Chromatin plays a key role in nearly all eukaryotic DNA templated processes such as mitosis, DNA repair, and transcription. Disruption of chromatin structure is intimately associated with various human diseases, such as cancer and several congenital syndromes including α-thalassemia/mental retardation and Rubinstein-Taybi syndromes 
. The molecular basis for chromatin function can be understood at level of the nucleosome, comprised of approximately 146 base pairs of DNA coiling around an histone octamer conformed by one histone H3–H4 tetramer and two histone H2A–H2B dimers 
. Chromatin domains are formed and maintained by the interaction and post-translational modification (PTM) of chromatin proteins, which can epigenetically alter gene expression 
. Within these domains, transcriptionally active regions constitute euchromatin, while transcriptionally inert regions constitute heterochromatin 
. Euchromatin is less condensed, and believed to be more accessible to transcription factors, whereas heterochromatin is more condensed and less accessible to the transcriptional machinery 
. This epigenetic and structural regulation, along with the genomic information, has been termed the “Chromatome” 
. Improved system-wide knowledge of the components of chromatin could provide a holistic insight into its higher-order structure and function.
Fully characterizing the chromatome is nontrivial as many chromatin proteins are expressed transiently, at low levels, or are difficult to extract from the nucleus 
. Furthermore, no purification method has arisen as the “gold standard” for chromatin extraction. Proteomic techniques have partly circumvented these difficulties and considerably accelerated studies on the chromatin proteomes from various species including Oryza sativa
, Saccharomyces cerevisiae
, Xenopus laevis
, and Caenorhabitis elegans 
. Several mass spectrometry (MS)-based proteomic studies have also made notable progress in characterizing human chromatin from mitotic chromosomes 
. In B lymphocytes, over 280 chromatin proteins were recently identified, however, with only 64 known to be nuclear, clearly illustrating the technical issues associated with purifying chromatin fractions 
. While a vast number of chromatin proteins has been detected, the total number of human chromatin proteins, including variants and isoforms, is likely to be much larger with over 2,000 hypothetical human genes encoding for transcriptional activators alone 
Another layer of chromatome complexity lies in the post-translational modification (PTM) of chromatin proteins. Several chromatin proteins are known to be highly modified, such as Heterochromatin Protein 1 (HP1) and High Mobility Group (HMG) proteins, where these PTMs may control protein function and regulate chromatin structure 
. Notable among this broad class of proteins, histone proteins exhibit extensive PTM patterns including methylation and acetylation at specific residues 
. Interestingly, histone PTMs are linked to various cellular events including apoptosis, cellular differentiation, and the cell cycle 
. Specific histone PTMs have been reported to associate with eu- and heterochromatin, and coexisting PTMs form and maintain those regions 
. The diversity and specificity associated with histone PTMs has led to the ‘Histone Code’ hypothesis, which proposes that PTMs act as binding sites for other chromatin proteins that “interpret” these modifications to regulate DNA-templated processes 
. Quantifying the full collection of histone PTMs involved in euchromatin and heterochromatin maintenance therefore may illuminate downstream and upstream mechanisms governing these genomic regions.
Here we present a large-scale proteomic mass spectrometry-based comparison of three selected chromatin extraction methods. Proteins enriched in each preparation were analyzed via a Bottom Up mass spectrometry based proteomics approach including separation by one-dimensional gel electrophoresis (1D-SDS-PAGE), in-gel tryptic digestion, and nanoflow LC-MS/MS performed in a high-resolution Orbitrap mass spectrometer. Our results indicate that, depending on the downstream application, a decision between biological specificity and broadness of characterization must be made in selecting a chromatin purification method. By way of this qualitative comparison, we also achieve an extensive proteomic catalog of human chromatin. This platform identified over 1,900 unique proteins from these fractions, the majority of which are annotated as nuclear proteins. We analyzed our datasets using a Normalized Spectral Abundance Factor (NSAF) approach to obtain a relative protein abundance profile and also detected numerous PTMs in our datasets, including acetylation, mono-, di- and trimethylation of lysines and arginine methylation 
. Moreover, we attempted to carry out proteomic investigations into euchromatin- and heterochromatin-enriched fractions and identified proteins seemingly enriched in either fraction. To corroborate the enrichment for these genomic regions, we also characterized histone PTMs in the euchromatin or heterochromatin enriched samples using a stable isotope labeling quantitative MS method. We hope our findings will act as a foundation for additional studies involving the higher-level structure of chromatin and its roles in basal or aberrant gene functions during dynamic or epigenetic processes.