Proteome-wide Identification of SUMO2 Modification Sites

Functional analysis of SUMO2^T90K

Digestion of ubiquitinated proteins with trypsin gives rise to a diGly remnant linked to the acceptor lysine, which can be used to enrich for modified peptides by affinity purification with a selective diGly-Lys specific monoclonal antibody (anti-K GG) (25). The Ubl family members, ubiquitin, NEDD8, and ISG15 each contain an Arg preceding a diGly sequence at the C-terminus (Fig. 1A). Thus, trypsin-mediated cleavage of proteins conjugated to these Ubls creates an identical branched side chain consisting of diGly conjugated to Lys. We mutated Thr⁹⁰ to Lys in SUMO2 (SUMO2^T90K) creating a Lys N-terminal to the diGly motif at the C-terminus (Fig. 1A) and generated HEK293 cells stably expressing 6His-SUMO2^T90K. Similar to parental cells, SUMO2^T90K-expressing cells had a comparable doubling time and morphology (Fig. S1) and responded to heat shock by increasing the abundance of SUMO modified proteins (Fig. 1B).

To assess whether the SUMO2^T90K mutation had any affect on SUMO2 conjugation or cleavage, we performed in vitro enzyme assays. We found that purified SUMO2 and SUMO2^T90K showed little difference in the rate of polymerization and conjugation to the substrate proteins SP100 (Fig. 1C), IRF2, RanGAP1, or PML (fig. S2A). Likewise, 6His-SUMO2 and 6His-SUMO2^T90K had similar affects on the rate of cleavage from these substrates by SENP1 (Fig. 1D and fig. S2B). Finally, SENP1 and SENP2 cleaved the inactive pro-forms of recombinant SUMO2 and SUMO2^T90K at a similar rate (Fig. S2C).

Proteome-wide identification of sumoylation sites in human cells

We established a workflow using 6His-SUMO2^T90K expressing cells that enabled us to select for SUMO modified proteins and enrich for peptides containing diGly conjugated lysines (Fig 2A). Cells (~ 3 × 10⁸) were heat stressed at 43°C for 30 minutes to enhance sumoylation (3) and lysed under denaturing conditions. Proteins conjugated to 6His-SUMO2^T90K were isolated by nickel affinity chromatography and subsequently digested with Lys-C, or Lys-C and Glu-C to truncate long substrate peptides. Peptides containing diGly-Lys were enriched with anti-K GG and analyzed by MS using parameters optimized for the identification of low abundance peptides. Using this strategy, we identified 2,747 peptides, of which 1217 (44%) contained a diGly-Lys motif (Table S1 and S2). These peptides represented 1,002 unique sumoylated sites in 539 proteins (Table S1).

To estimate the reproducibility of the SUMO2^T90K-based enrichment strategy, we repeated the experiment. We performed the SUMO2^T90K protein and peptide enrichment workflow in duplicate on a lysate of HEK293 culture. In this analysis, we identified 468 sumoylated sites in common with the 1,002 SUMO2^T90K modified sites found in the first experiment, of which 323 (69%) were common between replicate purifications (Fig. S3), indicating a high degree of reproducibility. In addition, this analysis suggested that relatively small scale experiments (~ 4 × 10⁷ cells) are sufficient to identify large numbers of sumoylated peptides. Moreover, we determined that setting the mass spectrometer to fragment and scan the most abundant peptide from the initial full MS scan (“top 1”) with a maximum one second fill time yielded almost twice the number of successful identifications of sumoylated peptides as using “top 10” data dependent acquisition with a 60 millisecond fill time (Fig. 2C). From these data we determined that antibody based enrichment of SUMO2^T90K modified peptides increased the percent of identified sumoylated peptides from 0.05% to greater than 31% compared to nickel affinity purification alone (Fig. 2C).

We also compared our data to published MS-based analyses of post-translational modifications. SUMO2^T90K modified sites overlapped with 55% of SUMO2 modifed sites detected in the largest MS-based study of sumoylation (29) and 39% of sites identified in all MS-based studies of sumoylation (34) (Fig 2B). Slightly less than half of SUMO2^T90K modified proteins overlapped with proteins identified in published studies involving protein-level SUMO substrate identification (fig. S4, A and B). However, the fact that these studies used different cell types, which likely contain different sets of sumoylated proteins, may, at least in part, explain the lack of overlap. . Comparison of functional annotations of sumoylated proteins discovered here and in other large-scale SUMO2 proteomic studies revealed similar enrichment of sumoylated proteins in cellular processes, including gene expression, RNA metabolism, DNA replication, repair and recombination, and cell proliferation, survival, and growth (Fig. S4C). Only 146 SUMO2^T90K modified sites (14.6%) overlapped with known sites of acetylation (34) (Table S1) and 378 SUMO2^T90K modified sites (37.8%) overlapped with known sites of ubiquitylation (34) (Table S1). Whereas the accuracy of the estimated percentage of overlap among these studies could be compromised by the relative completeness of the datasets, these observations at least suggest that ubiquitination and less commonly acetylation can occur on the same residues as sumoylation.

Sequence context of SUMO2 modification sites

Initial findings of a consensus motif for SUMO modification (ΨKxE or ΨKxD, where Ψ represents a hydrophobic residue and × represents any amino acid (35)) focused experimental investigation towards these sites, which may explain the fact that the majority of SUMO modified sites discovered to date conform to this consensus. Small scale proteomics studies confirmed this, but also identified a less common inverted consensus motif DxKΨ or ExKΨ (29). Our SUMO2^T90K modified peptide dataset enabled us to investigate the sequences of sumoylation sites using robust statistical analyses. We used pLogo (36) to analyze the sequence of diGly-Lys containing peptides. This analysis revealed a significant overrepresentation of Glu two amino acids C-terminal to the conjugated Lys (position +2), of hydrophobic residues Ile or Val at position −1, and to a lesser degree, of the acidic residues Asp or Glu at position −2 (Fig 3A). This result is consistent with a mixed population of sumoylated peptides containing forward and inverted sites. Unexpectedly, we also found that 90 sumoylated peptides had Glu or Asp at both positions +2 and −2 simultaneously (Table S1), suggesting that some sites contain “forward and inverted”-type consensus motifs. We separately analyzed sites containing forward, inverted, or “forward and inverted” consensus motifs for additional conserved residues. As expected, the majority of sequences with forward consensus motifs contained Glu at position +2 (90%) and a large hydrophobic residue at position −1; typically either Val (31.7%) or Ile (26.5%), although Leu, Phe, and Pro were also overrepresented (Fig. 3B). For sequences with inverted consensus motifs, there was an approximately equal likelihood of Glu or Asp at position −2, but unexpectedly, no preference for large hydrophobic residues at position +1 (Fig 3C). Instead, there was an overrepresentation of Val or Ile at position −1 and Glu at position +2, largely in peptides with the “forward and inverted” motif. Together, sites with forward or inverted consensus motifs represented 72.6% of all SUMO modification sites. The sequences of the remaining sumoylated sites did not show a strong consensus, although there was a small but significant overrepresentation of Gly at position +2 (Fig 3D).

Potential for multi-site modification

Reduced electrophorectic mobility of sumoylated proteins has led researchers to predict that proteins contain multiple SUMO modification sites (22). We found that about one third of SUMO2^T90K modified proteins had more than one sumoylation site (Fig. 4A and table S1). For example, PARP1 had 15 sites, hnRNPM had 14 sites, and hnRNPU, GTF2I, and Uba2 had 12 sites (Fig 4B and table S1). Because Uba2 is a component of the heterodimeric E1 SUMO activating enzyme (37, 38), this suggests that it may undergo auto-sumoylation. Sumoylation sites were clustered in some proteins (Fig 4B), implying that these regions are more susceptible to modification. We were not able to determine whether multiple sumoylation sites were simultaneously modified for the majority of proteins due to methodological limitations. However, because Lys-C is inhibited by post-translational modification of Lys we were able to identify a few peptides with simultaneous sumoylation of adjacent lysines (table S1 and SMfile1.pdf). SUMOs 1, 2 and 3 have multiple post-translational modifications, including acetylation at their N-terminus and sumoylation on internal lysines (table S1 and SMfile1.pdf). The major site involved in chain formation on SUMO2 and SUMO3 is Lys¹¹ (10), which we frequently detected as a SUMO2^T90K modified site. We also detected doubly modified peptides of SUMO2 and SUMO3, including one modified at Lys¹¹ and Lys⁷ and the other modified at Lys⁵ and Lys⁷ (Table S1 and SMfile1.pdf), suggesting that alternate branching pattern may occur.

Evidence for protein group modification

SUMO modification of diverse substrates is transiently increased in response to proteotoxic stress (3, 4, 6). Analysis of the functional annotations of 539 SUMO2^T90K modified proteins in our dataset derived from heat-shocked cells revealed a statistical overrepresentation of proteins involved in highly-interconnected complexes (Fig. 5) that regulate gene expression; DNA replication, recombination, and repair; and cell growth and proliferation (fig. S4C). Zinc finger transcription factors were frequently identified in our analysis with evidence for 85 SUMO2^T90K modified sites distributed among 52 different proteins (table S1). Several studies investigating individual zinc finger transcription factors identified sites of SUMO modification in these proteins by mutational analysis,(39) and demonstrated that sumoylation is generally associated with transcriptional repression (40). Similarily, 78 sumoylation sites were present in 14 heterogenous ribonucleoprotein particle proteins (hnRNPs) (Fig. 5), consistent with previous studies (41). Our data extended previous observations by demonstrating extensive SUMO modification of hnRNPs, including 14 sites on hnRNPM; 11 sites on hnRNPU; 9 sites on hnRNPAO; 9 sites on hnRNPUL1; and 8 sites on hnRNPC. In addition, as expected from a previous analysis of endogenous sumoylated proteins (22), we identified multiple SUMO2^T90K modified proteins involved in DNA replication, recombination and repair (Fig 5). Thus, these results suggest that groups of functionally related proteins are likely subjected to contemporaneous SUMO modification.

A database of virtual sites to search for modifications with wild type SUMO

Branched peptides generated from tryptic digestion of proteins modified by wild-type SUMOs produce complex MS2 fragmentation spectra that are not recognized by standard annotation algorithms (11, 28). To enable automated searching for these peptides, we used our list of SUMO2^T90K modified sites to generate virtual branched peptide databases of artificial peptide sequences. In this database the C-terminal tryptic peptide of either SUMO1 or SUMO2 was fused to the N-terminus of the predicted tryptic peptide corresponding to observed SUMO2^T90K modified sites (Fig. 6A and SUMO1.FASTA and SUMO2.FASTA).

Many SUMO2^T90K modified peptides were also phosphorylated (table S1). Therefore, we used the virtual branched peptide databases to search raw data files containing high-resolution spectra from an analysis of phosphopeptide enriched samples (42). We identified a branched peptide using the virtual branched SUMO2 database that contained the long tryptic remnant of wild-type SUMO2 conjugated to Lys⁶⁴⁸ of the transcription factor c-Myb (Fig. 6B). In the SUMO2^T90K data, we only detected the Ser⁶⁵³ phosphorylated form of the peptide (table S1), consistent with the fact that the sequence surrounding Lys⁶⁴⁸ on c-Myb corresponds to a phosphorylation dependent sumoylation motif (PDSM; KxExxSP). This observation is consistent with independent studies that show sumoylation ofLys⁶⁴⁸ (43, 44) and phosophorylation ofSer⁶⁵³ (45, 46).

To identify additional branched peptides modified by wild-type SUMO2, we used MS to re-analyze peptides from a proteomic study using exogenously expressed SUMO2 fused to a tandem affinity purification (TAP) tag (6) and searched the resultant spectra using the virtual branched SUMO2 peptide database. We identified 14 additional sumoylated peptides (SMfile2.pdf), including one containing sumoylation of Lys¹¹ of ubiquitin (Fig 6C). These data validated the SUMO2^T90K results and demonstrated that using virtual branched peptide databases based on the SUMO2^T90K data for re-analysis of existing MS2 spectra may be useful for identifying sites sumoylated by both endogenous and overexpressed wild-type SUMO2.

Generation of HEK293 N3S cells stably expressing 6His-SUMO2^T90K

pEFIRESpuro-6His-SUMO2 was created by cloning a PCR-generated 6His-SUMO2 fusion into the NheI and NotI sites of the plasmid vector pEFIRES-P-eYFP-C1 (51), replacing the coding sequence of eYFP (enhanced yellow fluorescent protein). The SUMO2^T90K mutation was introduced into pEFIRESpuro-6His-SUMO2 by site-directed mutagenesis. The SUMO2 coding regions of all plasmids were fully sequenced. HEK293 N3S cells (Sigma-Aldrich, 92052131) grown in suspension culture were transfected using Lipofectamine 2000 (Life Technologies) with pEFIRESpuro-6His-SUMO2^T90K and selected with puromycin at 2 g/ml. Thereafter, stable cell populations were maintained in growth media containing 1 g/ml puromycin.

Cell culture and protein extraction

HEK293 N3S cells stably expressing 6His-SUMO2^T90K were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco), supplemented with 10% dialyzed fetal calf serum (FCS), 1 μg/ml puromycin, and 100 U/ml penicillin and streptomycin. Cells were grown on five 175 cm² dishes to about 90% confluency prior to their transfer into Minimum Essential medium Eagle (spinner modification; Sigma-Aldrich) supplemented with 10% FCS, 1 g/ml puromycin, 2 mM L-glutamine, and 100 U/ml penicillin and streptomycin. 6 L of cell culture (~800 000 cells/ml) was stimulated by heat shock at 43 °C, harvested by centrifugation, washed twice with cold 1x DPBS (Dulbecco’s phosphate buffered saline; Gibco) and lysed in cell lysis buffer (6 M guanidinium-HCl, 100 mM sodium phosphate buffer pH 8.0, 10 mM Tris-HCl, 20 mM imidazole, 5 mM β-mercaptoethanol) (6 ml of lysis buffer per 1 g of cell pellet). DNA was disrupted by short pulses of sonication, insoluble particles were removed with 0.2 μm sterile filters (Sartorius) and protein concentration was determined by BCA (bicinchoninic acid) assay (Pierce). In the experiment designed to evaluate reproducibility, duplicate 750mL cultures of HEK293 6His-SUMO2^T90K corresponding to 4 × 10⁷ cells were processed in parallel.

Nickel affinity chromatography

Nickel affinity purification of 6His-SUMO2^T90K conjugates was performed with Ni²⁺-NTA agarose beads (Qiagen) according to a published protocol (52) with minor changes. 250 L Ni²⁺-NTA agarose beads were added to the cell lysate (286 mg total protein for the primary experiment or 48mg protein for the experiment designed to evaluate reproducibility) and mixed at 4 °C for 16 hours. Beads were washed with cell lysis buffer, wash buffer pH 8.0 (8 M urea, 100 mM sodium phosphate buffer pH 8.0, 10 mM Tris-HCl, 10 mM imidazole, 5 mM β-mercaptoethanol), wash buffer pH 6.3 (8 M urea, 100 mM sodium phosphate buffer pH 6.3, 10 mM Tris-HCl, 10 mM imidazole, 5 mM β-mercaptoethanol) and again with wash buffer pH 8.0. Proteins were eluted in three sequential steps with 1.5 column volumes of elution buffer (8 M urea, 100 mM sodium phosphate buffer pH 8.0, 10 mM Tris-HCl, 200 mM imidazole, 5 mM β-mercaptoethanol).

Filter aided sample preparation and protein digestion

Digestion of 6His-SUMO2^T90K proteins was performed on 30 kDa cutoff filter units (Sartorius) according to a published protocol (53) with minor changes. Samples were concentrated on filter units, washed twice with UA buffer (200 μl of 8 M urea, 100 mM Tris pH 7.5) and treated with 50 mM chloroacetamide in UA buffer for 20 minutes in the dark. Samples were then washed twice with UA buffer, three times with 200 μl of IP buffer (50 mM MOPS-NaOH pH 7.2, 10 mM Na₂HPO₄, 50 mM NaCl) and digested for 16 hours with Lys-C (Wako) in 50 μl IP buffer at 37 °C (enzyme to protein ratio 1:50). Sample were collected and the filters were washed with 50 μl of IP buffer to increase the yield of Lys-C peptides. Peptides retained on the filter units were subsequently digested with endoproteinase Glu-C for 16 hours in 50 μl of IP buffer at 20°C (enzyme to protein ratio 1:100) and following collection of peptides, the filter units were again washed with additional 50 μl of IP buffer. To analyse nickel affinity purifications directly a small fraction of the nickel column elution (~ 2 μg) was diluted tenfold in 8 M urea 100 mM Tris-HCl pH 7.5, treated with 50 mM chloroacetamide for 1.5 h at 20 °C, then diluted four times with 50 mM ammonium bicarbonate and mixed with Lys-C (enzyme to protein ratio 1:50), followed by digestion at 20 °C for 16 hours.

Immunopurification of diGly-Lys containing peptides

Anti-K-ε-GG conjugated to protein A beads (20 μl of beads; PTMScan, Cell Signaling Technology) was washed three times with 1 ml of 50 mM sodium borate pH 9.0, resuspended in 1 ml of fresh 20 mM dimethyl pimelimidate (DMP) and incubated for 30 min at room temperature while rotating. Beads were washed twice with 1 ml of 200 mM ethanolamine pH 8.0 for 2 h at 4 °C, three times with 1.5 ml of cold IP buffer and stored in IP buffer at 4 °C. Enrichment of diGly-Lys containing peptides with anti-KεGG was performed according to a published protocol (54) with minor changes. Anti-KεGG (19 g) cross-linked to protein A beads (3 L) was added to peptide mixtures (300 g for the primary experiment or 360 g for the experiment designed to evaluate reproducibility) in IP buffer and incubated at 4 °C for overnight while rotating. Beads were washed twice with 500 μl of cold 1x DPBS and peptides were eluted twice with 50 μl of 0.1% trifluoroacetic acid (TFA).

Mass spectrometric analysis and data processing

Prior to mass spectrometric analysis, all peptide samples were desalted on self-made reverse-phase C18 (Empore) stop-and-go-extraction tips (55) and analysed by liquid chromatography tandem MS on a Q Exactive mass spectrometer (Thermo Scientific) coupled to an EASY-nLC 1000 liquid chromatography system (Thermo Scientific) via an EASY-Spray ion source (Thermo Scientific). Purified peptides were loaded onto 75 μm × 500 mm EASY-Spray column (Thermo Scientific) at a maximum pressure of 800 bars and various gradient lengths from 90 minutes to 150 minutes were used with a linear gradient of 5 % to 22 % of solvent B (100 % acetonitrile, 0.1 % formic acid) in solvent A (0.1 % formic acid), followed by a ramp to 40% of solvent B. Flow rate was set to 250 nl/min and eluting peptides were injected online into the mass spectrometer. Various Q Exactive settings were tested depending on the complexity of the samples, however optimal data acquisition for low complexity diGly-Lys containing peptides was achieved with the following parameters: Precursor ion full scan spectra (m/z 300 to 1,600) were acquired with a resolution of 70,000 at m/z 400 (target value of 1,000,000 ions, maximum injection time 20 ms). Up to one data dependent MS2 spectrum was acquired with a resolution of 35,000 at m/z 400 (target value of 500,000 ions, maximum injection time 1,000 ms). Ions with unassigned charge state, and singly or highly (>8) charged ions were rejected. Intensity threshold was set to 2.1 × 10⁴ units. Peptide match was set to preferred and dynamic exclusion option was enabled (exclusion duration 40 s).

Data analysis and manual validation of the results

Raw mass spectrometric data files were processed using MaxQuant software (version 1.3.0.5) (56, 57) and searched against UniProtKB human proteome (canonical and isoform sequences; downloaded in April 2013). Enzyme specificity was set to cleave peptide bonds C-terminally to Lys residues for samples treated with Lys-C only or C-terminally to Glu, Asp, and Lys residues for samples digested with Lys-C and Glu-C. A maximum number of three or five missed cleavages were allowed for samples cleaved with Lys-C, or Lys-C and Glu-C, respectively. Carbamidomethylation of Cys was set as a fixed modification and oxidation of Met, acetylation of protein N-termini, phosphorylation of Ser, Thr, and Tyr, and diGly adduction to Lys (except in peptide C-terminus) were set as variable modifications. A minimum peptide length was set to seven amino acids and a maximum peptide mass was 10,000 Da. A false discovery rate (FDR) of 1 % was set as a threshold at both protein and peptide level, and a mass deviation of 6 parts per million was set for main search and 0.5 Da for MS2 peaks.

MS2 spectra (filtered at 1% FDR) of sumoylated peptides were manually validated and annotated using MaxQuant viewer expert system (58) based on following criteria: (a) good coverage of y-and b-ion series, (b) extensive identification rate of intensive fragment ion peaks, (c) mass error less than 2 ppm after mass recalibration or 4 ppm in case of unsuccessful recalibration, and (d) preferential fragmentation N-terminally to proline or C-terminally to Glu and Asp residues. All peptides identified from the reverse decoy database were removed and the probability of site localization was checked to be greater than 75%. Existence of a diagnostic peak corresponding to a fragment ion of diGly residue (GG⁺, m/z +115.0505 Th) or presence of an ion series with a neutral loss corresponding to single Gly (m/z 57.0215 Th) or an identification of various modified peptides corresponding to the same sumoylation site contributed to the confidence in assigned sumoylation sites. Finally, all accepted modified peptides corresponding to more than one protein were merged into one site identifier indicated in table S1. To investigate experimental reproducibility a SUMO site was considered “identified” if it matched a peptide from the first list of 1,002 modified peptides.

In vitro SUMO conjugation, deconjugation and processing assays

Recombinant substrate proteins used in these assays have been decribed previously (59). All reactions were buffered in 50 mM Tris-HCl pH 7.5. SUMO2 pro-form processing assays contained 150 mM NaCl, 0.5 mM TCEP, 600 M SUMO, and 100 nM SENP1 or 200 nM SENP2 recombinant catalytic domains (59), and reactions were incubated at 20 °C for 0, 5, 10, 20, 30, 60, 90, 120, 180, 240 or 960 minutes. Conjugation assays contained 5 mM DTT, 5 mM MgCl₂, 2 mM ATP, 110 nM SAE1 and SAE2, between 0.5 and 2 M Ubc9, ~10 M substrate protein, and a range of SUMO2 concentrations (0, 40, 80, or 200 M) and were incubated at 37 °C for 4 hours. Deconjugation assays were prepared as above using 200 M SUMO followed by addition of SENP1 to 10 nM and reactions monitored at 0, 0.5, 1, 2.5, 5 and 10 minutes at 20 °C.

Immunoblot analysis

HEK293 N3S cells either stably expressing or not 6His-SUMO2^T90K were grown on 75 cm² flasks in DMEM supplemented with 10% FCS, 1 μg/ml puromycin (where required), and 100 U/ml penicillin and streptomycin. Where indicated heat shock treatment at 43 °C for 30 minutes was applied. Cells were washed twice with 1x DPBS and lysed in protein sample buffer. Immunoblots were prepared using mouse antibody specific to 6His (Clontech No. 631212) or rabbit antibody specific to SUMO2 (Zymed, 91-5100).

TAP-SUMO2 sample preparation and MS analysis

TAP-SUMO2 protein samples remaining from a previous study (6) were fractionated by protein electrophoresis and the gel was cut into nine slices, trypsinized, and the tryptic peptides were extracted, as described previously (60). Peptide mixtures were analysed by MS as described with the following alterations: HPLC fractionation occurred over a linear 115 minute fractionation containing a gradient from 15-32% acetonitrile, with a loop count of 3, an m/z precursor scan range of 800-1800, +3 to +7 inclusion charge range, 5x10⁵ AGC target or 500 ms maximum injection time and 35,000 at 400 m/z resolution for MS2 scans.

Virtual branched peptide database

Virtual branched peptide databases were created as a modification of the procedure described in Matic et al. (11). A python script was created to extract protein sequence information for each SUMO modified protein and then determine the tryptic peptide encompassing each SUMO target lysine. Cleavages at KP and RP were ignored, and protein N-terminal methionines were omitted. Each tryptic peptide was appended at the N-terminus with either the tryptic fragement SUMO1 C-terminus (IADNHTPKELGMEEEDVIEVYQEQTGG) or that of SUMO2 C-terminus (FRFDGQPINETDTPAQLEMEDEDTIDVFQQQTGG), which include a single missed cleavage in this region of SUMO. Raw data files were searched against the databases (SUMO1.fasta, SUMO2.fasta) using MaxQuant version 1.3.0.5 (56, 57). Maximum peptide size was set to 10,000 Da. A whole human proteome database was used for first search and the branched peptide database(s) used for main search. Missed cleavages were set to at least 3 and no FDR filtering was used at the protein or peptide level. All spectra were computationally (58) and manually validated.

Bioinformatics analysis

Sequence analysis was performed using pLogo (36). As not all identified peptides could be assigned to a single protein, multiple 13 amino acid sequences were used for input in these cases. N- or C-terminal sequences that did not cover the 13 residue window were omitted from the output. Residues were scaled relative to their Bonferroni-corrected statistical significance using human proteome as a background data set (645,531 lysines). Protein functional annotation and network analysis was created using Ingenuity pathway analysis (Ingenuity Systems, QIAGEN, Redwood City, CA)