In eukaryotic cells, DNA is packaged in the form of chromatin. Approximately 147 base pairs of DNA wrap around an octomer composed of two H2A–H2B dimers and one H3–H4 tetramer to form nucleosomes, the fundamental repeating unit of chromatin 
. Because nucleosomes are organized into progressively higher-ordered structures, significant chromatin remodeling is necessary for the numerous DNA-templated processes that must occur for normal cellular function, such as transcription, DNA replication, DNA repair, and chromosome segregation.
One means by which alterations to chromatin structure is accomplished is through post-translational modifications (PTMs) of the histone proteins. The core histones are largely globular, with the exception of unstructured N-terminal tails that protrude from the surface of the core particle. Although numerous PTMs have been shown to occur on residues located on the histone tails 
, it is becoming increasingly evident that residues within the globular domain are also subject to modifications 
. The type of PTMs demonstrated to occur on histone proteins include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation, ADP ribosylation, proline isomerization, citrullination, butyrylation, propionylation and glycosylation 
. While the functional significance of some of the aforementioned modifications remains to be elucidated, it is well established that other histone PTMs function by at least one of the following mechanisms: (1) disruption of nucleosomal contacts between histones and their associated DNA or between histones in contiguous nucleosomes, or (2) recruitment of non-histone proteins 
. Acetylation of lysine residues is the best-characterized modification shown to affect higher-order chromatin structure, where this mark neutralizes the basic charge of the residue on which it occurs, thereby inhibiting histone-histone or histone-DNA interaction and thus chromatin compaction 
. With regard to the other means by which histone PTMs can function, the recruitment of non-histone proteins is facilitated by the ability of specialized domains to recognize and bind to defined marks 
. For example, methylation of specific lysine residues in a defined state (mono-, di-, or trimethyl) can serve as a binding platform for effector proteins containing one of the following types of methyl-binding domains: chromodomain, tudor domain, PHD finger, MBT, Ankyrin repeat, PWWP domain and WD40 repeats 
The complexity of the number and diverse types of PTMs has led to the hypothesis of a “histone code” 
, which posits that combinatorial patterns of histone PTMs lead to defined biological outcomes brought about by the recruitment of effector proteins necessary for function in DNA-templated processes. For example, TAF1 (the largest subunit of the TFIID complex which is involved in initiating the assembly of transcriptional machinery) contains a double bromodomain that preferentially binds to multiply acetylated histone H4 
, and itself can function as a histone acetyltransferase 
. There are numerous other examples of how defined combinations of histone modifications positively or negatively affect recruitment of specific proteins 
. Despite the identification of numerous histone PTMs to date, it is likely that other modifications still await discovery. Thus, of immediate importance in deciphering the histone code is the need for identifying all the PTMs that are present on histones, so that subsequent studies can be completed to determine the combinatorial patterns in which such modifications exist on physiological substrates and what the functional outcomes of such combinations are.
In recent history, mass spectrometry (MS) has widely been used as the primary method to identify histone PTMs. MS studies have commonly employed the bottom-up approach, in which short peptides derived from proteolytic cleavage of reverse-phase HPLC (RP-HPLC)-purified histones are analyzed by MS with peptide mass fingerprinting (PMF) or a combination of liquid chromatography (LC) and tandem MS (MS/MS) using electron transfer or collision-induced dissociation methods (ETD and CID, respectively) 
. While this technique is a highly effective means by which to determine the molecular mass (by MS-PMF) or the sequence of a protein (by LC-MS/MS), it is limited in that incomplete sequence coverage of the protein of interest often occurs, and proteins with multiple cleavage sites (including the histone core proteins, which are rich in lysine and arginine residues) result in peptide segments that are too small for effective retention and/or detection 
. More recently, advances in MS have led to the development of the top-down approach as a complementary method to bottom-up analysis as a highly useful means by which to identify PTMs on histones 
. Full-length proteins are analyzed with top-down MS, as samples are infused into the mass spectrometer by electrospray ionization (ESI), allowing for MS/MS fragmentation via ETD or electron capture dissociation (ECD) of intact proteins 
. A major advantage of top-down MS is that combinatorial patterns of modifications that exist on a single histone molecule can be identified 
, which is particularly valuable in outlining the global landscape of PTMs on histone proteins.
In this study, we sought to use top-down MS to analyze the global landscape of PTMs on histone H2B. From this analysis, we identified lysine 37 of histone H2B (H2BK37) as a novel site of methylation in the budding yeast Saccharomyces cerevisiae, and that this modification exists in the dimethyl state. We generated an antibody specific for dimethylated H2BK37 (H2BK37me2), with which we were able to confirm that this mark does in fact occur in vivo. Though our candidate approach to identify the methyltransferase responsible for placing this mark and phenotypic analysis to reveal a biological function did not offer conclusive results, we provide evidence that this modification is evolutionarily conserved supporting its overall importance as a novel histone modification. Furthermore, these results demonstrate that despite the numerous rounds of previous MS analysis, additional series of MS analyses employing recent technological advancements are necessary for continued identification of novel sites of modifications to generate a more complete atlas of the factors that putatively function in the context of the histone code.