Neil L. Kelleher

Proteoform: a single term describing protein complexity

genetic differences and not to variation at the protein level2. The term “protein species” was proposed in 2009 (ref. 2) but does not distinguish between proteins originating from different genes and those originating from a single gene, and thus we find it confusing. A similar issue arises with the term “protein variants.” The UniProt Knowledgebase (a definitive, gene-centric protein database)5 uses the term “isoform” in yet a different manner, one that denotes related forms of protein molecules arising from the same gene by alternative splicing or variable promoter usage (Fig. 1). Such events create a variable set of protein sequences that significantly change the numbering of amino acids for the protein as compared to the canonical sequence. These changes to the base primary sequence are referred to by some as “isoforms” and are denoted in UniProt by a –1, –2 and so on following the accession number (Fig. 1). However, genetic changes (for example, mutations and polymorphisms) are not covered by this terminology and create a conflict with the IUPAC definition of isoform2. Differences in IUPAC and UniProt definitions notwithstanding, the terms “variants” and “isoforms” were intended to describe proteins derived from distinct DNA or RNA; their use to describe modified proteins is confusing. Accordingly, we propose that the term ‘proteoform’ be used to designate all of the different molecular forms in which the protein product of a single gene can be found, including changes due to genetic variations, alternatively spliced RNA transcripts and posttranslational modifications (Fig. 1). Any gene or protein processing events such as those using inteins or RNA-editing mechanisms are now covered cleanly by the term ‘proteoform’. The term should include all post-translational modifications in the PSI-MOD ontology except those classified as reagent-derivatized or isotope-labeled residues (see the Supplementary Note for a precise definition). Products of multigene families should continue to be categorized on the basis of sequence identity (for example, >90%, >99% and so on). The term is compatible with a gene-centric approach for referring to proteins, which we support, because grouping related forms of proteins together even though they are the products of different genes leads to imprecision in protein identification5. We have begun to use the term ‘proteoform’ in our own writing and presentations, and we find it to be intuitive and readily grasped Proteoform: a single term describing protein complexity

Mapping intact protein isoforms in discovery mode using top-down proteomics

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 analysis has routinely involved digesting intact proteins followed by inferred protein identification using mass spectrometry. This ‘bottom-up’ process affords a high number of identifications (not always unique to a single gene). However, complications arise from incomplete or ambiguous 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 species. ‘Top-down’ interrogation of whole proteins can overcome these problems for individual proteins, 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 database, 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 research.

Decoding protein modifications using top-down mass spectrometry

Top-down mass spectrometry is an emerging technology which strives to preserve the post-translationally modified forms of proteins present in vivo by measuring them intact, rather than measuring peptides produced from them by proteolysis. The top-down technology is beginning to capture the interest of biologists and mass spectrometrists alike, with a main goal of deciphering interaction networks operative in cellular pathways. Here we outline recent approaches and applications of top-down mass spectrometry as well as an outlook for its future.

Precision proteomics: The case for high resolution and high mass accuracy

Proteomics has progressed radically in the last 5 years and is now on par with most genomic technologies in throughput and comprehensiveness. Analyzing peptide mixtures by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) has emerged as the main technology for in-depth proteome analysis whereas two-dimensional gel electrophoresis, low-resolution MALDI, and protein arrays are playing niche roles. MS-based proteomics is rapidly becoming quantitative through both label-free and stable isotope labeling technologies. The latest generation of mass spectrometers combines extremely high resolving power, mass accuracy, and very high sequencing speed in routine proteomic applications. Peptide fragmentation is mostly performed in low-resolution but very sensitive and fast linear ion traps. However, alternative fragmentation methods and high-resolution fragment analysis are becoming much more practical. Recent advances in computational proteomics are removing the data analysis bottleneck. Thus, in a few specialized laboratories, “precision proteomics” can now identify and quantify almost all fragmented peptide peaks. Huge challenges and opportunities remain in technology development for proteomics; thus, this is not “the beginning of the end” but surely “the end of the beginning.”

EZH2 is required for germinal center formation and somatic EZH2 mutations promote lymphoid transformation

The EZH2 histone methyltransferase is highly expressed in germinal center (GC) B cells and targeted by somatic mutations in B cell lymphomas. Here, we find that EZH2 deletion or pharmacologic inhibition suppresses GC formation and functions. EZH2 represses proliferation checkpoint genes and helps establish bivalent chromatin domains at key regulatory loci to transiently suppress GC B cell differentiation. Somatic mutations reinforce these physiological effects through enhanced silencing of EZH2 targets. Conditional expression of mutant EZH2 in mice induces GC hyperplasia and accelerated lymphomagenesis in cooperation with BCL2. GC B cell (GCB)-type diffuse large B cell lymphomas (DLBCLs) are mostly addicted to EZH2 but not the more differentiated activated B cell (ABC)-type DLBCLs, thus clarifying the therapeutic scope of EZH2 targeting.

Attomole protein characterization by capillary electrophoresis-mass spectrometry

Electrospray ionization with an ultralow flow rate (≤4 nanoliters per minute) was used to directly couple capillary electrophoresis with tandem mass spectrometry for the analysis and identification of biomolecules in mixtures. A Fourier transform mass spectrometer provided full spectra (>30 kilodaltons) at a resolving power of ≈60,000 for injections of 0.7 × 10−18 to 3 × 10−18 mole of 8- to 29-kilodalton proteins with errors of <1 dalton in molecular mass. Using a crude isolate from human blood, a value of 28,780.6 daltons (calculated, 28,780.4 daltons) was measured for carbonic anhydrase, representing 1 percent by weight of the protein in a single red blood cell. Dissociation of molecular ions from 9 × 10−18 mole of carbonic anhydrase gave nine sequence-specific fragment ions, more data than required for unique retrieval of this enzyme from the protein database.

Top Down Proteomics

The field of proteomics has reached a level of maturity where significant biological discoveries are being made with current technology. By far the largest numbers of proteomics experiments employ a “bottom-up” protein analysis. In this strategy proteins or protein mixtures are digested with a protease and then analyzed en masse in a “shotgun” format. Tandem mass spectrometry is then used to sequence peptides, and informatics tools are used to identify the sequences and assign them to proteins or genes. This same strategy can be used to identify modifications to peptides. This strategy works very well to identify the proteins present and their modifications. A challenge for bottom-up proteomics is the proper assignment of protein isoforms and patterns of modifications that create the many proteoforms present in cells. Why is this important? The functional sophistication of complex organisms, like humans, is driven by the ability to use a limited number of gene sequences in many different ways. Determining the precise proteoform involved in a specific function will be key for dissecting out the molecular mechanisms used by eukaryotic organisms. A key technology to determine this information is “top down” mass spectrometry. In this method, intact proteins are analyzed to both identify the protein and to determine the identity and location of modifications within the protein using a tandem mass spectrometer. The analysis of intact proteins by mass spectrometry is a major challenge. The first problem for top down analysis is fractionation of proteins. Traditional methods such as gel electrophoresis have limitations on the recovery of proteins in a form suitable for analysis by mass spectrometry. Extracting gel-separated proteins from the polyacrylamide matrix requires electroelution or electroblotting, frequently resulting in sample losses. Chromatographic methods to separate intact proteins can work well, but the resolving power for intact proteins with more than ~500 amino acids is poor. After introduction into the mass spectrometer, amide bonds in the protein backbone must be fragmented to create ladders of sequence information. Because proteins have many chemical bonds that can “soak up” energy put into ions, vigorous research into excitation methods to more efficiently and completely fragment intact protein ions is bearing fruit. The nonergodic methods for protein ion excitation are increasingly able to fragment larger and larger protein ions, which when coupled with improvements in mass resolving power are aiding in robust protein identification. A key technological change underway is the development of lower cost mass spectrometers that are able to analyze intact proteins. This change will put the technology into more hands, which will speed up innovations and developments. A recent American Society for Mass Spectrometry Sanibel Conference meeting for top down mass spectrometry was very well attended and the lectures demonstrated a vibrant and growing subfield of mass spectrometry-based proteomics and biopharmaceutical analysis. To complement this very successful meeting and highlight a growth area, the Journal of Proteome Research and Analytical Chemistry have assembled and published a joint thematic virtual issue of the papers published in these journals on top down proteomics. This thematic issue emphasizes the challenges and successes in the field from preparation and fractionation of intact proteins to methods for direct fragmentation of whole proteins and the informatics for their identification by database retrieval. Recent developments clearly show that top down proteomics is a vigorous and energized area of research as described in a recent Chemical and Engineering News article by Celia Arnaud.

A monovalent streptavidin with a single femtomolar biotin binding site

Streptavidin and avidin are used ubiquitously because of the remarkable affinity of their biotin binding, but they are tetramers, which disrupts many of their applications. Making either protein monomeric reduces affinity by at least 104-fold because part of the binding site comes from a neighboring subunit. Here we engineered a streptavidin tetramer with only one functional biotin binding subunit that retained the affinity, off rate and thermostability of wild-type streptavidin. In denaturant, we mixed a streptavidin variant containing three mutations that block biotin binding with wild-type streptavidin in a 3:1 ratio. Then we generated monovalent streptavidin by refolding and nickel-affinity purification. Similarly, we purified defined tetramers with two or three biotin binding subunits. Labeling of site-specifically biotinylated neuroligin-1 with monovalent streptavidin allowed stable neuroligin-1 tracking without cross-linking, whereas wild-type streptavidin aggregated neuroligin-1 and disrupted presynaptic contacts. Monovalent streptavidin should find general application in biomolecule labeling, single-particle tracking and nanotechnology.

The MMSET histone methyl transferase switches global histone methylation and alters gene expression in t(4;14) multiple myeloma cells

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.

Certain and Progressive Methylation of Histone H4 at Lysine 20 during the Cell Cycle

ABSTRACT 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 G2, M, and G1 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.