Conventional 'bottom-up' proteomics, in which mass spectrometry is used to analyse peptide mixtures made by tryptic digestion of target proteins, is a powerful way of characterizing complex proteomes. However, the technique has limitations when considering different protein isoforms and combinations of post-translational modifications. The 'top-down' approach is generally thought to be impractical because of the limitations of mass spectrometry and difficulties with automation. A new top-down system presented here avoids these problems by using a four-dimensional separation system that achieves greater proteome coverage than conventional methods. A proof-of-principle experiment shows that the method is capable of identifying previously undetected isoforms and isoform-specific post-translational modifications caused by cellular senescence. A full description of the human proteome relies on the challenging task of detecting mature and changing forms of protein molecules in the body. Large-scale proteome analysis1 has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry2. This ‘bottom-up’ process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous2 characterization of alternative splice forms, diverse modifications (for example, acetylation and methylation) and endogenous protein cleavages, especially when combinations of these create complex patterns of intact protein isoforms and species3. ‘Top-down’ interrogation of whole proteins can overcome these problems for individual proteins4,5, but has not been achieved on a proteome scale owing to the lack of intact protein fractionation methods that are well integrated with tandem mass spectrometry. Here we show, using a new four-dimensional separation system, identification of 1,043 gene products from human cells that are dispersed into more than 3,000 protein species created by post-translational modification (PTM), RNA splicing and proteolysis. The overall system produced greater than 20-fold increases in both separation power and proteome coverage, enabling the identification of proteins up to 105 kDa and those with up to 11 transmembrane helices. Many previously undetected isoforms of endogenous human proteins were mapped, including changes in multiply modified species in response to accelerated cellular ageing (senescence) induced by DNA damage. Integrated with the latest version of the Swiss-Prot database6, the data provide precise correlations to individual genes and proof-of-concept for large-scale interrogation of whole protein molecules. The technology promises to improve the link between proteomics data and complex phenotypes in basic biology and disease research7.

The multiple myeloma SET domain (MMSET) protein is overexpressed in multiple myeloma (MM) patients with the translocation t(4;14). Although studies have shown the involvement of MMSET/Wolf-Hirschhorn syndrome candidate 1 in development, its mode of action in the pathogenesis of MM is largely unknown. We found that MMSET is a major regulator of chromatin structure and transcription in t(4;14) MM cells. High levels of MMSET correlate with an increase in lysine 36 methylation of histone H3 and a decrease in lysine 27 methylation across the genome, leading to a more open structural state of the chromatin. Loss of MMSET expression alters adhesion properties, suppresses growth, and induces apoptosis in MM cells. Consequently, genes affected by high levels of MMSET are implicated in the p53 pathway, cell cycle regulation, and integrin signaling. Regulation of many of these genes required functional histone methyl-transferase activity of MMSET. These results implicate MMSET as a major epigenetic regulator in t(4;14)+ MM.

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.

Fibroblast growth factor receptors (FGFR) are cell surface tyrosine kinases that function in cell proliferation and differentiation. Aberrant FGFR signaling occurs in diverse cancers due to gene amplification, but the associated oncogenic mechanisms are poorly understood. Using a proteomics approach, we identified signal transducers and activators of transcription-3 (STAT3) as a receptor-binding partner that is mediated by Tyr⁶⁷⁷ phosphorylation on FGFR. Binding to activated FGFR was essential for subsequent tyrosine phosphorylation and nuclear translocation of STAT3, along with activation of its downstream target genes. Tyrosine phosphorylation of STAT3 was also dependent on concomitant FGFR-dependent activity of SRC and JAK kinases. Lastly, tyrosine (but not serine) phosphorylation of STAT3 required amplified FGFR protein expression, generated either by enforced overexpression or as associated with gene amplification in cancer cells. Our findings show that amplified FGFR expression engages the STAT3 pathway, and they suggest therapeutic strategies to attack FGFR-overexpressing cancers. Cancer Res; 70(8); 3391–401. ©2010 AACR.

Key Points: Data from Dnmt3a−/− mice implicate Dot1l as a critical mediator of the malignant gene expression program of Dnmt3a-mediated leukemia. Pharmacologic inhibition of DOT1L exerts potent antileukemic activity in DNMT3A-mutant human acute myeloid leukemia in vitro and in vivo.

Natural biological enzymes possess catalytic sites that are generally surrounded by a large three-dimensional scaffold. However, the proportion of the protein molecule that participates in the catalytic reaction is relatively small. The generation of artificial or miniature enzymes has long been a focus of research because enzyme mimetics can be produced with high activity at low cost. These enzymes aim to mimic the active sites without the additional architecture contributed by the protein chain. Previous work has shown that amyloidogenic peptides are able to self-assemble to create an active site that is capable of binding zinc and catalysing an esterase reaction. Here, we describe the structural characterisation of a set of designed peptides that form an amyloid-like architecture and reveal that their capability to mimic carbonic anhydrase and serve as enzyme-like catalysts is related to their ability to self-assemble. These amyloid fibril structures can bind the metal ion Zn²⁺via a three-dimensional arrangement of His residues created by the amyloid architecture. Our results suggest that the catalytic efficiency of amyloid-like assembly is not only zinc-dependent but also depends on an active centre created by the peptides which is, in turn, dependent on the ordered architecture. These fibrils have good esterase activity, and they may serve as good models for the evolution of modern-day enzymes. Furthermore, they may be useful in designing self-assembling fibrils for applications as metal ion catalysts. This study also demonstrates that the ligands surrounding the catalytic site affect the affinity of the zinc-binding site to bind the substrate contributing to the enzymatic activity of the assembled peptides.