IsoForma: An R Package for Quantifying and Visualizing Positional Isomers in Top-Down LC-MS/MS Data.

Proteoforms, the different forms of a protein with sequence variations including post-translational modifications (PTMs), execute vital functions in biological systems, such as cell signaling and epigenetic regulation. Advances in top-down mass spectrometry (MS) technology have permitted the direct characterization of intact proteoforms and their exact number of modification sites, allowing for the relative quantification of positional isomers (PI). Protein positional isomers refer to a set of proteoforms with identical total mass and set of modifications, but varying PTM site combinations. The relative abundance of PI can be estimated by matching proteoform-specific fragment ions to top-down tandem MS (MS2) data to localize and quantify modifications. However, the current approaches heavily rely on manual annotation. Here, we present IsoForma, an open-source R package for the relative quantification of PI within a single tool. Benchmarking IsoForma's performance against two existing workflows produced comparable results and improvements in speed. Overall, IsoForma provides a streamlined process for quantifying PI, reduces the analysis time, and offers an essential framework for developing customized proteoform analysis workflows. The software is open source and available at https://github.com/EMSL-Computing/isoforma-lib.

An Improved Top-Down Mass Spectrometry Characterization of Chlamydomonas reinhardtii Histones and Their Post-translational Modifications.

We present an updated analysis of the linker and core histone proteins and their proteoforms in the green microalga Chlamydomonas reinhardtii by top-down mass spectrometry (TDMS). The combination of high-resolution liquid chromatographic separation, robust fragmentation, high mass spectral resolution, the application of a custom search algorithm, and extensive manual analysis enabled the characterization of 86 proteoforms across all four core histones H2A, H2B, H3, and H4 and the linker histone H1. All canonical H2A paralogs, which vary in their C-termini, were identified, along with the previously unreported noncanonical variant H2A.Z that had high levels of acetylation and C-terminal truncations. Similarly, a majority of the canonical H2B paralogs were identified, along with a smaller noncanonical variant, H2B.v1, that was highly acetylated. Histone H4 exhibited a novel acetylation profile that differs significantly from that found in other organisms. A majority of H3 was monomethylated at K4 with low levels of co-occuring acetylation, while a small fraction of H3 was trimethylated at K4 with high levels of co-occuring acetylation.

Quick and facile preparation of histone proteins from the green microalga Chlamydomonas reinhardtii and other photosynthetic organisms.

The development of universal, broadly applicable methods for histone extraction from animal cells and tissues has unlocked the ability to compare these epigenetic-influencing proteins across tissue types, healthy and diseased states, and cancerous versus normal cells. However, for plants and green algae, a quick and easily implemented histone extraction method has yet to be developed. Here, we report an optimized method that provides a unified approach to extract histones for the green microalgal species Chlamydomonas reinhardtii and Scenedesmus dimorphus as well as for maize (corn) leaf tissue. Histone extraction methods include treatment with high salt concentrations and acidification. Preparations of nuclei can be made in ∼3.5 h and histones extracted in ∼3.5 h either immediately or nuclei may be frozen and histone proteins can be later extracted without a change in histone PTM patterns. To examine the efficiency of the new methods provided, we performed both qualitative and quantitative analysis of salt and acid-extracted whole histone proteins (SAEWH) via SDS-PAGE gel electrophoresis and intact protein mass spectrometry. SDS-PAGE analysis indicated that histone yields decrease when using walled Chlamydomonas strains relative to cell-wall-less mutants. Using top-down mass spectrometry (TDMS) for intact protein analysis, we confirmed the presence of H4K79me1 in multiple algal species; however, this unique modification was not identified in corn leaf tissue and has not been reported elsewhere. TDMS measurements of SAEWH extracts also revealed that oxidation which occurs during the histone extraction process does not increase with exposure of harvested algal cells, their nuclei and the extracted histone samples to light.

How many human proteoforms are there?

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Characterization of Chlamydomonas reinhardtii Core Histones by Top-Down Mass Spectrometry Reveals Unique Algae-Specific Variants and Post-Translational Modifications.

The unicellular microalga Chlamydomonas reinhardtii has played an instrumental role in the development of many new fields (bioproducts, biofuels, etc.) as well as the advancement of basic science (photosynthetic apparati, flagellar function, etc.). Chlamydomonas' versatility ultimately derives from the genes encoded in its genome and the way that the expression of these genes is regulated, which is largely influenced by a family of DNA binding proteins called histones. We characterize C. reinhardtii core histones, both variants and their post-translational modifications, by chromatographic separation, followed by top-down mass spectrometry (TDMS). Because TDMS has not been previously used to study Chlamydomonas proteins, we show rampant artifactual protein oxidation using established nuclei purification and histone extraction methods. After addressing oxidation, both histones H3 and H4 are found to each have a single polypeptide sequence that is minimally acetylated and methylated. Surprisingly, we uncover a novel monomethylation at lysine 79 on histone H4 present on all observed molecules. Histone H2B and H2A are found to have two and three variants, respectively, and both are minimally modified. This study provides an updated assessment of the core histone proteins in the green alga C. reinhardtii by top-down mass spectrometry and lays the foundation for further investigation of these essential proteins.

Cdc45 (cell division cycle protein 45) guards the gate of the Eukaryote Replisome helicase stabilizing leading strand engagement.

DNA replication licensing is now understood to be the pathway that leads to the assembly of double hexamers of minichromosome maintenance (Mcm2-7) at origin sites. Cell division control protein 45 (Cdc45) and GINS proteins activate the latent Mcm2-7 helicase by inducing allosteric changes through binding, forming a Cdc45/Mcm2-7/GINS (CMG) complex that is competent to unwind duplex DNA. The CMG has an active gate between subunits Mcm2 and Mcm5 that opens and closes in response to nucleotide binding. The consequences of inappropriate Mcm2/5 gate actuation and the role of a side channel formed between GINS/Cdc45 and the outer edge of the Mcm2-7 ring for unwinding have remained unexplored. Here we uncover a novel function for Cdc45. Cross-linking studies trace the path of the DNA with the CMG complex at a fork junction between duplex and single strands with the bound CMG in an open or closed gate conformation. In the closed state, the lagging strand does not pass through the side channel, but in the open state, the leading strand surprisingly interacts with Cdc45. Mutations in the recombination protein J fold of Cdc45 that ablate this interaction diminish helicase activity. These data indicate that Cdc45 serves as a shield to guard against occasional slippage of the leading strand from the core channel.

DNA binding polarity, dimerization, and ATPase ring remodeling in the CMG helicase of the eukaryotic replisome.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.

Histone H1 phosphorylation is associated with transcription by RNA polymerases I and II.

Histone H1 phosphorylation affects chromatin condensation and function, but little is known about how specific phosphorylations impact the function of H1 variants in higher eukaryotes. In this study, we show that specific sites in H1.2 and H1.4 of human cells are phosphorylated only during mitosis or during both mitosis and interphase. Antisera generated to individual H1.2/H1.4 interphase phosphorylations reveal that they are distributed throughout nuclei and enriched in nucleoli. Moreover, interphase phosphorylated H1.4 is enriched at active 45S preribosomal RNA gene promoters and is rapidly induced at steroid hormone response elements by hormone treatment. Our results imply that site-specific interphase H1 phosphorylation facilitates transcription by RNA polymerases I and II and has an unanticipated function in ribosome biogenesis and control of cell growth. Differences in the numbers, structure, and locations of interphase phosphorylation sites may contribute to the functional diversity of H1 variants.

Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins.

MCM2-7 proteins provide essential helicase functions in eukaryotes at chromosomal DNA replication forks. During the G1 phase of the cell cycle, they remain loaded on DNA but are inactive. We have used recombinant methods to show that the Drosophila MCM2-7 helicase is activated in complex with Cdc45 and the four GINS proteins (CMG complex). Biochemical activities of the MCM AAA+ motor are altered and enhanced through such associations: ATP hydrolysis rates are elevated by two orders of magnitude, helicase activity is robust on circular templates, and affinity for DNA substrates is improved. The GINS proteins contribute to DNA substrate affinity and bind specifically to the MCM4 subunit. All pairwise associations among GINS, MCMs, and Cdc45 were detected, but tight association takes place only in the CMG. The onset of DNA replication and unwinding may thus occur through allosteric changes in MCM2-7 affected by the association of these ancillary factors.

Combinatorial modification of human histone H4 quantitated by two-dimensional liquid chromatography coupled with top down mass spectrometry.

Quantitative proteomics has focused heavily on correlating protein abundances, ratios, and dynamics by developing methods that are protein expression-centric (e.g. isotope coded affinity tag, isobaric tag for relative and absolute quantification, etc.). These methods effectively detect changes in protein abundance but fail to provide a comprehensive perspective of the diversity of proteins such as histones, which are regulated by post-translational modifications. Here, we report the characterization of modified forms of HeLa cell histone H4 with a dynamic range >10(4) using a strictly Top Down mass spectrometric approach coupled with two dimensions of liquid chromatography. This enhanced dynamic range enabled the precise characterization and quantitation of 42 forms uniquely modified by combinations of methylation and acetylation, including those with trimethylated Lys-20, monomethylated Arg-3, and the novel dimethylated Arg-3 (each <1% of all H4 forms). Quantitative analyses revealed distinct trends in acetylation site occupancy depending on Lys-20 methylation state. Because both modifications are dynamically regulated through the cell cycle, we simultaneously investigated acetylation and methylation kinetics through three cell cycle phases and used these data to statistically assess the robustness of our quantitative analysis. This work represents the most comprehensive analysis of histone H4 forms present in human cells reported to date.

Interpreting top-down mass spectra using spectral alignment.

Recent advances in mass spectrometry instrumentation, such as FTICR and OrbiTrap, have made it possible to generate high-resolution spectra of entire proteins. While these methods offer new opportunities for performing "top-down" studies of proteins, the computational tools for analyzing top-down data are still scarce. In this paper we investigate the application of spectral alignment to the problem of identifying protein forms in top-down mass spectra (i.e., identifying the modifications, mutations, insertions, and deletions). We demonstrate how spectral alignment efficiently discovers protein forms even in the presence of numerous modifications and how the algorithm can be extended to discover positional isomers from spectra of mixtures of isobaric protein forms.

Preferential dimethylation of histone H4 lysine 20 by Suv4-20.

Post-translational modifications of histone tails direct nuclear processes including transcription, DNA repair, and chromatin packaging. Lysine 20 of histone H4 is mono-, di-, or trimethylated in vivo, but the regulation and significance of these methylations is poorly understood. The SET domain proteins PR-Set7 and Suv4-20 have been implicated in mono- and trimethylation, respectively; however, enzymes that dimethylate lysine 20 have not been identified. Here we report that Drosophila Suv4-20 is a mixed product specificity methyltransferase that dimethylates approximately 90% and trimethylates less than 5% of total H4 at lysine 20 in S2 cells. Trimethylation, but not dimethylation, is reduced in Drosophila larvae lacking HP1, suggesting that an interaction with HP1 regulates the product specificity of Suv4-20 and enrichment of trimethyllysine 20 within heterochromatin. Similar to the Drosophila enzyme, human Suv4-20h1/h2 enzymes generate di- and trimethyllysine 20. PR-Set7 and Suv4-20 are both required for normal levels of methylation, suggesting they have non-redundant functions. Alterations in the level of lysine 20 methylation following knock-down or overexpression of Suv4-20 did not affect lysine 16 acetylation, revealing that these two modifications are not competitive in vivo. Depletion of Suv4-20h1/h2 in HeLa cells impaired the formation of 53BP1 foci, suggesting dimethyllysine 20 is required for a proper DNA damage response. Collectively, the data indicate that Suv4-20 generates nearly ubiquitous dimethylation that facilitates the DNA damage response and selective trimethylation that is involved in heterochromatin formation.

Certain and progressive methylation of histone H4 at lysine 20 during the cell cycle.

Methylation of histone H4 at lysine 20 (K20) has been implicated in transcriptional activation, gene silencing, heterochromatin formation, mitosis, and DNA repair. However, little is known about how this modification is regulated or how it contributes to these diverse processes. Metabolic labeling and top-down mass spectrometry reveal that newly synthesized H4 is progressively methylated at K20 during the G(2), M, and G(1) phases of the cell cycle in a process that is largely inescapable and irreversible. Approximately 98% of new H4 becomes dimethylated within two to three cell cycles, and K20 methylation turnover in vivo is undetectable. New H4 is methylated regardless of prior acetylation, and acetylation occurs predominantly on K20-dimethylated H4, refuting the hypothesis that K20 methylation antagonizes H4 acetylation and represses transcription epigenetically. Despite suggestions that it is required for normal mitosis and cell cycle progression, K20 methylation proceeds normally during colchicine treatment. Moreover, delays in PR-Set7 synthesis and K20 methylation which accompany altered cell cycle progression during sodium butyrate treatment appear to be secondary to histone hyperacetylation or other effects of butyrate since depletion of PR-Set7 did not affect cell cycle progression. Together, our data provide an unbiased perspective of the regulation and function of K20 methylation.

Mild performic acid oxidation enhances chromatographic and top down mass spectrometric analyses of 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 posttranslational modification localization after MS/MS. We also describe an optimized performic acid oxidation procedure that circumvents these problems without catalyzing additional oxidations or altering the levels of posttranslational modifications common in histones. MS and MS/MS of HeLa cell core histones confirmed that Met and Cys were the only residues oxidized and that complete 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.

Pervasive combinatorial modification of histone H3 in human cells.

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.

Quantitative analysis of modified proteins and their positional isomers by tandem mass spectrometry: human histone H4.

Here we show that fragment ion abundances from dissociation of ions created from mixtures of multiply modified histone H4 (11 kDa) or of N-terminal synthetic peptides (2 kDa) correspond to their respective intact ion abundances measured by Fourier transform mass spectrometry. Isomeric mixtures of modified forms of the same protein are resolved and quantitated with a precision of

Gene-specific characterization of human histone H2B by electron capture dissociation.

The basis set of protein forms expressed by human cells from the H2B gene family was determined by Top Down Mass Spectrometry. Using Electron Capture Dissociation for MS/MS of H2B isoforms, direct evidence for the expression of unmodified H2B.Q, H2B.A, H2B.K/T, H2B.J, H2B.E, H2B.B, H2B.F, and monoacetylated H2B.A was obtained from asynchronous HeLa cells. H2B.A was the most abundant form, with the overall expression profile not changing significantly in cells arrested in mitosis by colchicine or during mid-S, mid-G2, G2/M, and mid-G1 phases of the cell cycle. Modest hyperacetylation of H2B family members was observed after sodium butyrate treatment.

Precise characterization of human histones in the H2A gene family by top down mass spectrometry.

Top Down analysis revealed that at least fourteen genes encoding histone H2A are coexpressed in HeLa cells. Characterization of these species revealed that all except H2A.Z and H2A.F/Z were alpha-N-acetylated, H2A.O and H2A.C,D,I,N,P were the most abundant, and those exceeding approximately 10% abundance lacked post-translational modifications. This unequivocal identification of H2A forms illustrates the advantages of Top Down Mass Spectrometry and provides a global perspective of H2A regulation through the cell cycle.

Phylogenetic investigations of Antarctic notothenioid fishes (Perciformes: Notothenioidei) using complete gene sequences of the mitochondrial encoded 16S rRNA.

The Notothenioidei dominates the fish fauna of the Antarctic in both biomass and diversity. This clade exhibits adaptations related to metabolic function and freezing avoidance in the subzero Antarctic waters, and is characterized by a high degree of morphological and ecological diversity. Investigating the macroevolutionary processes that may have contributed to the radiation of notothenioid fishes requires a well-resolved phylogenetic hypothesis. To date published molecular and morphological hypotheses of notothenioids are largely congruent, however, there are some areas of significant disagreement regarding higher-level relationships. Also, there are critical areas of the notothenioid phylogeny that are unresolved in both molecular and morphological phylogenetic analyses. Previous molecular phylogenetic analyses of notothenioids using partial mtDNA 12S and 16S rRNA sequence data have resulted in limited phylogenetic resolution and relatively low node support. One particularly controversial result from these analyses is the paraphyly of the Nototheniidae, the most diverse family in the Notothenioidei. It is unclear if the phylogenetic results from the 12S and 16S partial gene sequence dataset are due to limited character sampling, or if they reflect patterns of evolutionary diversification in notothenioids. We sequenced the complete mtDNA 16S rRNA gene for 43 notothenioid species, the largest sampling to-date from all eight taxonomically recognized families. Phylogenetic analyses using both maximum parsimony and maximum likelihood resulted in well-resolved trees with most nodes supported with high bootstrap pseudoreplicate scores and significant Bayesian posterior probabilities. In all analyses the Nototheniidae was monophyletic. Shimodaira-Hasegawa tests were able to reject two hypotheses that resulted from prior morphological analyses. However, despite substantial resolution and node support in the 16S rRNA trees, several phylogenetic hypotheses among closely related species and clades were not rejected. The inability to reject particular hypotheses among species in apical clades is likely due to the lower rate of nucleotide substitution in mtDNA rRNA genes relative to protein coding regions. Nevertheless, with the most extensive notothenioid taxon sampling to date, and the much greater phylogenetic resolution offered by the complete 16S rRNA sequences over the commonly used partial 12S and 16S gene dataset, it would be advantageous for future molecular investigations of notothenioid phylogenetics to utilize at the minimum the complete gene 16S rRNA dataset.

Shotgun annotation of histone modifications: a new approach for streamlined characterization of proteins by top down mass spectrometry.

Eukaryotic histones serve as prototypical examples of posttranslational complexity with diverse modifications (PTMs) on many different residues that comprise a "Histone Code". To help crack this code more efficiently, we demonstrate a new strategy for protein characterization wherein complete PTM descriptions are obtained by database retrieval instead of manual interpretation of information-rich data from high-resolution tandem mass spectrometry (MS/MS). A database of nearly 50 000 modified histone H4 sequences was created and queried with 91 fragment ions from electron capture dissociation of a histone form +112 Da (versus unmodified mass) selectively accumulated in a quadrupole Fourier transform hybrid mass spectrometer. The correct form atop the retrieval list indicated dimethylation at Lys20, acetylation at the N terminus, and acetylation at Lys16 (resolved from trimethylation, Deltam = 0.036 Da). A statistical evaluation reveals the critical role of mass accuracy and that PTM "isomers" are retrieved as next-best matches. The applicability of shotgun annotation to forms of H4 with up to six PTMs is demonstrated, with extensibility to other histones (e.g., H2A, H2B, H3) and other protein classes projected.