PMC:4996381 / 39996-49279 JSONTXT

{"project":"2_test","denotations":[{"id":"27600229-22464331-69479431","span":{"begin":417,"end":418},"obj":"22464331"},{"id":"27600229-22464331-69479432","span":{"begin":2203,"end":2204},"obj":"22464331"},{"id":"27600229-22464331-69479433","span":{"begin":3162,"end":3163},"obj":"22464331"},{"id":"27600229-22464331-69479434","span":{"begin":5222,"end":5223},"obj":"22464331"}],"text":"3. Results and Discussion\nWe previously employed peptide SPOT arrays to probe all possible acetylation sites found on human histones, as well as the effect of neighboring PTMs (including phosphorylation on serine and threonine residues, acetylation of lysine residues and methylation states (mono-, di- and tri-) of lysines) on the recognition of a central acetyl-lysine epitope by human bromodomain protein modules [3]. Although the technology has been in use for several years we had to adapt our own experimental procedures to the system used in order to probe BRD/acetylated lysine interactions. First, we implemented a protocol for detecting BRDs employing hexa-histidine (His6) tagged proteins, as our recombinant platform allowed for rapid scale up of highly pure proteins carrying such an affinity tag. We found His6 to be sufficient both for affinity purification of the protein as well as for detection on SPOT membranes. We did note that GST-fused bromodomains are also soluble and can be recombinantly expressed and purified to high yields, but we decided to avoid using the bulky GST tag as it does introduce dimerization as well as steric hindrance in our detection experiment. We then explored the effects of peptide density in the generated arrays, by using well established protocols for density reduction based on mixing Fmoc-β-Alanine with Ac-β-Alanine during the first step of synthesis, effectively reducing the capacity of each SPOT within the array. This was particularly crucial for control peptides (in our case His8 peptides) that tend to saturate the antibody readout quickly, resulting in un-interpretable results with SPOT overlap.\nWe synthesized histone peptides that were 15 amino acids long onto cellulose membranes using an INTAVIS MultiPep-RSi-Spotter System. Our design covered previously screened peptides from histones H2A, H2B, H3 and H4, focused on the N-terminal part of the histone sequences and carrying one or more acetylated lysine residues (Table S1). We previously observed that strongly interacting peptides resulted in rapid signal saturation using commercial membranes (Amino-PEG500-UC540 sheets optimized for use with the INTAVIS MultiPep instruments) [3] and therefore attempted to reduce the loading capacity on our arrays to account for this effect. Interestingly, control peptides (His8) systematically generated the highest intensity in our detection, however the variability of the control signal within the same array was small (Figure 1A) regardless of the amount of density reduction used during synthesis. We decided that a ratio of Fmoc-β-Alanine to Ac-β-Alanine of 1:6 yielded the best results in terms of control SPOTs (i.e., clear and defined signal with no overlap with adjacent SPOTs) although this is subject to personal preferences rather than numerical justification (Figure S1).\nNext, we used the recombinant first bromodomain of bromodomain containing protein 4 (BRD4(1)), which we previously found to strongly interact with two adjacent acetyl-lysine marks and validated its interaction with histone in solution, employing isothermal titration calorimetry as well as by structural biology [3]. We found a large number of peptides carrying one or more acetylated lysine residues on all four histones to interact with BRD4(1). As expected the intensity of the interaction after quantification varied between membranes but there was no clear correlation between SPOT-capacity reduction and binding. Peptides that we previously found to bind in a weak fashion to this protein domain showed a general reduction of SPOT intensity as we reduced the amount of SPOT density (Figure 1B–E). Interestingly, peptides that carried multiple acetyl-lysine marks and were previously found to bind in a 1:1 ratio to BRD4(1) with low double to single digit μM affinity, also exhibited a gradual loss of SPOT intensity as we decreased the SPOT loading (Figure 1F). However several peptides, often carrying a single acetylation site, had non-consistent patterns of SPOT intensity in our entire membrane panel, suggesting that non-specific binding may be taking place (Figure 1C).\nFigure 1 Comparison of SPOT intensity differences following SPOT loading capacity reduction. Peptide arrays were prepared using a ratio of Fmoc-β-Alanine to Ac-β-Alanine that ranged from 1:0 to 1:10 in the first step of synthesis, effectively reducing the amount of peptide found on each SPOT of the array. (A) Binding of an His-tag® Antibody HPR conjugated (Novagen) to control peptides carrying 8 histidine residues (His8). Reduction of SPOT density did not show a dramatic difference in SPOT intensity, however SPOTs were much smaller and better defined as the SPOT loading was reduced. (B–F) Normalized SPOT intensity of histone H2A (B), H2B (C), H3 (D) and H4 (E,F) peptides bound to BRD4(1). Weaker peptides exhibited a drop in intensity as the SPOT density decreased, while non-specific peptides showed large variations. Interestingly, strongly binding peptides carrying 2 acetyl-lysine modifications (e.g., shown in F) exhibited overall a decrease in intensity when the SPOT loading was decreased and the binding was 1:1 (as previously determined by in solution binding studies in [3]), however this was not clear when the peptide:protein binding ratio was not 1:1, as in the case of H4K12ac/K16ac. In order to better understand this data we used a commercial peptide array (AltaBiosciences Histone Set 4) which contained biotinylated 20-amino-acid long peptides carrying single or multiple acetyl-lysine modifications on histones H2A, H2B, H3 and H4, with significant overlap with our membrane SPOT design (Table S1). Using biolayer interferometry we measured the ability of recombinant BRD4(1) to bind to these peptides. Comparison of the normalized BLI data to the SPOT results revealed good overlap and explained the non-specific strong signal seen with many peptides on the SPOT assay (Figure 2). For example peptides carrying a K23ac or K24ac mark on H2B systematically showed high intensity profiles by SPOT, however they displayed very weak binding by BLI. Interestingly, the histone H4 peptide K8ac/K12ac which was previously found to bind to two protein modules (both by in solution isothermal titration calorimetry as well as by co-crystallizing with BRD4(1)) exhibited strong binding intensity in both the BLI and SPOT assays and its signal was reduced as a function of SPOT loading, suggesting that a secondary orthogonal assay is necessary in order to introduce confidence in the SPOT results.\nFigure 2 Comparison of BRD4(1) binding to peptides using SPOT arrays and BLI. Membranes carrying histone H2A, H2B, H3 and H4 15-amino acid long peptides with single or multiple lysine acetylation modifications were prepared on cellulose support. Peptide density was controlled using a ratio of Fmoc-β-Alanine to Ac-β-Alanine that ranged from 1:0 to 1:10 in the first step of synthesis, effectively reducing the amount of peptide found on each SPOT of the array. His6 tagged BRD4(1) was used to identify binding after incubating membranes overnight at 4 °C. Bound protein was detected using an anti-his antibody (His-tag® Antibody HPR conjugated, Novagen, #71841). SPOT intensity was measured on a luminescent image analyser (Luminescent Image Analyser LAS-4000 Fujifilm) using the KODAC 1D software package (Kodak 1D Scientific Imaging System V.3.6.2.). His8 peptides were used as controls for antibody binding and array intensities were normalized between 0 and 100 using the control peptide intensity. The last column on the graphs depicts binding of BRD4(1) to the same peptides derived from a commercial set (AltaBioSciences Histone array, Set 4 Histone Acetyl-Lysine library) carrying biotinylated histone peptides used in a biolayer interferometry experiment (BLI). Binding was normalized for this experiment and the scale is given in the inset. Strongly bound peptides showed binding in both methods while weaker binding peptides were over-represented in the peptide array. Our results highlight the utility of SPOT assays in covering large libraries of potential interacting acetylation motifs for rapid screening against bromodomains. We did not see an obvious advantage of reduced SPOT density for most peptides screened, however SPOT density did affect the intensity of the control peptide signal so we would recommend reducing the loading capacity for control peptides using a 1:6 ratio of Fmoc-β-Alanine to Ac-β-Alanine. Furthermore, using normal Whatman™ paper (Whatman™ Chromatography paper Grade 1CHR, GE Healthcare Life Sciences #3001-878) that we functionalize prior to membrane synthesis we found that most strong interactions were retained when compared to commercial “super-membranes” (data not shown), and we only found small amounts of “corona” effects (which we were able to detect by simple manipulation of intensities in Microsoft Excel, since most “corona” affected SPOTs showed about 80%–85% of the intensity between the two profiles chosen for quantification). Lastly, we used the same home-made membranes to test antibodies capable of recognizing phosphorylated residues (pS, pT and pY) and obtained very clean signal to noise ratios which we used to define the quality of these reagents (Figure S2)."}