoic acid. The m͞z values for the resulting singly and doubly charged ions are used to read a sequence of 15-40 aa at both the N and C termini of the protein. This information, with the measured mass of the intact protein, is used to search protein or nucleotide databases for possible matches, detect posttranslational modifications, and determine possible splice variants.electron transfer dissociation ͉ fragmentation ͉ ion trap ͉ ion͞ion reactions ͉ top down P erhaps one of the most influential concepts in protein mass spectrometry has been the notion of enzymatic protein digestion to render a collection of peptides of suitable size for conventional tandem mass spectrometry, i.e., bottom-up proteomics. Doubtless this methodology has enabled significant progress for global protein identification (1, 2); however, many investigators now realize this approach has significant limitations (3). First, protein posttranslational modifications (PTMs) on multidomain proteins often work in concert; to determine their biological relevance, these patterns must be detected within the context of one another (across the whole protein). And second, mRNA editing (alternative splicing) has recently gained credit for significantly increasing the protein repertoire of complex organisms, up to three-fourths of all human genes have at least one variant (4-6). Thus, the use of short peptides as proxy markers for genes is inadequate and often misleading (7).To detect these biological events, several laboratories are now pursuing mass spectrometry-based methods to analyze whole proteins. Intact proteins are directly dissociated, either by electron capture (ECD) or collision activation (CAD), and the products are measured with the high resolving power of a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) (3,(8)(9)(10)(11)(12). McLuckey and coworkers (13-15) have used ion trap-based instrumentation to collisionally activate intact protein ions followed by spectral simplification with ion͞ion charge reduction reactions. In either case, the intact protein mass is measured while the product ions are used for sequencing and locating sites of modification. Unfortunately, mainstream implementation of this ''top-down''-type analysis is not routine because CAD typically generates only a handful of fragments (cleavage is directed at a few weak linkages, mak...

BackgroundWithin chromatin, the histone variant H2A.Z plays a role in many diverse nuclear processes including transcription, preventing the spread of heterochromatin and epigenetic transcriptional memory. The molecular mechanisms of how H2A.Z mediates its effects are not entirely understood. However, it is now known that H2A.Z has two protein isoforms in vertebrates, H2A.Z-1 and H2A.Z-2, which are encoded by separate genes and differ by 3 amino acid residues.ResultsWe report that H2A.Z-1 and H2A.Z-2 are expressed across a wide range of human tissues, they are both acetylated at lysine residues within the N-terminal region and they exhibit similar, but nonidentical, distributions within chromatin. Our results suggest that H2A.Z-2 preferentially associates with H3 trimethylated at lysine 4 compared to H2A.Z-1. The phylogenetic analysis of the promoter regions of H2A.Z-1 and H2A.Z-2 indicate that they have evolved separately during vertebrate evolution.ConclusionsOur biochemical, gene expression, and phylogenetic data suggest that the H2A.Z-1 and H2A.Z-2 variants function similarly yet they may have acquired a degree of functional independence.

Purified histone H2A.Z from chicken erythrocytes and a sodium butyrate-treated chicken erythroleukemic cell line was used as a model system to identify the acetylation sites (K4, K7, K11, K13, and K15) and quantify their distribution in this vertebrate histone variant. To understand the role played by acetylation in the modulation of the H2A.Z nucleosome core particle (NCP) stability and conformation, an extensive analysis was conducted on NCPs reconstituted from acetylated forms of histones, including H2A.Z and recombinant H2A.Z (K/Q) acetylation mimic mutants. Although the overall global acetylation of core histones destabilizes the NCP, we found that H2A.Z stabilizes the NCP regardless of its state of acetylation. Interestingly and quite unexpectedly, we found that the change in NCP conformation induced by global histone acetylation is dependent on H2A/H2A.Z acetylation. This suggests that acetylated H2A variants act synergistically with the acetylated forms of the core histone complement to alter the particle conformation. Furthermore, the simultaneous occurrence of H2A.Z and H2A in heteromorphic NCPs that most likely occurs in vivo slightly destabilizes the NCP, but only in the presence of acetylation.In the nucleus of the living cell, DNA interacts with a set of highly basic proteins called histones to form a nucleoprotein complex, which is called chromatin (1). Histones are grouped into two major types: core histones (H2A, H2B, H3, and H4) and linker histones (histone H1). The former organize the protein core around which approximately 146 bp of DNA is wrapped to produce the basic chromatin subunit, the nucleosome core particle (NCP). 1 Histones of the H1 family bind to the linker DNA regions connecting adjacent nucleosomes. Both core and linker histones consist of nonallelic variants. Some of these are expressed exclusively during S phase, and the mRNAs transcribed from their genes are not polyadenylated (replication-dependent variants). They are also often termed canonical variants. Other variants are expressed throughout the cell cycle; their mRNAs are polyadenylated, and they replace the replicationdependent variants to specify function or supplement their loss during cell cycle turnover (replacement variants).

Background: The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/ Z or H2A.V) that differ by three amino acids.