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Supramolecular affinity chromatography for methylation-targeted proteomics Graham A.E. Garnett, Melissa J. Starke, Alok Shaurya, Janessa Li, and Fraser Hof Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04508 • Publication Date (Web): 13 Mar 2016 Downloaded from http://pubs.acs.org on March 14, 2016

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Analytical Chemistry

Supramolecular affinity chromatography for methylationtargeted proteomics Graham A.E. Garnett, Melissa J. Starke, Alok Shaurya, Janessa Li, and Fraser Hof* Department of Chemistry, University of Victoria, Victoria, BC, V8W3V6, Canada *[email protected]

Keywords. Post-translational modification, methylation, affinity chromatography, host-guest chemistry, proteomics, histones ABSTRACT. Proteome-wide studies of post-translationally methylated species using mass spectrometry are complicated by high sample diversity, competition for ionization among peptides, and mass redundancies. Antibody-based enrichment has powered methylation proteomics until now, but the reliability, pan-specificity, polyclonal nature, and stability of the available pan-specific antibodies are problematic and do not provide a standard, reliable platform for investigators. We have invented an anionic supramolecular host that can form host-guest complexes selectively with methyllysine-containing peptides, and used it to create a methylysine-affinity column. The column resolves peptides on the basis of methylation—a feat impossible with a comparable commercial cation-exchange column. A proteolyzed nuclear extract was separated on the methyl-affinity column prior to standard proteomics analysis. This experiment demonstrates that such chemical methyl-affinity columns are capable of enriching and improving the analysis of methyllysine residues from complex protein mixtures. We discuss the importance of this advance in the context of biomolecule-driven enrichment methods.

INTRODUCTION Post-translational modification (PTM) is a critical component of many cellular pathways. Prominent examples include methylation, phosphorylation, acetylation, and ubiquitylation. Posttranslationally methylated lysine and arginine residues are central players in epigenetic pathways and are the subject of intense research into their roles in development, stem cell pathways, and disease.4 DNA-packaging histones were the first proteins whose methylation was intensely studied, but it is now clear that all plant and animal proteomes have many hundreds of proteins that are regulated by methylation.5-8 Methylation stands apart from other post-translational modifications in multiple ways. It is the smallest group that can be added to a biomolecule. While many PTMs change a protein’s bulk properties by installing charge on a neutral site (phosphorylation, sulfation) or rendering a charged residue neutral (acetylation, citrullination), methylation does not significantly change the charge or pKa’s of lysine or arginine side chains. Unlike all other PTM’s, methyl groups are installed and removed by enzymes that must control the number of resulting methyl groups installed with high specificity. Lysine can be mono-, di-, or trimethylated, and arginine can be monomethylated or dimethylated (with dimethylarginine existing as two isomeric marks) (Figure 1a). Even when they occur at the same site, each kind of methyl mark encodes distinct epigenetic signalling outcomes. Protein methylation therefore defines an entire class of PTMs that generate enormous diversity in biochemical structure and function, without generating a large change in physicochemical properties. Analysis of methyl marks remains difficult. Standard proteomics analysis—proteolysis followed by LC-MS/MS—allows one to identify high-abundance methylated residues, but compe-

tition for ionization in complex samples suppresses signals for low-abundance analytes. Selective pre-enrichment is therefore a critical step. A variety of antibodies have been developed that can bind specific analytes, but pan-specific reagents (those that bind to a certain modification only, but without regard for its surrounding peptide sequence) remain the most desirable and powerful reagents for discovery (see Discussion, below). The well-known shortcomings of antibodies that target methyl PTMs continue to hinder progress in this field.9-12 Reproducibility and pan-specificity are difficult to achieve. All antibodies give different results from each other,13 and the field has not settled on a single, standard antibody-based affinity enrichment tool. Chemical affinity reagents are often intrinsically pan-specific, have superior lab-to-lab reproducibility, and have dominated the study of certain other PTMs. The chemical affinity enrichment of phospho-proteins has grown into an entire, rich field of research.14 Immobilized metal affinity chromatography (IMAC) is the main technique that is widely used for phosphorylated proteins,15 and has spawned many daughter technologies.14 An IMAC variant selective for pyrophosphorylated peptides was recently reported.16 Molecular imprinted polymers (MIPs) (‘plastic antibodies’) have been demonstrated for pan-specific enrichment tasks in phospho-proteomics17,18 and in sulfoproteomics.19 Citrullination has also been targeted, in this case by using a glyoxal-based affinity reagent that undergoes covalent reaction with the unique, urea-type citrulline side chain.20,21 Each of the above examples achieves pan-specificity in a chemical way that allows them to achieve PTM discovery and/or quantification from complex proteomics samples. In spite of intense interest in methylation biology, no chemically based enrichment of methylated peptides exists. We and others

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have created supramolecular hosts that can bind methylated amino acids, peptides, and proteins in solution.22-34 Our favoured scaffold for this task is an anionic sulfonated calix[4]arene. This synthetic binding pocket binds weakly to cationic patches on peptides and proteins,35-42 and we have shown that it can be synthetically modified to tune affinities strongly toward methyllysines.43,44 Neri et al. have tuned a calixarene scaffold to provide a high specificity ligand for a single target in the proteome.45 Our idea at the outset of this study was diametrically opposed to this high-specificity, medicinal chemistrylike approach—we aim to create broadly useful, pan-specific tools for methyllysine marks. We report here the first chemical affinity column for methyllysines, and demonstrate that it enriches for methyllysines when used within proteomics workflows.

EXPERIMENTAL SECTION Peptide synthesis, calixarene synthesis, and indicator displacement assays are described in the Supporting Information. The affinity columns were prepared as follows: AffiGel-102 cross-linked agarose resin (Bio-Rad) was coupled to calixarene 2 using EDC by the instructions provided. To monitor the progress of the coupling, the supernatant of the coupling reaction was sampled and monitored by LC-MS using a Phenomenex Luna C18 analytical column. After coupling, the resin was filtered over a glassfritted funnel and rinsed with 2 M NH4Cl and diH2O before being stored in a 1.5 mL microfuge tube suspended in diH2O. Empty 1 mL columns (35 x 6.2 mm, Agarose Bead Technologies) were loaded with resin 2-aga following the instructions provided. Capillary columns (1 x 600 mm) were prepared from 1/16” OD, 0.040” ID PFA tubing (IDEX). Tubing was cut to the desired length and packed with resin using a syringe pump, with the ends of the capillary sealed with 10 µm pore frits enclosed in a union assembly. For each type of column, thorough washing of the resin with 50-100 CV of elution buffer was performed prior to first sample injection. The 1 mL HiTrap SPXL ion exchange column (GE Healthcare) was used as purchased. Elution protocols were adapted from normal ion exchange stepgradient elution protocols, involving running buffer (RB) for sample loading and elution buffer (EB) for elution of strongly retained material. Either NaCl or NH4Cl were used as high ionic strength component of the elution buffer (see main text for the RB and EB composition for each experiment). A general gradient consisted of an initial 5-10 column volumes (CV) of low ionic strength running buffer, such as 50 mM aqueous phosphate buffer, pH 7.5. A switch of up to 2 M NH4Cl elution buffer would then be applied over 5 CV and held for 5 CV to ensure the elution of all bound species. Flow rates were chosen to limit back pressure to ≤1 bar, preventing collapse of the agarose bead bed. Detailed elution programs are provided in Supporting Information. Calf thymus histone (Worthington Chemicals) were incubated at 100:1 (w/w) with ArgC protease (Sigma Aldrich) at 37°C for 18 h in 100 mM NH4HCO3. After incubation, digested samples were frozen at –4°C and thawed immediately before use. Fractions as eluted from column 2-aga were collected in 1 mL aliquots in Eppendorf tubes and pooled into two samples: flow-through fraction (S2), and retained fraction (S3). Along with the input sample (S1), these fractions were submitted for analysis at the UVic-Genome BC Proteomics Centre. Full details of Orbitrap LC-MS/MS analysis and data processing are provided in Supporting Information.

RESULTS The parent p-sulfonatocalix[4]arene (1) binds trimethyllysines, but cannot easily be connected to solid support. Modification of the lower rim of sulfonato calixarenes can be detrimental to binding,27 just as connection of another macrocycle to an anchoring biotin group affected that host’s methyllysine selectivity.33 Compound 2 has an upper-rim aryl group attached via sulfonamide linkage, (Figure 1c) while also including a dis-

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tal carboxy group for conjugation to solid supports. We determined the solution-phase affinities of 2 for various methyllysine and methylarginine containing peptides by fluorescence indicator displacement assay (Figure 1d).1,2,44 Host 2 shows submicromolar affinities for Kme2, Kme3, and aDMA-containing peptides, and weaker binding to unmethylated controls and Kac containing peptides. It can bind both H3K4me3 and H3K9me3 peptides, showing that it is less sensitive to the sequence surrounding a particular methylation site than it is to the absence/presence of the trimethyllysine residue itself. Compound 2 was conjugated to a cross-linked agarose solid support by reaction with aminopropyl-functionalized AffiGel102 (BioRad) using the peptide coupling agent EDC (Figure 1c). After coupling was complete, the resin 2-aga was filtered, washed extensively with MeOH and H2O, and packed into a standard 1 mL glass column (6.2 x 35 mm) or a teflon capillary column (1 x 600 mm). We performed chromatography using a test set of peptides representing varied lengths, charges, and methylation states that would be expected in a typical methylation-driven proteomics experiment. Our 7-mer peptides represented histone 3 residues 24-30, bracketing well known methylation site H3K27 and having net charge of +2. Our 12-mer peptides represent histone 3 residues 1-12, home to both H3K4 and H3K9 methylation sites and bearing an overall charge of +5. The sulfonates of the affinity reagent 2-aga provide a solidphase reagent with strong cation-exchange functionality. In order to provide a baseline for understanding its behaviour, we first used a commercial, sulfonate-based strong cation exchange resin (SPXL, GE Healthcare), and found that ion exchange alone is incapable of resolving methylated and unmethylated peptides (Supp. Info.). Column 2-aga, containing multiple sulfonates on its affinity handle, retained peptides much more strongly than did SPXL. We found that histone-derived peptides would not elute from column 2-aga using the SPXL running buffer/elution buffer program. The test peptides eluted with reasonable retention times and peak shapes only when the elution buffer was changed from its original value of 1 M NaCl to 2 M NH4Cl. Figures 2a and 2b show that the 1 mL column of 2-aga retains peptides differently on the basis of their methylation. The 7-mer peptides based on H3K27 and H3K27me3 (having net charge of +2 each) elute separately, and are baseline separated when co-injected. The longer peptides based on H3(1-12) have net charge of +5 each, and are only partially resolved under the same running conditions. Figures 2c and 2d show the impact of changing to the longer, skinnier capillary column (1 x 600 mm). Chromatograms for 7mer (+2) peptides are still cleanly resolved, and the 12-mer peptides H3K4me3 and H3K9me3 are better resolved from their unmethylated counterpart. The traces in Figures 2e and 2f show how more subtly different methylation states behave on column 2-aga. Acetyllysine, as expected of a charge-neutralizing modification, causes dramatic reduction in retention times of analyte H3K4ac. This is significant because of the known problems with isobaric Kme3 and Kac peptide fragments in proteomics analysis.46 Mono-, di-, and trimethyllysine provide incrementally longer retention times than the unmethylated control. Asymmetric dimethyl arginine (aDMA) is retained better than is symmetric dimethylarginine (sDMA), in line with the solution phase affinities determined for these two analytes. We conclude that the affinity column operates via methylspecific affinity layered on top of a background of very strong electrostatic attraction (ion exchange). Tuning the elution condi-


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tions provided a series of chromatograms that suggest some degree of ‘pan-specific’ affinity for many kinds of posttranslational methylations. Analytes with lower affinities as determined in the simple phosphate buffer solution (Figure 1d) are less well retained by the column under forcing salt concentrations. We next tried to demonstrate methylation-discovery proteomics experiments. The net charge on peptides in proteomics samples would likely span the whole range of our test set of peptides from +2 (in which case we expect excellent resolution of methylated analytes) to +5 (in which case we would expect reduced discrimination, with some highly cationic unmethylated analytes eluting along with the retained fraction). But we would expect based on our control studies that all methylated analytes would be retained, and that this would provide an improvement in their observation in the retained fraction due to the removal of most (if not all) competing unmethylated analytes. In order to test the column’s performance we chose ‘calf thymus histones,’ a fractionated tissue extract that, in spite of its name, actually contains dozens of important histone and nonhistone nuclear proteins bearing every major kind of posttranslational modification. We identified 38 different nuclear proteins at ≥50% confidence under the conditions of our proteomics experiments (Supp. Info.). We treated the sample with the protease ArgC prior to running the resulting peptide mixtures on capillary column 2-aga. The input sample (S1), the unretained fraction (S2), and the retained fraction (S3), were subjected to LC-MS/MS analysis (see Supporting Information). Peptide sequences and posttranslational modification states/sites were determined by molecular weight and MS/MS fragmentation patterns using Mascot and Scaffold. Peptide identities were assigned by comparison to the Bos taurus proteome. A complete list of proteins and PTMs identified in each fraction are provided in the supporting information. Several conclusions can be drawn from these data: - The column behaves as predicted by studies with pure peptides even when handling complex mixtures. The trace (Figure 3a) is identical to those observed for purified peptides. There is no significant retention bias based on peptide size or molecular weight (Figure 3b). From the differences in peptide charges between unretained and retained fractions we conclude that the column is acting partially as a cation exchanger, but the strongly overlapping ranges of net charges observed in each fraction show that its retention of analytes is not exclusively governed by charge. - Figure 3b shows that enriched fraction S3 has spectral counts—a crude measure of abundance used in proteomics experiments—that are lower than input overall (indicating some selection of peptide content) but proportionally higher in PTMs (generally showing enrichment in PTMs relative to input). Some highly cationic, but unmethylated analytes are expected from ArgC cleavage of histones (Supp. Info.) and are also expected to be retained alongside methylated analytes in this fraction. Globally, the proof of enrichment operating successfully is shown by the fraction of unmethylated analytes in S3 being much lower in enriched fraction S3 than in either input control S1 or unretained fraction S2. - The column 2-aga enables observation of unique lysine methylation marks. Together, Kme1, Kme2, Kme3 modifications are more often observed in the retained fraction (S3; total of 26 sites) relative to the unretained fraction (S2, total of 5

sites, none of which are uniquely observed in S2) (Figure 3c). Figure 3d shows the redundancy or uniqueness for each observed methyllysine site in the three fractions, and proves that the methyllysine sites observed in S3 are most often unique sites not otherwise observed in S1 or S2. Kme3 sites are only observed in the retained S3 fraction. This shows that methylated analytes that are present, but whose ionization is inefficient in the complex input mixture (S1) are excluded from unretained fraction S2 but are identifiable after enrichment in fraction S3. - Column 2-aga does not improve observation of methylarginine containing peptides. Rme1/Rme2 modifications are more readily seen in the input control S1 than in either of the eluted fractions S2 or S3 (Figure 3c). - Many canonical histone methylation sites are identified in the samples (Table S1, ESI). H3K27 methylation is expected to be abundant and is visible in both the input control and the enriched fraction. H3K9, H3K36, and less well-known (and presumably less abundant) H3K37 methylations are visible only in enriched sample S3 and not in the input S1. Opposite to this general trend, H3K79 methylation is observed in S1 and S2, but not S3 fractions. Even in this proof-of-concept experiment on a relatively simple tissue extract, some never-before-seen methylation sites were suggested to exist with high probabilities (Table S1, ESI). Histone 1.3 is trimethylated at K16 and monomethylated at K34. Histone 1.3 was not previously reported as methylated at these residues according to UniProt, PhosphositePlus (a comprehensive PTM database),8 or a recently published antibodyenrichment-driven data set.6 HP1-binding-protein-3 (HP1BP3) is methylated at multiple sites in our data set, but has not otherwise been identified as a methylated protein in other data sets. Most importantly for the goal of this work—establishing the physico-chemical properties and performance of a new affinity medium—the lysine methylations seen for H1.3 and HP1BP3 are only visible after enrichment, are not visible in the input control, and were not visible when we used a commercial panmethyl antibody kit to enrich an identical sample of calf thymus histones (Supp. Info.).

DISCUSSION Chemical enrichment is fundamentally different from biomolecule-driven enrichment. Antibodies can be produced using more-or-less generic haptens, bearing a certain PTM in a structural context that aims not to be recognized by the host animal’s immune system. These efforts provide researchers with valuable ‘pan-specific’ antibodies—those that recognize a given kind of PTM with no regard for the surrounding sequence. Efforts to do so for methyllysines have been frustrating. After years of development effort, one monoclonal, pan-specific methyllysine antibody was recently released for sale, but the product data sheet suggests it has a preference for Kme1 over Kme2 or Kme3.47 Because of a lack of monoclonals, all published methyllysine antibody papers to date have used polyclonal reagents.5,6,13 Cross-reactivity rates are known to be especially high for anti-PTM antibodies in general,11,12 and moreso for pan-specific polyclonal antibodies. Only one pan-specific polyclonal antibody—claiming antiKme1Kme2 activity (Abcam Ab23366)—has had test results posted in a new online database;11 only three of its top 20 targets from among an array of hundreds of PTM peptides actually have Kme2 residues, and zero of the top 20 hits have Kme1 residues. As of the writing of this paper, other pan-specific antibodies had not yet had results from this rigorous peptide array


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method posted in the database. One other useful analysis of the current state of technology comes from a side-by-side comparison of five pan-methyl polyclonals.13 Of the 53 methylated analytes observed in that study, only 2 were identified in common by all five antibodies. In spite of such issues, pan-specific polyclonal antibodies have driven a huge expansion of our knowledge of the methylated proteome beyond the few canonical histone methylation sites. In recent examples, cell-lysate enrichments were performed with pan-specific antibody mixtures developed for each of mono-, di-, trimethyllysine, monomethylarginine, and/or asymmetric dimethylarginine prior to proteomics analysis.5,6,13 The authors of each study report the discovery of hundreds of new methyllysine and methylarginine sites, demonstrating the breadth of proteome methylation and the value of enrichment tools in general. A few other biomolecule-driven approaches to PTM enrichment have been developed. In a ‘chemical proteomics’ variation on antibody-driven approaches, cell lysates were chemically treated with propionic anhydride prior to digestion and enrichment with an antibody specific for the propionylated Kme1 side chain.48 The authors identified 446 Kme1 sites. Reactivitydriven approaches like this cannot be applied to Kme2/Kme3 because of the lack of reaction between electrophilic reagents with the tertiary/quaternary amines on Kme2/Kme3 side chains. Other efforts have gotten rid of antibodies altogether by using methyl reader proteins as a basis for engineering affinity reagents.49 In one example, a reader protein MBT domain—known to care little for the surrounding sequence of its Kme2 targets in nature—was engineered to create a pan-specific Kme1Kme2 prospecting reagent.7 Again, the value of pre-enrichment was demonstrated by the resulting identification of large numbers of never-before-seen methylation sites in the proteome. Supramolecular affinity reagents like 2-aga are different. Their small binding pockets mean that they have limited ability to interact with their targets compared to the extensive binding surfaces present on antibodies or engineered proteins. This makes them inherently well suited to pan-specific recognition of PTMs, simply because they do not have other binding elements that could engage residues surrounding the PTM of interest. A chemical affinity reagent is expected also to provide superior batch-to-batch reproducibility and stability. Compound 2 is easily purified to homogeneity on HPLC before coupling to solid support, and provided identical data even when a separate batch of 2 was synthesized, coupled to new agarose beads, and packed into a column by two different students several months apart from each other. We have not torture-tested its chemical stability, but it is synthesized under harsh conditions (including a penultimate step in hot H2SO4), meaning that the limiting shelf stability of the affinity reagent 2-aga will almost certainly arise from the solid support, as opposed to the affinity handle itself.

CONCLUSIONS Our data prove the concept that solution-phase supramolecular host can successfully operate as a methyllysine-targeting affinity column, and allow us to understand the molecular mechanisms operating when eluting peptides as pure or mixed samples. We will seek in the future to reduce the background binding of highly cationic unmethylated analytes, but even this first-generation column shows that the chemical affinity approach can be applied in a way that improves proteomics analysis of methyllysines. The approach is compatible with existing proteomics workflows, and offers a new tool that is different from the antibodies that dominate modern epigenetics research. This applied supramolecular tool will drive new experiments in

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discovery science (prospecting for new methylation sites), in diagnostics (enrichment to improve quantification of methylated analytes), and other basic tasks in epigenetics research. Supporting Information Synthetic methods, indicator displacement data, elution time programs, LC-MS/MS protocols, complete tables of proteins and modification sites/states identified by proteomics experiments, comparison to antibody-based enrichment data. The Supporting Information is available free of charge on the ACS Publications website.

Acknowledgements We thank Darryl Hardie and Leonard Foster for helpful discussions. MS thanks the BC Proteomics Network and AS thanks UVic for fellowship support. FH thanks the Canada Research Chairs program.

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Figure 1. (a) Post-translational methylations of lysine and arginine. (b) p-sulfonatocalix[4]arene 1 forms a host-guest complex in which the methylated sidechains of trimethyllysine-containing peptides are bound within its cup-shaped binding pocket. (c) Compound 2 is attached to an agarose support to make solid-linked affinity reagent 2-aga, which can be packed into standard 1 mL columns or capillary columns. (d) Solution-phase equilibrium dissociation constants (Kd) of 1 and 2 for a panel of PTM-containing peptides. All values were determined in 10 mM phosphate buffer at pH 7.4 and are averages of duplicate determinations by an indicator displacement assay originally created by Nau for related analytes.1-3 Uncertainties are reported as standard deviations. *All H3K4 peptides are variations of ARTXQTAY and all H3R2 peptides are variations of AXTKQTAY. **H3K9 is TARXSTGY. See Supporting information for details. (e) Photo of 2-aga 1 mL column. (f) Photo of 2-aga capillary.


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Figure 2. (a, b) The affinity column 2-aga can resolve on the basis of methylation, with better resolution for peptides with lower net charge. Chromatograms arising from 1-mL column (6.2 x 35 mm) containing stationary phase 2-aga treated with 7-mer (+2 charge) peptides (a) and 12-mer (+5 charge) peptides (b) with or without trimethyllysine marks. (c, d) A capillary column form factor improves resolution. Chromatograms arising from capillary column (1 x 600 mm) containing stationary phase 2-aga treated with 7-mer (+2 charge) peptides (c) and 12-mer (+5 charge) peptides (d) with or without trimethyllysine marks. (e, f) Elution profiles for a complete set of lysine and arginine PTMs within the same background peptide sequence. Chromatograms arising from capillary column (1 x 600 mm) containing stationary phase 2-aga treated with H3(1-7) peptides bearing no modification, Kme1, Kme2, or Kme3 marks (e) or bearing no modification, Kac, aDMA, or sDMA marks (f). All H3(1-7) peptides are +3 overall charge, except H3K4ac which is +2. All chromatograms collected with running buffer (RB = 50 mM phosphate, pH 7.5), followed by elution buffer (EB = running buffer plus 2 M NH4Cl). Change from RB to EB is by linear gradient during 10-15 minutes (a,b) or 40-60 minutes (c-f). See Supporting Information for detailed time programs.


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Figure 3. The column 2-aga can enhance analysis of methyllysines. (a) Chromatographic trace arising from ArgC-proteolyzed commercial extract ‘calf thymus histones’ eluted on the 2-aga capillary column. See Methods. (b) Analysis of peptide sets identified in each fraction reveal column’s basic properties. Uncertainties are reported as standard deviations. * The mean solution-phase charges on the peptides at pH 7.4 were predicted by totalling the number of cationic and anionic side chains. See Supp. Info. for predicted charges of ArgC cleavage products. Total spectral counts as reported by Scaffold demonstrate that the retained fraction has fewer peptides with a greater proportion having PTMs. (c) Occurrence of post-translational modification sites in peptides arising from ArgC-proteolysis of calf thymus histones before and after separation on column 2-aga. (d) Separation on 2-aga helps identify otherwise unobserved modification sites. Unique and shared observations of particular Kme1, Kme2, and Kme3 sites in each fraction are shown. The PTM sites that are only observed in the retained fraction S3 after enrichment are in the shaded sector. See Supporting Information for complete lists of identified proteins and modifications.


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Supramolecular Affinity Chromatography for ... - ACS Publications

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