Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation

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Technical Note

Urea artifacts interfere with immuno-purification of lysine acetylation Ana Martinez-Val, Fernando Garcia, Pilar Ximenez-Embun, Ailyn Martínez Teresa-Calleja, Nuria Ibarz, Isabel Ruppen, and Javier Muñoz J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00463 • Publication Date (Web): 09 Jan 2017 Downloaded from http://pubs.acs.org on January 9, 2017

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Journal of Proteome Research

UREA ARTIFACTS INTERFERE WITH IMMUNO-PURIFICATION OF LYSINE ACETYLATION Ana Martinez-Val1, 2, Fernando Garcia1, 2, Pilar Ximénez-Embún1, Ailyn Martínez Teresa-Calleja1, Nuria Ibarz1, Isabel Ruppen1, Javier Munoz*1 1

Proteomics Unit, Spanish National Cancer Research Centre (CNIO), 28029 Madrid, Spain. ISCIII-ProteoRed. 2

These authors contribute equally to this work

*Correspondence to: [email protected], (34) 917 328 000 3110

Abstract

Comprehensive analysis of post-translational modifications (PTM) often depends on the purification of modified peptides prior to LC-MS/MS. The implementation of these enrichment methods requires thorough knowledge of the experimental conditions in order to achieve optimal selectivity and sensitivity. In this regard, large-scale analysis of lysine acetylation, a key PTM for multiple cellular processes, makes use of monoclonal pan-antibodies designed against this moiety. In this technical note, we report that the immuno-purification of lysine-acetylated peptides is hampered by the co-purification of lysine carbamylated peptides, a frequent urea artifact. This specific interaction can be explained by the similar chemical structures of lysine acetylation and lysine carbamylation. As an alternative, we propose a sample preparation protocol based on sodium deoxycholate that eliminates these artifacts and dramatically improves the selectivity and sensitivity of this immuno-purification assay. Keywords: lysine acetylation, mass spectrometry, carbamylation, urea, immuno-purification, antibody, sodium deoxycholate.

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Introduction Post-translational modifications (PTMs) of proteins constitute an essential regulatory mechanism for multiple biological processes1. Recent technological advances in mass spectrometry enable the comprehensive characterization of PTMs at unprecedented levels of sensitivity and depth2–4. However, these studies are far from trivial and several issues might hamper the analysis of PTMs. On the one hand, computational issues which can cause, for instance, assigning the wrong modification to a peptide (reviewed recently in Kim et al.5). This is the case of isobaric modifications, such as the 2-acetamidoacetamide covalent adduct (H6C4N2O2, 114.042927 Da) with the ubiquitin di-glycine remnant (H6C4N2O2, 114.042927 Da)6 and the mono-methylation (H2C, 14.01565 Da) with certain amino acid substitutions7. On the other hand, experimental issues which mainly affect the MS sensitivity in detecting PTMs. Owing to the extreme complexity of proteomes and the low stoichiometry of PTMs, enrichment of modified peptides prior to mass spectrometric analysis is mandatory8,9. This purification relies on highly selective affinity tools directed against specific chemical moieties

10–12

. A common problem in most of these enrichment methods is the

presence of non-specific interactions with the affinity matrix (i.e. unmodified peptides). These undesired interactions can be minimized by modifying the experimental conditions (e.g. preclearing, more stringent washing, competitive elution buffers) so as to reach as high selectivity as possible without compromising sensitivity. However, undesired specific interactions with the affinity matrix can also occur and, besides the PTM of interest, other moieties might be co-purified. For instance, metal oxides (e.g. titanium dioxide, TiO2), commonly used as affinity chromatographic sorbents to purify phosphopeptides12, possess a wide selectivity for other acidic modifications, such as sialydated glycopeptides or phospholipids13 which may compete for binding and reduce the enrichment of phosphopeptides. Modification of the protocol, either by addition of phosphatases or PNGase F prior to TiO2 enrichment might direct the selectivity towards sialydated glycopeptides or phosphopeptides respectively14. More recently, antibodies designed against specific histone modifications have been shown to possess significant cross-reactivity with other off-targets which might have important implications in ChIP-seq experiments15. 2 ACS Paragon Plus Environment

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These examples highlight the importance of determining the selectivity of an affinity method. Indeed, the unexpected co-purification of other modifications can lead to a decrease in the sensitivity of the analysis and/or to a misinterpretation of the biological conclusions. However, finding out whether other modifications are co-enriched with the PTM of interest is not straightforward. Including several variable modifications in the database search engine has limitations as it increases the search space, leading to longer processing time and an increase of the false positive rate

16,17

. Besides, there are more than 200 PTMs described to date

(www.unimod.org), making this survey a very inefficient task. In this regard, several strategies have been proposed for the unbiased identification of modified peptides. One of them is ModifiComb that looks for unidentified spectra whose precursor mass shows a recognizable delta mass with an identified PSM18. However, this approach requires that both un-modified and modified peptides coexist in the sample. Recently, it has been described a mass-tolerant database search 19 which makes use of very wide precursor mass windows (up to ±500 Da). Modified peptides are identified with a precursor mass error that should correspond to a given modification without the need of an unmodified counterpart. Lysine acetylation is a post-translational modification important for several biological processes such as chromatin remodeling, cell cycle, splicing and nuclear transport3. Dysregulation of acetylation has been linked to several disorders and de-acetylases/acetyl-transferases are considered as potential therapeutic targets. Compared to other PTMs like phosphorylation or ubiquitination, lysine acetylation is less abundant 20, which makes the purification of this modification especially challenging. However, the development of pan-specific antibodies designed against peptides carrying this mark

3,21–23

allows probing the acetylome comprehensively. In these studies, the

specificity of the antibody is critical to the downstream mass spectrometric read out. Here, using an unbiased mass-tolerant database search

19

we uncover that antibodies directed against lysine

acetylated peptides co-purify significant levels of lysine carbamylated peptides. Urea-based proteomics protocols can induce carbamylation of peptides24,19. However, the extent of this artifact can vary depending of multiple factors, such as urea concentration, time of exposure and



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temperature25. Therefore, we show that replacing urea for sodium deoxycholate during the IP procedure eliminates these undesired artifacts and dramatically improves the sensitivity of the assay.


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Experimental Procedures Sample Preparation Human embryonic kidney 293T cells were lysed and reduced using four different approaches. (i) Urea, HEPES, 56ºC reduction: 7M urea, 2M thiourea, 100 mM HEPES pH 7.5, 5 mM sodium butyrate, 15 mM TCEP, 1:100 (v/v) HaltTM phosphatase and protease inhibitor cocktail, 1:1000 (v/v) benzonase. Reduction was performed at 56ºC for 30 minutes. (ii) Urea, HEPES: 7M urea, 2M thiourea, 100 mM HEPES pH 7.5, 5 mM sodium butyrate, 15 mM TCEP, 1:100 (v/v) HaltTM phosphatase and protease inhibitor cocktail, 1:1000 (v/v) benzonase. Reduction was performed at room temperature for 30 minutes. (iii) Urea, Tris-HCl: 7M urea, 2M thiourea, 100 mM Tris-HCl pH 8.0, 5 mM sodium butyrate, 15 mM TCEP, 1:100 (v/v) HaltTM phosphatase and protease inhibitor cocktail, 1:1000 (v/v) benzonase. Reduction was performed at room temperature for 30 minutes. (iiii) Sodium deoxycholate: 5% (w/v) sodium deoxycholate (SDC), 100 mM Tris HCl pH 8.0, 5 mM sodium butyrate, 15 mM TCEP, 1:100 (v/v) HaltTM phosphatase and protease inhibitor cocktail, 1:1000 (v/v) benzonase. This extract was heated during 10 minutes at 80ºC as described in León et al.26. In all four approaches, alkylation was performed using 5mM chloroacetamide for 1h at room temperature in darkness. First digestion using Lys-C (1:200 w/w, Wako) was performed during 4h at RT, followed by dilution 8-fold. Second digestion using trypsin (1:100 w/w, Promega) was carried out overnight at 37⁰C. Digestions were stopped by acidification to a final concentration of 2% TFA. SDC was removed from the fourth extract by acid precipitation with TFA and clarified by centrifugation (15 minutes at 16,000g). Samples were desalted with C18 Sep-Pack and digested peptides were lyophilized. Immuno-purification of lysine acetylated peptides 12 mg of lyophilized peptides from each extract were used to perform each immuno-affinity purification with the PTMScan® Acetyl-Lysine Motif [Ac-K] kit (Cell Signaling Technology®). Similar protein amounts have been used in several other studies (listed in Table S1). For each extract, two immuno-purifications were performed in parallel. Peptides were dissolved in 1.2 ml of



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IAP buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na2HPO4, 50 mM NaCl). For each IP, 20 µl of antibody-bead slurry were used. Beads were washed three times with 500 µl of PBS and once with 500 µl of IAP buffer. Peptides were added to the beads, and samples were incubated for 2 h in a rotator at 4ºC. Afterwards, samples were washed twice with 1 ml of IAP buffer, once with 1 ml of water and, finally, once with 400 µl of water. Elution of purified peptides was performed in two steps of 50 µl of 0.5% TFA (10 min, 25ºC in agitation). Finally, samples were desalted with C18 stage tips. Eluate was dried in vacuum and re-suspended in 0.1% formic acid for subsequent analysis by LC-MS/MS in a Q-q-TOF Impact (Bruker Daltonics).

High pH reverse phase fractionation Besides, 500 µg of each protein extract was subjected to high pH reverse phase fractionation. Peptides were dissolved in 100 µl of phase A (10mM NH4OH). The peptides were eluted at a flow rate of 500 µl/min onto an XBridge BEH130 C18 (3.5 µm, 4.6 x 250 mm) column (Waters) during 60 minutes using the following gradient of phase B (10mM NH4OH, 90% CH3CN): 0-50 min 25% B, 50-54 min 60% B, 54-61 min 70% B. 50 fractions were collected, and concatenated into 15 fractions. The first 6 fractions were analysed by LC-MS/MS in a Q-q-TOF Impact (Bruker Daltonics). LC-MS/MS The Impact (Bruker Daltonics) was coupled online to a nanoLC Ultra system (Eksigent), equipped with a CaptiveSpray nanoelectrospray ion source supplemented with a CaptiveSpray nanoBooster operated at 0.2 bar/minute with isopropanol as dopant. Samples were loaded onto a reversed-phase C18, 5 µm, 0.1 x 20 mm trapping column (NanoSeparations) and washed for 10 min at 2.5 µl/min with 0.1% FA. The peptides were eluted at a flow rate of 300 nl/min onto a home-made analytical column packed with ReproSil-Pur C18-AQ beads, 3 µm, 75 µm x 50 cm, heated to 50 °C. Solvent A was 4% ACN in 0.1% FA and Solvent B acetonitrile in 0.1% FA. The gradient used was: 0-2 min


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2% B, 3-164.5 min 30%B, 165-175 min 98% B, 176-180 min 2% B. The MS acquisition time used for each sample was 180 min. The Q-q-TOF Impact was operated in a data dependent mode. The spray voltage was set to 1.35 kV (1868 nA) and the temperature of the source was set to 160oC. The MS survey scan was performed at a spectra rate of 2.5 Hz in the TOF analyzer scanning a window between 80 and 1600 m/z. The minimum MS signal for triggering MS/MS was set to a normalized threshold of 500 counts. The 30 most abundant isotope patterns with charge ≥2 and m/z > 350 from the survey scan were sequentially isolated and fragmented in the collision cell by collision induced dissociation (CID) using a collision energy of 23 – 56 eV as function of the m/z value. The m/z values triggering MS/MS with a repeat count of 1 were put on an exclusion list for 30 s using the rethinking option. Data processing All files were analysed using MaxQuant 1.5.3.30 with Andromeda as the search engine against a Uniprot Homo sapiens database (20,187 sequences). Carbamidomethylation of cysteine was included as fixed modification. Oxidation of methionine, acetylation of protein N-terminal, acetylation of lysine, carbamylation of lysine and peptide N-termini were included as variable modifications for the analysis of immuno-purified samples. For high pH fractions of total extracts, oxidation of methionine, acetylation of protein N-terminal, carbamylation of lysine and peptide Ntermini were included as variable modifications. Precursor mass tolerance was 30 ppm for the first search, and 7 ppm for the main search. Fragment mass tolerance was set to 40 ppm. The false discovery rate for peptide, protein, and site identification was set to 1%. In order to count the number of acetylated or carbamylated peptides, the msmsScans.txt table was used. Peptides that contained both acetylation of lysine and carbamylation of either N-terminal or lysine were not taken into account for further analysis. Peptides that contain lysine carbamylation in the peptide Nterminal were considered in the subset of N-terminal carbamylated peptides. In addition, all the raw data was processed with Proteome Discoverer 1.4 (Thermo) with Sequest HT as the search engine using the same settings as in MaxQuant. Then, results were filtered to 1% FDR with Percolator.



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Mass-tolerant database search The mass-tolerant database search19 was performed using Comet (2015.02 rev.1)27,28 for the four immuno-purifications using the first extract (Urea, HEPES and 56ºC reduction). A precursor mass tolerance of 100 Da and fragment mass tolerance of 0.02 Da were set. As input for this search, the calibrated peak.apl and sil0.apl files generated by MaxQuant were used. No variable modifications were included in the search, and only carbamidomethylation of cysteine was used as fixed modification. Mass accuracy was excluded as a feature for post-search filtering by Percolator (v208). From all the features used by Percolator, mass accuracy related ones should be discarded in this type of searches, otherwise, Percolator would penalize hits with high mass shifts. Results were filtered by a q-value lower than 0.05. Mass shifts between 41.9 – 42.1 and 42.9 – 43.1 Da were clustered using the densityMclust function of the mclust package for R as described in Chick et al.19. The experimental mass shift was determined as the mean of the Gaussian distribution modelled with the highest probability and the lowest variance.

Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD003701 (Username: [email protected], Password: 3PWLVQkS).


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Results and discussion Anti-lysine acetylation antibodies co-purify lysine carbamylation in a specific manner We performed a series of lysine acetylation (K-ac) immuno-purifications (IPs) on a human cell lysate (see Experimental Procedures) and found a relatively low number of lysine acetylated peptides (only 5% of the identified peptides were K-acetylated) (data not shown). In principle, this low yield could be explained by a poorly efficient IP because, among other factors, an inadequate input-antibody ratio22. However, we also noticed that a significant fraction of high-quality spectra had not being assigned to peptide sequences by the database search engine algorithm. We hypothesized that these spectra could belong to peptides carrying other type of modifications besides acetylation. To investigate the origin of these unmatched MS/MS spectra, we performed a mass-tolerant database search19 on our IPs data sets (Figure 1a). Increasing the precursor mass tolerance allows the identification of modified peptides in an unbiased way (see Experimental Procedures). As expected, we found a distribution at +42.0103 Da that we assigned to K-ac peptides (mass error = 0.00026 Da) (Figure 1b). In addition, a second distribution at +43.00543 Da was also observed (Figure 1c). Among all the modifications (or combinations of several of them) that could explain this shift in mass, carbamylation (CHNO) fit the best (mass error = 0.00038 Da) (Figure 1c). Furthermore, carbamylated peptides are known to undergo neutral loss during MS/MS fragmentation 29. Indeed, the presence of CHNO neutral losses (-43.00581 Da) in the spectra from the second distribution (Figure 2) unequivocally confirmed the presence of this peptide modification in our K-ac IPs. Carbamylation is a common artifact in urea-based proteomic preparations. In aqueous solutions, urea is slowly degraded into isocyanate and ammonium, and this reaction is enhanced by high temperatures 25. Primary amines, such as those in peptide N-termini and ε-amines groups of lysines, can react with free isocyanate and artifactually form carbamylated peptides24,30. Indeed, the



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experimental conditions most frequently used in K-ac immuno-purification studies involve the use of urea-based buffers3,22,31–33. In our K-ac purifications, following the indications of the antibody manufacturer, all steps prior to trypsin digestion were performed in 7M urea including protein reduction at 56ºC during 30 minutes (See Experimental Procedures). To test whether these conditions caused abnormal levels of carbamylation and, hence, could explain the presence of this artefact in our IPs, we examined the peptide input by LC-MS/MS (i.e. prior to the immunopurification step). To determine the fraction of carbamylated peptides in our input samples more accurately, we reduced sample complexity by high pH reverse phase pre-fractionation34. Then, data was analysed using a regular database search strategy, including both lysine carbamylation (K-cam) and N-terminal peptide carbamylation (Nter-cam) as competitive variable modifications (See Experimental Procedures). In total, 35,102 PSMs were identified, from which 2,017 PSMs (5.8%) were found to be carbamylated (Figure 3a). Next, we compared these results with other available proteome data sets that were obtained under similar experimental conditions to ours (i.e. protein extraction in the presence of high urea concentration followed by pre-fractionation prior to LCMS/MS analysis) 19,20,31,35–37. After re-analysis, we found significant amounts of carbamylation in all of these data sets, ranging from 1-8% (Figure S1 and Table S2). Interestingly, the highest levels of carbamylation were found in data sets in which protein reduction was carried out at 56ºC whereas the FASP protocol (reduction is performed in the absence of urea) seemed to minimize these artifacts (Figure S1 and Table S2). Most importantly, we always found a much higher proportion of carbamylation in the peptide N-termini than in lysine residues. In our peptide inputs, 1,731 PSMs (5%) were assigned to Nterm-cam and 286 PSMs (0.8%) to K-cam (Figure 3a). Similar proportions were found in the re-analysed proteome data sets (Figure S1 and Table S2). This observation agrees with previous reports

24

which showed that isocyanate preferentially reacts with the free amine

groups of peptides N-termini. Strikingly, when we examined our K-ac immuno-purifications, the inverted Nter-cam/K-cam proportion was found. In these samples, 462 and 3,005 Nter-cam and K-cam peptides were identified respectively (Figure 3b and Tables S4, S5). Whilst K-cam represented only 14% of the


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carbamylated peptides found in the input (Figure 3a), after immuno-purification this proportion increased 6-fold to 86% (Figure 3b and Tables S4, S5). These percentages were confirmed using a second search engine, Sequest HT (see Experimental procedures and Table S6). These results suggested that lysine carbamylated peptides were co-purified by the K-ac antibody in a specific manner. To confirm this hypothesis, we examined the levels of lysine carbamylation in other published large-scale acetylome studies, including data sets from the two most frequently used K-ac antibodies (Table S3). Significant levels of K-cam were detected in all of them: between 8% and 30% of the modified peptides found in these K-ac IPs were assigned to K-cam peptides (Figure S2). As expected, Nterm-cam peptides were present in a much lower proportion than K-cam. Taken together, these results demonstrate that currently available anti-acetyl lysine antibodies possess selectivity towards both lysine-acetylated and lysine-carbamylated peptides. This finding might be explained by the similarity of the chemical structure of these modifications (Figure 4). Acetyl lysine only differs from carbamyl-lysine in the radical of the chemical group bound to the ε-amine. Whilst acetyl-lysine presents a methyl group with tetrahedral geometry, carbamyl-lysine presents a primary amine, with a trigonal planar geometry (Figure 4c and 4d). The stereochemistry of both modifications is highly similar, which could explain the specificity of these antibodies for both Kcam and K-ac modified peptides. Furthermore, motif analysis of both acetylated and carbamylated peptides sequences showed that the antibody did not have any apparent bias towards any particular peptide sequence (Figure S3). Taking advantage of the titration assays performed by Svinkina et al.

22

, we next assessed the

binding affinities of the antibody towards either K-cam or K-ac. In such experiments, different ratios of peptide input and antibody were tested. Thus, we re-analysed all these data sets to account for K-cam levels. On the one hand, we found that increasing the amount of antibody from 25 to 125 µg (using a fixed input amount) resulted in the co-purification of more K-cam peptides (from 916 to 2,088 PSMs) and less K-ac peptides (from 7,246 to 5,613 PSMs) (Figure S4a, S4c). On the other hand, increasing the input from 1 to 15 mg (using a fixed antibody amount) yielded more K-ac (from 2,742 to 7,405 PSMs) while K-cam subtle decreased its levels (from 2,000 to 1,300 PSMs)



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(Figure S4b, S4d) and this effect was particularly evident using 15 mg. These observations imply that the antibody used by Svinkina (the same as in this report) possess a subtle higher specificity towards K-ac than K-cam and underpin the importance of determining the optimal input/antibody ratio

22

to reduce both un-specific (i.e. unmodified peptides) and specific (i.e. lysine carbamylated

peptides) interactions.

Sodium deoxycholate eliminates carbamylation artefacts and improves selectivity of acetyl lysine immuno-purifications In view of these results, we decided to investigate alternative sample preparation strategies to minimize carbamylation levels, as this should consequently improve the specificity of the downstream K-ac immuno-purification. As explained previously, high temperatures catalyse urea degradation into isocyanate. Hence, we evaluated the effect of reducing disulfide bonds at room temperature (instead of 56 ºC as employed in the initial procedure) (see Experimental Procedures). These new conditions led to a noticeable decrease, from 5.8% to 1.1%, in the levels of carbamylation in the input samples (Figures 3a, 3c). In the subsequent IPs, a reduction of nearly 6fold in K-cam peptides was observed (from 3,005 to 562 PSMs) which resulted in a 2-fold improvement in the number of K-ac peptides (from 1,492 to 3,026 PSMs) (Figure 3d and Tables S4, S5, S6). We next considered the substitution of the HEPES buffer for a Tris-HCl buffer, performing protein reduction also at room temperature (see Experimental Procedures). Tris is an organic compound containing a primary amine, and thus should compete with the reactive peptide amino groups for isocyanate. Under these conditions, one should expect a further reduction in the levels of peptide carbamylation. Using this approach, carbamylation levels in the input were found to be as low as 0.8% (below FDR) (Figure 3e). However, despite these marginal levels of carbamylation in the input, still 16% (435 PSMs) of the modified peptides found in the immuno-purifications were carbamylated in lysines (Figure 3f and Tables S4, S5, S6). Taken together, these results proved that carbamylation, even at very low concentrations, can yet interfere during acetyl-lysine purification. In consequence, we replaced the causal agent of carbamylation and tested the efficacy of urea-free


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buffers for the immuno-purification of K-ac peptides. Recently, Proc et al.38 and Leon et al.26 evaluated different reagents used for protein solubilisation and denaturation and concluded that sodium deoxycholate (SDC) was more efficient than those protocols based on surfactants such as Rapigest26. Thus, we performed the IP using a SDC-based sample preparation protocol. As expected, we found negligible (below FDR limits) carbamylation levels in our input samples (Figure 3g). Accordingly, the selectivity of the immuno-purification improved remarkably, since 96% (4,792 PSMs) of the modified peptides were found to be lysine-acetylated (Figure 3h). Interestingly, the percentage of modified peptides (i.e. sum of Nter-cam, K-cam and K-ac) found in the IP using SDC (44%) (Figure 3h and Tables S4, S5, S6) was significantly higher than in the other protocols tested (13-20%) (Figures 3b, 3d, 3f and Tables S4, S5, S6). This further improvement in the selectivity of the IP might be explained by the presence of traces of SDC during the incubation of the peptide input with the antibody. This is in agreement with a previous report showing that the use of detergents (n-octyl glucoside) dramatically improves the selectivity of the immunopurification of tyrosine phosphorylated peptides by reducing non-specific interactions with unmodified peptides39.

Conclusion Large scale analysis of PTMs using affinity methods requires the optimization of experimental conditions in order to achieve maximum selectivity and sensitivity. Here, we show that the selectivity of antibodies directed against lysine acetylation can be compromised by the undesired co-purification of a common urea artefact. A simple modification in the protocol solves this issue and significantly improves the results of the assay. Our findings suggest that the tools used for the enrichment of modifications in proteins/peptides should be carefully evaluated. The strategy used here for the unbiased identification of modified peptides may reveal similar problems with other PTMs and be useful to further refine existing enrichment protocols.



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Acknowledgments The CNIO Proteomics Unit belongs to ProteoRed, PRB2-ISCIII, supported by grant PT13/0001. Part of this work was funded by SAF2013-45504-R (MINECO). J.M. is also supported by Ramon y Cajal Programme (MINECO) RYC-2012-10651, A.M.V. is supported by BES-2014-070098 (MINECO).

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Figure S1. Carbamylation levels found in other “total proteome” datasets. Figure S2. Carbamylation levels found in other K-acetylation immuno-purificationsdata sets. Figure S3. Motif analysis (IceLogo) of acetylated and carbamylated peptides. Figure S4. Titration curves for K-acetylation and K-carbamylation in the immunopurifications from Svinkina et al. Table S1. Protein amounts used as input in other studies. Table S2. “Total proteome” data sets re-processed in this study. Table S3. Lysine acetylation immuno-purifications data sets re-processed in this study. Table S4. MaxQuant’s ‘evidence’ table for the four IP conditions evaluated (Excel file). Table S5. Summary table of PSMs identified in each one of the four conditions evaluated, with the PTMs identified and mentioned in the manuscript highlighted using the same color code as in the text (Excel file). Table S6. Comparison of acetyl-lysine and carbamylated (K- or N-) levels found using Andromeda (MaxQuant) and Sequest HT (Proteome Discoverer 1.4).

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Figure legends Figure 1. Mass-tolerant search of K-acetylation immuno-purifications. (a) Histogram of identified PSMs in our K-acetylation immuno-purifications using the recommended protocol (i.e. urea extraction and reduction at 56ºC). A mass error window of ±100 Da was used (bin width=0.05 Da). Acetylation and carbamylation distributions are highlighted in grey. (b) Detail showing the histogram and density plot of the PSMs identified in the mass range between 41.975 and 42.04 Da. Red line indicates the theoretical monoisotopic mass of acetylation as annotated in UniMod. Blue line indicates the experimentally observed mass. (c) Histogram and density plot of the PSMs identified in the mass range between 42.975 and 43.040 Da. Red line indicates the theoretical monoisotopic mass of carbamylation as annotated in UniMod. Blue line indicates the experimentally observed mass. Density plots were generated in R 3.1.2 using the function geom_density from the ggplot2 package with default parameters. Bin width used for both histograms was 0.0015 Da. Figure 2. Neutral loss of lysine carbamylated peptides. (a), (b) Tandem mass spectra of two peptides containing carbamylated lysines. A neutral loss of -43.0058 Da in y-ions series is observed (indicated with an asterisk). Fragments highlighted in yellow correspond to y-series with ammonia and water neutral losses. Annotation was done with MaxQuant 1.5.3.30. Figure 3. Lysine carbamylation is co-immunopurified with lysine acetylation. (a) Carbamylation levels found in input sample (prior to IP) using the protocol with urea-HEPES and reduction at 56ºC. (b) Proportion of unmodified (light grey) and modified peptides (dark grey) 20 ACS Paragon Plus Environment

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found in the lysine acetylation immuno-purification using the protocol with urea-HEPES and reduction at 56ºC. Nested pie chart shows the proportion of Nter-cam, K-cam and K-ac modified peptides. (c) Same as in (a) with the urea-HEPES and reduction at RT protocol. (d) Same as in (b) using the protocol with urea-HEPES and reduction at RT. (e) Same as in (a) with the urea-Tris and reduction at RT protocol. (f) Same as in (b) using the protocol with urea-Tris and reduction at RT. (g) Same as in (a) with the SDC extraction protocol. (h) Same as in (b) using the protocol with the SDC extraction protocol. Two independent IPs were performed, and each sample was analysed twice by LC-MS/MS. Absolute numbers of PSMs (sum of four analyses) and relative percentages are shown. Data shown above was obtained from immune-purifications using 12 mg of protein input. A more detailed analysis using different protein amounts from Svinkina et al. is shown in Figure S4b, S4d. Figure 4. Chemical structures of acetylated lysine and carbamylated lysine. Both moieties only differ in the tetrahedral geometry of the methyl group (acetylation) (a) (c) and planar geometry of the amino group (carbamylation) (b) (d) which are highlighted in green in the figure.



Figure 1

a

Count

1.5E4

1E4

5E3

0 -100

b

-50

0 50 Mass error (Da)

100

c

Observed = 42.01030 Da Theoretical = 42.01056 Da Error = 0.00026 Da

Observed = 43.00543 Da Theoretical = 43.00581 Da Error = 0.00038 Da

100 100 Density

75 50 25

50

Mass error (Da)

ACS Paragon Plus Environment

Mass error (Da)

43.0350

43.0250

43.0150

43.0050

42.9950

42.9850

42.0400

42.0300

42.0200

42.0100

42.0000

41.9900

42.9750

0

0 41.9800

Density

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 2

a

120

Raw file 20151130_FG_AMV AcKCamK C1_2_2-B,3_01_5645

Method TOF; CID

Score 205.68

m/z 856.39

Gene names RANBP1

y₄

80

100

624.33

y₂

187.14

y₇

995.5

40

y₃*

b₂

y₅*

y₄*

581.32

b₅

434.25

215.14

y₉

y₈

1124.54

473.22

175.12

20

737.41

306.16

b₄

y₁

y₆

866.46

y₅

477.26

b₃

344.18

a₂

60

y₃

588.25

b₆

y₇*

y₆*

694.41

1497.65

1454.65

1368.61

y₁₀*

1081.53

975.38

y₁₁*

y₁₀

y₈*

b₈

717.29

y₉*

1196.56

952.49

823.45

y₁₁

1239.57

1325.6

Counts (a.u.)

Relative Intensity

Scan 30144

6000 5000 4000 3000 2000 1000

0

0

100

200

300

400

500

-

T

L

b₂

600

700

y₁₁

y₁₀

y₉

y₈

y₇

y₆

y₅

y₄

E

E

D

E

E

E

L

F

b₃

800

b₄

b₅

900 1000 1100 1200 1300 1400 1500 1600 1700

b₆

b₈

y₃

y₂

y₁

K

M

R

ca

-

b Scan 21509

Method TOF; CID

Score 318.94

m/z 895.49

Gene names CA2

100

y₁₀

80

1264.7

0

20

40

60

y₁₂

y₁

b₂

227.1 147.11 199.11

100

a₂

200

y₃

y₂

450.28

b₄

303.21

b₃

427.22

b₅

734.47

y₆

833.54

b₆ 613.32 y₅* y₆* 790.53 y₄* 691.46 b₇ b₈ 578.38 700.35

526.29

340.19

829.39

300

400 y₁₄

-

y₅

y₄

621.38

E

P

b₂

500 y₁₃

I

b₃

600

700

y₁₂

S

b₄

y₁₁

V

b₅

800 y₁₀

S

b₆

y₇

y₈

y₉

y₁₁

1177.67

1450.8

y₁₃

y₁₄

1660.94

1090.64 y₁₀* 1363.77 1563.89 y₁₄* y₉* 1221.69 y₁₁* b₁₀ 1134.66 1617.93 1320.76 y₁₂* 1056.52 b₉ b₁₁ 1407.8 y₁₃* b₁₄ 957.45 y₈* 1169.6 b₁₂ 1643.88 1520.88 1340.71 961.59

y₇*

918.59

1047.63

900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 y₉

S

b₇

y₈

y₇

E

Q

b₈

b₉

y₆

V

b₁₀

y₅

L

b₁₁


y₄

y₃

K

F

ca b₁₂

y₂

y₁

R

K

b₁₄

-

Counts (a.u.) [1e4]

Relative Intensity

120

Raw file 20151130_FG_AMV AcKCamK C1_2_2-B,3_01_5645

5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Figure 3

Unmodified peptides Modified peptides

N-terminal carbamylated (Nter-cam) peptides K-carbamylated (K-cam) peptides K-acetylated (K-ac) peptides

IMMUNO-PURIFICATION

INPUT SAMPLE Urea-HEPES 56 ºC reduction

Urea-HEPES RT reduction

Urea-TrisHCl RT reduction

Sodium deoxycholate

a

c

e

g

33,085 (94%)

286 (0.8%) 1731 (5%)

24,458 (99%)

33 (0.1%) 251 (1%)

28,027 (99%)

37 (0.1%) 187 (0.7%)

27,382 (100%)

15 (0.1%) 22 (0.1%)

b

d

f

h

19,605 (80%)

3,005 (61%) 462 (9%) 1,492 (30%)

18,128 (83%)

562 (15%) 89 (2%) 3,026 (82%)

17,622 (87%)

435 (16%) 42 (2%) 2,206 (82%)

6,132 (56%)

177 (4 %) 8 (0.2%) 4,792 (96%)


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Figure 4

a

b

c

d



For TOC only

UREA Cam K

LYSIS-DIGESTION

Ac K

ELUTION

Cam-K Ac-K Unmodified

ELUTION

Unmodified

Ac

K

Cam

K

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

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Ac

CARBAMYLATION

K

Cam K

Ac

K

Ac-K IMMUNO-PURIFICATION

SDC Ac

K

LYSIS-DIGESTION Ac

Ac

K

K

Ac

Ac K

K

Ac K


Ac-K

Urea Artifacts Interfere with Immuno-Purification of Lysine Acetylation

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