Arginine-Selective Chemical Labeling Approach for Identification and Enrichment of Reactive Arginine Residues in Proteins.

Modification of arginine residues using dicarbonyl compounds is a common method to identify functional or reactive arginine residues in proteins. Arginine undergoes several kinds of posttranslational modifications in these functional residues. Identifying these reactive residues confidently in a protein or large-scale samples is a very challenging task. Several dicarbonyl compounds have been utilized, and the most effective ones are phenylglyoxal and cyclohexanedione. However, tracking these reactive arginine residues in a protein or large-scale protein samples using a chemical labeling approach is very challenging. Thus, the enrichment of modified peptides will provide reduced sample complexity and confident mass-spectrometric data analysis. To pinpoint arginine-labeled peptide efficiently, we developed a novel arginine-selective enrichment reagent. For the first time, we conjugated an azide tag in a widely used dicarbonyl compound cyclohexanedione. This provided us the ability to enrich modified peptides using a bio-orthogonal click chemistry and the biotin-avidin affinity chromatography. We evaluated the reagent in several standard peptides and proteins. Three standard peptides, bradykinin, substance P, and neurotensin, were labeled with this cyclohexanedione-azide reagent. Click labeling of modified peptides was tested by spiking the peptides in a myoglobin protein digest. A protein, RNase A, was also labeled with the reagent, and after click chemistry and biotin-avidin affinity chromatography, we identified two selective arginine residues. We believe this strategy will be an efficient way for identifying functional and reactive arginine residues in a protein or protein mixtures.

Introduction

Arginine is one of the basic amino acids of proteins and functionally very important for protein structures, enzyme activities, and protein interactions.[1−4] Arginine goes through several kinds of posttranslational modifications (PTMs) during cellular processes. Among them, glycation is a nonenzymatic modification of proteins, which is the result of the addition of a sugar molecule to a protein by Maillard reaction.[5] In this reaction, nucleophilic amino groups of amino acids react with carbonyl groups of the sugar to form a Schiff base product. This unstable Schiff base intermediate rearranges to a stable adduct known as Amadori product, which is a keto-amine compound. This irreversible covalent modification can occur particularly at protein arginine residues by carbonyl compounds, resulting in the formation of advanced glycation end products (AGEs).[5−7] AGEs lead to many diseases including diabetes mellitus, alzheimer’s disease, and atherosclerosis.[8−10]

AGEs can be formed not only from the reducing sugars but also from dicarbonyl compounds such as glyoxal and methylglyoxal, which are intermediates of several cellular processes. These dicarbonyl compounds are more selective to guanidino, amino, and thiol group of proteins and nucleic acids.[11] Methylglyoxal modifications have also been observed in arginine residues in recombinant antibody developed for protein therapeutics.[12] Methylglyoxal reaction with arginines and arginyl residues in proteins was reported by Takashi in 1977, Cheung and Fonda in 1979, and Selwood and Thornalley in 1993.[13−15] Brock and his group addressed the site specificity of AGE formation on ribonuclease and they found that the main site of carboxymethylation was at lysine-4[16] Apart from that, Cotham et al. in 2004 reported that the glyoxal-derivative formation takes place mainly at arginine-39 and arginine-85.[17]

Identifying the specific binding sites of proteins plays a significant role in drug discovery.[18,19] Chemical modification is a powerful technique for identifying these reactive residues.[20,21] The reactivity of arginine toward dicarbonyl groups was evaluated thoroughly by our group recently.[22] In cellular processes, these dicarbonyl compounds modify mainly arginine residues, which are functional, reactive, or surface-accessible. Locating these modifications in a protein and large-scale samples is very difficult because of sample complexity after digestion. The present study aimed at the development of an enrichment strategy for the reactive arginine residue containing peptides. In this study, we introduced an azide tag in a widely known arginine-reactive reagent, cyclohexanedione (CHD), hence reactive peptides can be affinity-purified utilizing bio-orthogonal click chemistry and biotin–avidin chromatography. This will help studying protein surface topology’s targeting arginines, PTM status in those residues, and profiling enzyme activities confidently.

Incorporating an affinity tag in dicarbonyl compounds is very challenging because of the difficulty of synthesis. To the best of our knowledge, for the first time, we are reporting an arginine-selective reagent with an azide functionality. Azides are known to be bio-orthogonal reagents, which do not undergo any side reactions with the functional groups present in the proteins.[23,24] They selectively react with alkynes to form a triazole product during an azide–alkyne cycloaddition reaction called click chemistry.[25,26] In this study, we performed the chemical labeling of the arginine residues in peptides and proteins using our arginine-selective reagent. Finally, chemically modified peptides were affinity-purified by avidin–biotin coupling after click reaction. We demonstrated selective labeling of reactive arginine residues in ribonuclease A (RNase A) protein using this cyclohexanedione-azide (CHD-Azide) compound. We believe that with efficient clickable reagent, this chemical labeling approach will help us to study reactive or functional arginines in proteins in large-scale studies. Further selective profiling of these arginines in diseased samples using a targeted proteomics approach will provide information about the PTM status of these residues.

Results and Discussion

Modification of Peptides by the CHD-Azide Reagent

A scheme of the chemical labeling and affinity purification of reactive arginine residues containing peptides is shown in Figure . At first, chemical labeling of arginine residues in peptides/proteins using CHD-Azide was performed. The structure of CHD-Azide is shown in the same scheme (see also Figure S1). After that, proteins were digested and resultant peptides were coupled using a clickable biotinylated reagent biotin-PEG4-alkyne (Figure S1). With subsequent click labeling, the CHD-Azide-labeled peptides were enriched using biotin–avidin affinity chromatography. Liquid chromatography–mass spectrometry (LC–MS)/MS analysis of enriched peptides provided the identity and the location of the CHD-Azide reacted products.

Figure 1

Chemical labeling and affinity purification strategy of reactive arginine residues by arginine-selective CHD-Azide reagent and bio-orthogonal click chemistry.

Electrospray ionization (ESI)–MS analysis of the modified model peptides is shown in Figure . The three model peptides we used in our studies include bradykinin (RPPGFSPFR, monoisotopic mass 1059.5614 Da), substance P (RPKPQQFFGLM, monoisotopic mass 1346.7281 Da), and N-terminal blocked peptide, neurotensin (pyroglu-LYENKPRRPYIL, monoisotopic unmodified mass 1671.9097 Da). These three model peptides contained two arginine residues at the two terminal ends, single arginine at the N-terminal end, and two adjacent arginine residues in the middle of the peptide, respectively. CHD-Azide-modified bradykinin mass was given in Figure A at m/z 749.92 [M + 2H]2+ and at m/z 500.34 [M + 3H]3+. Details of mass addition are shown with ChemDraw in Figure S2A,B. We also compared the fragmentation pattern of the unmodified and modified peptides (see the Supporting Information for comparison, Figure S3). The modified peptide clearly showed complete labeling of two arginines by this CHD-Azide reagent.

Figure 2

ESI–MS and MS/MS spectra of modified peptides with CHD-Azide reagent. (A) CHD-Azide-modified bradykinin at m/z 749.92 (M + 2H + CHD-Azide)2+ and m/z 500.34 (M + 3H + CHD-Azide)3+. (B) MS/MS spectrum of CHD-Azide-modified bradykinin at m/z 749.92 (M + 2H + CHD-Azide)2+. (C) CHD-Azide-modified neurotensin at m/z 1056.60 (M + 2H + CHD-Azide)2+ and m/z 704.76 (M + 3H + CHD-Azide)3+. (D) MS/MS spectrum of CHD-Azide-modified neurotensin at m/z 704.76 (M + 3H + CHD-Azide)3+. See the Supporting Information for unmodified spectra for comparison. * denotes CHD-Azide-modified sites.

Furthermore, the modification with neurotensin yielded two peaks at m/z 1056.60 for the doubly charged ion and for the triply charged ion at m/z 704.76 (Figure C). The mass difference between the unmodified neurotensin mass at m/z 837.45 and m/z 558.66 confirmed dual CHD-Azide incorporation to the two adjacent internal arginine residues in the peptide (Figures S2B and S3B). The results were further confirmed by tandem MS studies after careful inspection of all MS/MS spectra from modified peptides.

Tandem MS Studies of the Peptides

CHD-Azide modification at the two-arginine-containing peptide bradykinin R*PPGFSPFR* (Figure A) was characterized by the CID tandem MS spectra (Figure B). Fragmentations of the doubly charged molecular ion at m/z 749.92 (Figure B) and unmodified parent ion at m/z 530.85 are given in the Supporting Information (Figure S3), respectively. The mass difference 438.14 Da revealed that the two arginine residues are modified with the CHD-Azide. Note that for the formation of a single CHD-Azide adduct, the mass addition is 219.07 Da. The annotated MS/MS spectrum in Figure B gave the modified b and y ion series (detailed m/z of fragment ions are provided in an Excel file). It is notable that the addition of 219.14 Da to modified ion fragments corresponds to the formation of two CHD-Azide adducts with the two arginine residues.

Figure D shows the MS/MS spectrum obtained after reaction between CHD-Azide and neurotensin. These data further supported the conclusion that the two arginine residues are modified by CHD-Azide. Most of the b and y ion fragments were observed with high abundance (please see the Excel file for the fragment ion mass). The above deduction was confirmed by the mass difference between the unmodified fragments and the modified fragment ions (see Supporting Information, Figure S3B). The MS2 data confirmed the selectivity of this reagent toward arginine residues and their locations with high confidence.

Feasibility of Click-Chemistry-Based Peptide Enrichment

Next, we tested click labeling and biotin–avidin affinity purification of CHD-Azide-modified peptides using a commercially available biotin-PEG4-alkyne reagent. Biotin-PEG4-alkyne is not a suitable reagent for peptide-level mass-spectrometric fragmentation because of poly(ethylene glycol) (PEG) and biotin groups. This kind of reagent was widely used for the enrichment of proteins. Because of the solubility, we decided to use this reagent and to provide feasibility of affinity purification at the peptide level. To test the feasibility, a substance P-modified peptide was spiked in a mixture of myoglobin digest. After click labeling in this complex mixture, we affinity-purified the peptide by avidin. The base peak chromatogram (BPC) of spiked myoglobin digests and corresponding BPC after CHD labeling and click enrichment are shown in Figure A,B, respectively. We also searched the myoglobin peptides before and after enrichment and found that a few myoglobin peptides were identified with reduced PSMs in the enriched samples (Supporting Information, excel file). It is clearly shown that affinity purification significantly reduced the MS data. Further analysis of MS/MS data clearly indicated that CHD-modified peptide was successfully labeled by biotin-PEG4-alkyne. MS/MS was performed on the [M + 2H]2+ ion at m/z 1012.88. The fragmentation behaviors of the click-modified and affinity-purified peptide are shown in Figure (see excel files for fragments m/z’s). The CHD-Azide-modified peptide containing the clickable part gave most of the b fragment ions (Figure C). The modified fragment ion series indicated that the addition is 457.58 Da, which corresponds to the biotin-PEG4-alkyne attached to the CHD-Azide modification site. This together with the unmodified ions b2, b7, b8, and b10 and CHD-modified fragments b2*, b7*, b8*, and b10* (*-CHD-Azide-modified) as well as the CHD-Azide-click-modified fragments b2#, b7#, b8#, and b10# (#-CHD-Azide-click-modified) can be used to justify the modification at the arginine residue (Figure S4A,B). The mass difference between b2 and b2* is 219.14 Da, which clearly showed that the CHD-Azide modification is at the arginine residue. The mass shift between b2* and b2# fragment ion of the enriched peptide is 457.58 Da, giving a strong evidence of the reaction of biotin-PEG4-alkyne with the azide group. As mentioned before, biotin and PEG functionalities complicate the MS/MS fragmentation spectra. Our future strategy is also to develop or use a cleavable click reagent to remove these groups before MS analysis. The above spiking studies clearly demonstrated the feasibility of click-chemistry-based labeling and purification of arginine-modified peptides in a protein digest.

Figure 3

Feasibility of click-chemistry-based enrichment using spiking studies. (A) BPC of a myoglobin digest with a spiked substance P. (B) BPC after CHD labeling, click chemistry reaction, and biotin–avidin purification. (C) MS/MS spectra of spiked CHD-Azide-modified and click-labeled substance P peptide at m/z 1012.88 (M + 2H + CHD-Az + CLICK)2+. #-denotes both CHD-Azide and click labeling.

From the outcome noted on the model peptides, we decided to carry out the CHD-Azide modification reaction with an enzyme RNase A. This model protein system was studied to demonstrate the applicability of this chemical labeling method for locating functional arginine residues in the enzyme.

Modification of RNase A Protein by the CHD-Azide Compound

To identify the amino acids modified by CHD-Azide, the digested peptides of the modified protein sample were analyzed by nano LC–MS/MS. We were able to identify the modified arginine residues in RNase A protein. Two amino acid sequences were identified with the chemical modification at the arginine residues. Specifically, we found two peaks at m/z 469.28 [M + 2H]2+ and m/z 1003.17 [M + 3H]3+, which corresponded to a peptide with amino acid sequence SR*NLTK and a large peptide, DR*C!KPVNTFVHESLADVQAVC!SQK (*-CHD-Azide modified, !-cysteine −SH blocked by carbamidomethylation) with two carbamidomethyl modification at two cysteine residues, respectively.

To confirm the modification sites, we investigated the tandem MS data of the CHD-Azide-treated RNase A protein. The results in Figure A demonstrated the MS/MS fragmentation of the unmodified peptide SRNLTK. The difference between untreated and treated RNase A peptide SRNLTK was 219.14 Da, which suggested that Arg-33 was modified by CHD-Azide. As presented in Figure B, the MS/MS of the peptide SR*NLTK confirmed the Arg-33 modification by the CHD-Azide compound. The precursor ion at m/z 469.93 that generated CID fragment ions corresponds to b2*-NH3, b3*, b4*, b5*, y1, y2, y3, y4, and y5*-H2O. In addition, we saw that the unmodified peptide SRNLTK at m/z 360.90 gave fragment ions b4, b5, y1, y2, y3-H2O, and y6-NH3. A comparison of a fragment ion at m/z 471.27 (b4) of the unmodified peptide with m/z 690.41 (b4*) of the modified peptide SR*NLTK showed that the mass addition was 219.14 Da. Closer examination of another fragment ion b5* of the modified peptide at m/z 791.45 with b5 of the unmodified peptide at m/z 572.32 revealed the characteristic mass addition of 219.13 Da. These data clearly suggested that the CHD-Azide modification is at Arg-33. After click labeling, we observed the modified peptides in the enriched sample at m/z 465.72. This is a small peptide and was observed with +3 charge states with the tag. It has two serine and threonine residues and contained a PEG biotin, so loss of water was anticipated from the fragmented residues (Figure S6). However, we were able to confirm the modification on this arginine residue. We observed #b23+ (#CHD-Azide and click-modified) fragment with high intensity. The difference between this peak and the *b2 (*-CHD-Azide-modified) is 456.44 Da, which corresponds to the click-modified mass.

Figure 4

MS/MS spectra of unmodified and modified RNase A peptides. (A) MS/MS spectrum of peptide SRNLTK at m/z 360.90 (M + 2H)2+. (B) MS/MS spectrum of CHD-Azide-modified peptide SR*NLTK at m/z 469.93 (M + 2H + CHD-Az)2+. For example, the difference between b4* and b4 corresponds to the addition of 219.14 Da, the CHD-Azide modification mass. * denotes CHD-Azide-modified sites.

Under identical tandem MS conditions, we saw Arg-39 modification in peptide DR*C!KPVNTFVHESLADVQAVC!SQK (*-CHD-Azide, !-carbamidomethylation of cysteine) (Figure S7A). As expected, CID data of the precursor ion peak at m/z 1003.17 produced few fragment ions of this large peptide with several modifications. In this peptide, the 219.16 Da increases in the peptide mass between the unmodified peptide and CHD-Azide-modified peptide confirmed that the CHD-Azide compound also modified Arg-39. The tandem MS data produced by both sequences were matched against the molecular calculator software (omics.pnl.gov) to confirm the identity of the peptides. After click chemistry labeling and affinity purification by avidin, we have seen significant reduction of MS data (see the BPC in Supporting Information S5, A and B). The most abundant b and y ions of peptide DR#C!KPVNTFVHESLADVQAVC!SQK (# CHD-Azide click-modified after click modification) (Figure S7B) confirmed the modification at Arg-39. Extracted ion chromatogram (XIC) and retention time of these modified peptides were provided in pages 15–17 at the Supporting Information. However, the quality of the tandem MS data was poor because of the incomplete fragmentation of this highly modified large peptide and side-chain PEG and biotin group. As mentioned earlier, PEG–biotin is a very good reagent for the purification of modified proteins by biotin–avidin chromatography, but in peptide label, their fragmentations complicate the tandem MS data. Thus, cleavable biotinylated reagents to release the PEG and biotin from the conjugated peptides are necessary to assign all fragment ions.[27,28] As displayed in Figure S7B, some of the b and y ions are absent or they occur at very low intensity m/z values because the peptide contained two carbamidomethyl-modified cysteine residues along with the CHD-Azide-click modification. The difference between click-modified fragment ion b173+ at m/z 882.56 (Figure S7B) and the CHD-Azide-modified b173+ at m/z 730.68 (Figure S7A) confirmed a mass addition of 457.58 Da, which corresponds to the molecular weight (MW) of the biotinylated click reagent. Because of the large tag, these peptides shifted to longer retention time. Retention time and XIC for these peptides are provided in the Supporting Information pages 15–17.

Our data showed that Arg-39 and Arg-33 are the most reactive arginine residues present in RNase A. Functional probing of arginine residues in RNase A protein was done previously. This labeling study will pinpoint solvent-accessible arginine residues and their local environment. If there are strong interactions with other residues, they will not be available for reactions. Our findings agreed with Laszlo Patthy’s studies on the identification of functional arginine residues in RNase A and lysozyme.[29] The data also agreed with Brock et al. mass spectrometric studies on the detection and identification of arginine modifications on methylglyoxal-modified ribonuclease.[30] These findings were displayed with the crystal structure of RNase A protein with all arginine residues and the selective arginine residues labeled and enriched with our CHD-Azide and bio-orthogonal chemical labeling strategy (Figure A,B).

Figure 5

Crystal structure of RNase A protein (PDB file 4J5Z). (A) All arginine residues in RNase A. (B) Modified sites of RNase A with CHD-Azide.

Another challenge associated with the CHD-type compounds is the high pH reaction condition. Several researchers frequently used CHD because of its superior labeling efficiency compared to phenyl and methylglyoxal. Fortunately, it is found that even with high pH reaction environment, it is quite capable of labeling functional arginine residues.[29,31,32] Screening the reactive residues will also help in understanding the targets, which will be utilized further for activity assays. We believe that this structural information will be extremely useful for the structural biologist for further exploration of new enzyme activities by mutating these residues.

Conclusions

In this study, we utilized a chemical labeling approach for evaluating selective purification of reactive arginine residues in proteins. To the best of our knowledge, for the first time, we developed an arginine-selective reagent with enrichment functionality utilizing a cyclic 1,2 dicarbonyl compound. We incorporated an azido group in 1,2 CHD so that arginine-labeled peptides can be enriched with a clickable biotinylated reagent. We tested the chemical labeling methods in several standard peptides with arginine residues in different positions in the sequence. Complete labeling was observed in those peptides. We demonstrated affinity purification with a commercially available clickable biotinylated reagent. A peptide spiked in a protein digest was successfully affinity-purified after click labeling. Furthermore, we tested the labeling efficiency of this reagent on RNase A protein to identify and validate the reactive arginine residues. Two functional arginine residues were selectively modified and click-labeled using this approach. We utilized a commercially available clickable biotinylated reagent to evaluate the chemical labeling of CHD-Azide-labeled peptides. Because of PEG and biotin functionality in this compound, we have seen poor fragmentation in the click-modified large and small peptides. Nevertheless, we have successfully demonstrated the attachment of CHD-Azide and click adducts after affinity purification. We are in the process of employing a cleavable clickable biotinylated reagent so that the modified peptide can be removed from the avidin beads by chemical cleavage. Thus, fragmentation complexity can be reduced from the click-labeled mass spectra. In this study, we provided a proof-of-concept of an arginine-selective reagent with enrichment functionality for identifying reactive arginine residues in large-scale samples. Our approach demonstrated that CHD-Azide labeling and click-based enrichment method is a reliable analytical technique to determine the reactive/functional arginine residues. We believe this study will pinpoint the reactive arginine targets with high confidence for studying protein structures and biomarker identifications.

Methods

Materials

Ribonuclease A from bovine pancreas (RNase A), ubiquitin from bovine erythrocytes, ammonium bicarbonate (NH4HCO3), formic acid (FA), sodium hydroxide (NaOH) pellets, acetonitrile, and iodoacetamide (IAM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Biotin-PEG4-alkyne was from Click Chemistry Tools, Scottsdale, AZ. For proteolysis, sequencing-grade-modified trypsin was purchased from Promega (Madison, WI, USA). For disulfide bond reduction, dithiothreitol was obtained from Bio-Rad (Hercules, CA). Pierce C18 Tips from Thermo Fisher Scientific (Rockford, IL, USA) were used to desalt the samples. Phosphate-buffered saline (PBS) was from VWR, Suwanee GA. Three model peptides bradykinin, neurotensin, and substance P were purchased from AnaSpec (Fremont, CA). To remove excess CHD-Azide, Pierce concentrators [3k MW cutoff (MWCO)] were utilized. Pierce UltraLink monomeric avidin was obtained from Thermo Scientific (Rockford, IL). All sodium dodecyl sulfate-polyacrylamide gel electrophoresis supplies were purchased from Bio-Rad (Hercules, CA).

Sample Preparation

Chemical Modification of Arginine Residues in Peptides

Bradykinin, neurotensin, and substance P were purchased from AnaSpec (Fremont, CA). Recently, we have reported an assessment of chemical labeling method to identify functional/reactive arginine residues of proteins by MS utilizing two widely used arginine-reactive reagents.[22] We followed the same labeling protocol mentioned in our recently published article.[22] For each peptide sample (1 mM, 5 μL), we added 5 μL of 30 mM CHD-Azide reagent in 200 mM sodium hydroxide solution. The reaction was allowed to continue for 2 h at 37 °C under agitation. After the reaction, the samples were dried in a SpeedVac at 30 °C for 1 h. The two dried samples were then reconstituted in 0.1% FA. After that, samples were vortexed and desalted using Thermo Scientific Pierce C18 tips. Desalted samples were then used for the analysis after diluting with 1:1 MeOH/H2O with 2% acetic acid.

Chemical Modification of Arginine Residues in Proteins

Protein was reduced and alkylated using 2 μL of 10 mM dithiothreitol (Bio-Rad, CA) and 10 μL of 10 mM IAM (Sigma-Aldrich, MO), respectively. The sample was then diluted with 200 mM sodium hydroxide, followed by the addition of 5 μL of 30 mM CHD-Azide solution. The reaction was carried out at 37 °C for 24 h.

Samples were transferred into the pierce concentrators (3 kDa MWCO) for centrifugal ultrafiltration to remove the excess CHD-Azide reagent. Cleared protein samples were digested with trypsin, using 1:100 enzyme-to-protein ratio, for 16 h at 37 °C with frequent mixing.

After trypsin digestion, the peptide mixture was fully dried and reconstituted in PBS buffer. At the same time, the azide-containing peptide sample was prepared for the click chemistry reaction by adding 2 μL of 100 mM water-soluble tris(3-hydroxypropyltriazolylmethyl)amine click ligand, 400 μL of 50 mM CuSO4, and 80 μL of 50 mM Tris(2-carboxyethyl)phosphine.[24] Click reagent biotin-PEG4-alkyne was added in a 1:20 molar ratio of peptide and allowed to react for 2 h at room temperature in a rotor.

The click-labeled sample was then incubated with 30 μL of monomeric avidin beads. Prior to incubation, the avidin beads were washed thrice with 1× PBS buffer. The sample was then incubated with the avidin beads at room temperature for 4 h. The beads were washed again thrice with PBS buffer, twice with 25 mM NaCl, and thrice with water. Finally, the captured peptides were eluted from the avidin beads using the elution solution, which contained 50:50 acetonitrile/H2O and 0.4% trifluoroacetic acid (TFA).

Feasibility of Click-Chemistry-Based Peptide Enrichment

To study the feasibility of click-chemistry-based peptide enrichment, we performed a spiking study. The CHD-Azide-modified substance P (1 mM, 5 μL) was spiked into a tryptic digest of myoglobin (1 mM, 5 μL). After the click-labeling reaction, the CHD-Azide-modified peptides were captured by avidin beads following the same protocol mentioned above. Then, the click-labeled biotinylated peptide was eluted using the same elution solution containing 50% acetonitrile/50% H2O and 0.4% TFA.

Instrument Setup

The samples were analyzed using a Thermo Scientific Velos Pro dual-pressure linear ion trap mass spectrometer operated in a positive-ion mode and controlled by Xcalibur software (Thermo Fisher Scientific, San Jose, CA, USA). Peptides were loaded onto a nanoViper Acclaim PepMap C18 column with an inner diameter of 75 μm, a particle size of 2 μm, a pore size of 100 Å, and a length of 15 cm bed for separation. Reverse-phase chromatographic separations of the loaded peptides were performed on a Dionex Ultimate 3000 RSLC nanochromatography (Thermo Fisher Scientific) system across a 65 min run with a flow rate of 300 nL/min. A multistep gradient was used, which consisted of 4% B for 3 min, 4–30% B for 30 min, 30–60% B for 55 min (A, 0.1% FA in water; B, 95%: 5%: 0.1% acetonitrile: water: FA).

The mass spectrometer was operated in normal scan modes, and a full MS spectrum was obtained. Peptides were identified in data-dependent acquisition (DDA) mode to obtain the tandem mass spectra (MS/MS) for the 10 most abundant ions (collision energy of 40, activation Q of 0.25, and activation time of 10 ms). In DDA analysis, singly charged ions were rejected. To observe the fragmentation behavior of the CHD-Azide modification at the peptide level, a direct infusion analysis was performed for untreated and CHD-Azide-treated peptide samples using ESI-thermo Velos-MS. The HESI source was operated in the positive-ion mode using ESI probe voltage at 2.5 kV, a capillary temperature of 250 °C, sheath gas = 5, and S-lens RF = 62. Before injection, the peptide samples were diluted with 1:1 MeOH/H2O: 2% acetic acid and were injected into the mass spectrometer through the syringe pump.

Identification of CHD-Azide Adducts

The software Proteome Discoverer (version 1.3, Thermo Scientific, USA) was used to extract mass spectral data and search them against RNase A sequence (Uniprot P02769). Trypsin was selected as the cleaving proteases, allowing a maximum of three missed cleavages. Peptide and fragment-ion tolerances were set to 5 ppm and 0.7 Da, respectively. Cysteine carbamidomethylation was set as fix modification (+57.02147). The MS/MS spectra of modified peptides were manually inspected for the confident mapping of the modification sites.

Synthesis

Detailed synthesis is described in the supplementary sections with NMR, IR, and high-resolution MS data. Briefly, 1,6-dibromohexane was first reacted with p-methoxy benzyl alcohol, under basic conditions, to prepare 1-((6-bromohexyloxy)methyl)-4-methoxybenzene. After this, 4-(6-((4-methoxybenzyl)oxy)hexyl)-2-oxocyclohexyl acetate was synthesized by coupling 2-oxocyclohex-3-en-1-yl acetate with the freshly prepared ((6-bromohexyloxy)methyl)-4-methoxybenzene. This new coupling product was deprotected under oxidative conditions to expose its terminal hydroxyl group, which was tosylated and subsequently substituted with sodium azide to produce the expected alkyl azide intermediate. Finally, an acetyl deprotection was performed, followed by a Swern oxidation reaction to produce the CHD-Azide final compound (see Supporting Information data, pages 11–14).