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RESEARCH PROFILES Decoding the chromosome core with top-down MS The histone family of proteins—H2A, H2B, H3, and H4—form a core around which DNA is wrapped and ultimately packaged into chromosomes. In addition, histones have been implicated in DNA replication and repair. Modifications to histones that are in nucleosomes—the basic DNA–histone units— have been linked to cancer, and posttranslational modifications (PTMs) of histones affect DNA binding and may play a central role in eukaryotic gene transcription. Histones are also amazingly heterogeneous, with >15 different genetic sequences typically coding for a single protein sequence. In a series of four papers, Neil Kelleher and colleagues at the University of Illinois at Urbana–Champaign have illustrated the utility of top-down MS (TDMS) in differentiating among histone isoforms, characterizing histone modifications, and, in this issue of Analytical Chemistry (pp 4271– 4280), quantitating PTMs of histones. The importance of histones in transcriptional regulation was recognized in the late 1990s, and the histone code hypothesis was developed to explain the regulation of transcription and chromatin remodeling. According to this model, histone modifications, alone or in combination, on the same or neighboring histones, actively direct specific nuclear processes such as transcriptional activation. Kelleher’s goal was to use TDMS to establish a “basis set” of expressed histones in human cells and determine the nature of posttranslational codes. “We wanted to look at the combinations of posttranslational modifications and variant histones in the most unbiased fashion possible,” he says. Classical methods to approach this type of analysis include either the use of antibodies or a bottom-up approach, in which proteins are enzymatically digested into a set of peptides. MS/MS is then used to deduce the peptide sequences, and the peptides are linked back to their parent proteins via com4244
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parisons with a protein database. “Although these methods can be more sensitive,” Kelleher states, “you can’t get a bird’s-eye perspective on the bulk chromatin at the molecular level.” H2A
H2B
H4
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11,373.5 1 Ac, 2 Me 62%
11,359.5 1 Ac, 1 Me 38%
0 Ac 1 Ac 41% 59% 2+
C15
m/z 811
812
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815
816
817
Top-down MS provides a bird’s-eye view of the histone variants that comprise the nucleosome (top) and their modifications.
In the top-down approach, the analysis is performed at the level of intact proteins, rather than peptides. The researchers used ion cyclotron resonance MS and an 8.5-T magnet to perform exquisitely accurate measurements of the molecular weights of the histone variants; these data provided a picture of sample diversity. In contrast, most bottom-up measurements use quadrupolebased or TOF-based instruments, which have moderate mass resolution; that makes it difficult to discriminate among protein forms that may differ only by fractions of an atomic mass unit. After each mass measurement, Kelleher’s group isolated one histone isoform of interest in the mass spectrometer and used electron capture dissociation (ECD) to fragment it. ECD is a relatively new method for fragmenting peptides and proteins, and it generates a rich set of product ions. Unlike more traditional techniques, such as collision-induced dissociation and IR multiphoton dissociation, ECD fragmentation generally can
be used to localize PTMs to precise sites on the protein. Histone variability, therefore, is a perfect test case. Kelleher notes that his group has used TDMS to “identify both histone isoforms and their modifications, getting a largely unbiased picture of the core proteins that comprise chromatin.” In particular, he adds, “we found there is a hierarchy of modifications on the tails of histone H4, as described in the classical literature, where acetylation of lysine residues proceeds in the C- to N-terminal direction. But we have detected definite exceptions.” Kelleher’s group also sought to quantify the abundances of histone variants and their PTMs. Quantification in MS has typically relied on measuring the relative abundances of peptides by using a bottom-up approach to project the proportional abundances of associated proteins. When Kelleher and colleagues dissociated modified histone peptides, they observed a correlation between the relative abundances of the fragment ions and the parent ion. However, the measured ion abundances correlated poorly with the known solution concentrations. For instance, a 1:1 solution mixture of unmodified and triacetylated peptides had a measured ratio near 1:4. However, fragmentation of intact proteins revealed much greater consistency between the relative ratios of the fragment ions and the parent ion and the solution concentrations. For example, when intact recombinant proteins were mixed at a ratio of ~59:41, the observed ratio was ~58:42 with a precision of 5.7% (2). Kelleher’s hypothesis is that ionization efficiency and instrument effects are far more pronounced at the peptide level, whereas analysis of intact proteins ameliorates these systematic biases. Histone quantitation by TDMS may reveal new perspectives for chromatin biochemistry and also, he suggests, within the broader world of quantitative proteomics. “When you build a new telescope, you might find some new moons,” says Kelleher. a —Karen R. Jonscher
