Mass spectrometry (MS) research has revolutionized modern biological and biomedical fields. At the heart of the majority of mass spectrometry experiments is the use of Bottom Up mass spectrometry methods where proteins are first proteolyzed into smaller fragments before MS interrogation. The advent of electron capture dissociation and, more recently, electron-transfer dissociation, however, has allowed Top Down (analysis of intact proteins) or middle down (analysis of large polypeptides) mass spectrometry to both experience large increases in development, growth, and overall usage. Nevertheless, for high-throughput large-scale proteomic studies, Bottom Up mass spectrometry has easily dominated the field. As Top Down mass spectrometry methodology and technology continue to develop, will it genuinely be able to compete with Bottom Up mass spectrometry for whole proteome analysis? Discussed here are the current approaches, applications, issues, and future view of high-throughput Top Down mass spectrometry.

Methylation of specific histone residues is capable of both gene activation and silencing. Despite vast work on the function of methylation, most studies either present a static snapshot of methylation or fail to assign kinetic information to specific residues. Using liquid chromatography-tandem mass spectrometry on a high-resolution mass spectrometer and heavy methyl-SILAC labeling, we studied site-specific histone lysine and arginine methylation dynamics. The detection of labeled intermediates within a methylation state revealed that mono-, di-, and trimethylated residues generally have progressively slower rates of formation. Furthermore, methylations associated with active genes have faster rates than methylations associated with silent genes. Finally, the presence of both an active and silencing mark on the same peptide results in a slower rate of methylation than the presence of either mark alone. Here we show that quantitative proteomic approaches such as this can determine the dynamics of multiple methylated residues, an understudied portion of histone biology.

Tri-methylation of histone H3 lysine 9 is important for recruiting heterochromatin protein 1 (HP1) to discrete regions of the genome, thereby regulating gene expression, chromatin packaging and heterochromatin formation. Here we show that HP1alpha,-beta, and -gamma are released from chromatin during the M phase of the cell cycle, even though tri-methylation levels of histone H3 lysine 9 remain unchanged. However, the additional, transient modification of histone H3 by phosphorylation of serine 10 next to the more stable methyl-lysine 9 mark is sufficient to eject HP1 proteins from their binding sites. Inhibition or depletion of the mitotic kinase Aurora B, which phosphorylates serine 10 on histone H3, causes retention of HP1 proteins on mitotic chromosomes, suggesting that H3 serine 10 phosphorylation is necessary for the dissociation of HP1 from chromatin in M phase. These findings establish a regulatory mechanism of protein-protein interactions, through a combinatorial readout of two adjacent post-translational modifications: a stable methylation and a dynamic phosphorylation mark.

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immuno-biochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as S. cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared to mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (K) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously-grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, me2, me3 in Tet. On histone H4, methylation at K20 showed a similar pattern as H3 methylation at K9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined, suggesting that the more complex and species-specific PTM patterns observed on H3 might have originated from the development of additional enzyme systems that govern the steady-state balance of these marks and from the presence of more H3 variants in mammals.

Chromatin is regulated at many different levels, from higher-order packing to individual nucleosome placement. Recent studies have shown that individual histone modifications, and combinations thereof, play a key role in modulating chromatin structure and gene activity. Reported here is an analysis of Arabidopsis histone H3 modifications by nanoflow-HPLC coupled to electrospray ionization on a hybrid linear ion trap-Fourier transform mass spectrometer (LTQ/FTMS). We find that the sites of acetylation and methylation, in general, correlate well with other plants and animals. Two well-studied modifications, dimethylation of Lys-9 (correlated with silencing) and acetylation of Lys-14 (correlated with active chromatin) while abundant by themselves were rarely found on the same histone H3 tail. In contrast, dimethylation at Lys-27 and monomethylation at Lys-36 were commonly found together. Interestingly, acetylation at Lys-9 was found only in a low percentage of histones while acetylation of Lys-14 was very abundant. The two histone H3 variants, H3.1 and H3.2, also differ in the abundance of silencing and activating marks confirming other studies showing that the replication-independent histone H3 is enriched in active chromatin.

Platelet-derived microparticles are the most abundant type of microparticle in human blood and contribute to many biologically significant processes. Here, we report the first proteomic analysis of microparticles generated from activated platelets. Using 1D SDS-PAGE and liquid chromatography coupled to a linear ion trap mass spectrometer, the identification of 578 proteins was accomplished using a minimum of 5 MS/MS detections of at least two different peptides for each protein. These microparticles displayed many proteins intrinsic to and well-characterized on platelets. For example, microparticles in these experiments were found to contain membrane surface proteins including GPIIIa, GPIIb, and P-selectin, as well other platelet proteins such as the chemokines CXCL4, CXCL7, and CCL5. In addition, approximately 380 of the proteins identified were not found in two previous studies of the platelet proteome. Since several of the proteins detected here have been previously implicated in microparticle formation and/or pathological function, it is hoped that this study will help fuel future work concerning the possible role of microparticles in various disease states.

Covalent histone modifications and the incorporation of histone variants bring about changes in chromatin structure that in turn alter gene expression. Interest in non-allelic histone variants has been renewed, in part because of recent work on H3 (and other) histone variants. However, only in mammals do three non-centromeric H3 variants (H3.1, H3.2 and H3.3) exist. Here, we show that mammalian cell lines can be separated into two different groups based on their expression of H3.1, H3.2 and H3.3 at both mRNA and protein levels. Additionally, the ratio of these variants changes slightly during neuronal differentiation of murine ES cells. This difference in H3 variant expression between cell lines could not be explained by changes in growth-rate, cell-cycle stages or chromosomal ploidy, but rather suggests other possibilities, such as changes in H3 variant incorporation during differentiation and tissue- or species-specific H3 variant expression. Moreover, quantitative mass spectrometry analysis of human H3.1, H3.2, and H3.3 showed modification differences between these three H3 variants, suggesting that they may have different biological functions. Specifically, H3.3 contains marks associated with transcriptionally active chromatin, while H3.2, in contrast, contains mostly silencing modifications that have been associated with facultative heterochromatin. Interestingly, H3.1 is enriched in both active and repressive marks, although the latter marks are different from those observed in H3.2. Although the biological significance as to why mammalian cells differentially employ three highly similar H3 variants remains unclear, our results underscore potential functional differences between them and reinforce the general view that H3.1 and H3.2 in mammalian cells should not be treated as equivalent proteins.

We developed a platform using hydrophilic interaction chromatography and high-resolution tandem mass spectrometry (MS) for analyses of histone H3 that allows comprehensive characterization of 'histone codes' at the molecular level. We identified over 150 differentially modified forms of histone H3.2 in asynchronously grown and butyrate-treated HeLa cells, revealing pervasive combinatorial modification previously unaccounted for by other techniques and providing a clarified estimate of the molecular diversity of histone H3 in mammals.

Histone H1 isoforms isolated from asynchronously grown HeLa cells were subjected to enzymatic digestion and analyzed by nano-flow reversed-phase high performance liquid chromatography (RP-HPLC) tandem mass spectrometry (MS/MS) on both quadrupole ion trap and linear quadrupole ion trap-Fourier transform ion cyclotron resonance mass spectrometers. We have observed all five major isoforms of histone H1 (H1.1, H1.2, H1.3, H1.4, and H1.5) as well as a lesser studied H1, isoform H1.X. MS/MS experiments confirmed N-terminal acetylation on all isoforms plus a single internal acetylation site. Immobilized metal affinity chromatography in combination with tandem mass spectrometry was utilized to identify 19 phosphorylation sites on the five major H1 isoforms plus H1.X. Fourteen of these phosphorylation sites were located on peptides containing the cyclin dependent kinase (CDK) consensus motif (S/T)-P-X-Z (where X is any amino acid and Z is a basic amino acid). Five phosphorylation sites were identified in regions that did not fit the consensus CDK motif. One of these phosphorylation sites was found on the serine residue on the H1.4 peptide KARKSAGAAKR. The adjacent lysine residue to the phosphoserine was also shown to be methylated. This finding raises the question of whether the hypothesized "methyl/phos" switch could be extended to linker histones, and not exclusive to core histones.

Recent developments in Top Down mass spectrometry have enabled closely-related histone variants and their modified forms to be identified and quantitated with unprecedented precision, facilitating efforts to better understand how histones contribute to the epigenetic regulation of gene transcription and other nuclear processes. It is therefore crucial that intact MS profiles accurately reflect the levels of variants and modified forms present in a given cell type or cell state for the full benefit of such efforts to be realized. Here, we show that partial oxidation of Met and Cys residues in histone samples prepared by conventional methods, together with oxidation that can accrue during storage or during chip-based automated nanoflow electrospray ionization, confounds MS analysis by altering the intact MS profile as well as hindering PTM localization after MS/MS. We also describe an optimized performic acid oxidation procedure which circumvents these problems without catalyzing additional oxidations or altering the levels of PTMs common in histones. MS and MS/MS of HeLa cell core histones confirmed that Met and Cys were the only residues oxidized and that oxidation restored true intact abundance ratios and significantly enhanced MS/MS data quality. This allowed for the unequivocal detection, at the intact molecule level, of novel combinatorially modified forms of H4 that would have been missed otherwise. Oxidation also enhanced the separation of human core histones by reverse phase chromatography and decreased the levels of salt adducted forms observed in ESI-FTMS. This method represents a simple and easily automated means for enhancing the accuracy and sensitivity of Top Down analyses of combinatorially modified forms of histones that may also be of benefit for Top Down or Bottom Up analyses of other proteins.

Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized.

A new method of sample preparation was developed for MALDI-TOF-MS analysis of oligonucleotides. First, aqueous DNA samples are dispensed and allowed to dry. Then 6-aza-2-thiothymine matrix dissolved in nonaqueous volatile solvents is applied on top of the DNA residue to form a thin homogeneous film. MALDI-TOF analysis shows such preparation generates much better shot-to-shot and sample-to-sample reproducibility and essentially eliminates the need to search for "hot" spots. The increased homogeneity of the matrix/analyte crystal distribution results in significant improvement for quantitative and high-throughput analyses of DNA. Using this method, isotopically resolved oligonucleotide spectra up to a 24-mer can also be easily obtained in a reflectron instrument. Due to the ease of preparation, this method could be widely useful for a number of applications such as for assays that are performed on surface in vitro, as the thin-layer matrix could be applied directly for MALDI analysis.