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Detection of Alkynes via Click Chemistry with A Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry Lihua Yang, Chris Chumsae, Jenifer B. Kaplan, Kevin Ryan Moulton, Dongdong Wang, David H Lee, and Zhaohui Sunny Zhou Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00354 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 26, 2017

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Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Bioconjugate Chemistry

The following graph will be used For Table of Contents Only

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Detection of Alkynes via Click Chemistry with A Brominated Coumarin Azide by Simultaneous Fluorescence and Isotopic Signatures in Mass Spectrometry Lihua Yang,†‡* Chris Chumsae,†* Jenifer B. Kaplan,† Kevin Ryan Moulton,‡ Dongdong Wang,†∥ David H. Lee,†§ Zhaohui Sunny Zhou‡ †

AbbVie Bioresearch Center, 100 Research Drive, Worcester, MA 01605, United States

‡

Barnett Institute of Chemical and Biological Analysis, Department of Chemistry and Chemical

Biology, Northeastern University, 360 Huntington Ave, Boston, Massachusetts 02115, United States ABSTRACT: Alkynes are a key component of click chemistry and used for a wide variety of applications including bioconjugation, selective tagging of protein modifications and labeling of metabolites and drug targets. However, challenges still exist for detecting alkynes because most 1,2,3-triazole products from alkynes and azides do not possess distinct intrinsic properties that can be used for their facile detection by either fluorescence or mass spectrometry. To address this critical need, a novel brominated coumarin azide was used to tag alkynes and detect alkyneconjugated biomolecules. This tag has several useful properties: first, it is fluorogenic and the click-chemistry products are highly fluorescent and quantifiable; second, its distinct isotopic pattern facilitates identification by mass spectrometry; and third, its click-chemistry products form a unique pair of reporter ions upon fragmentation that can be used for quick screening of data. Using a monoclonal antibody conjugated with alkynes, a general workflow has been developed and examined comprehensively. INTRODUCTION Click chemistry is a class of biocompatible reactions that effectively introduces a functional group and reporter to biomolecules.1-3 One major type of click chemistry is based on alkynes.4-7 Due to their absence in almost all natural biomolecules and exhibition of biorthogonal reactivity, alkynes are commonly introduced for broad use in biological, chemical and medical applications as tags, labels and intermediates.8-10

However, bioconjugation can lead to heterogeneous

products or unexpected by-products;11-12 therefore, the challenge is to identify and quantify all 1 ACS Paragon Plus Environment

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possible conjugation sites.

Once a biomolecule is conjugated or modified by a species

containing an alkyne, an azide tag can be used to label the alkyne1-2, 13-14 as the reaction between an alkyne and an azide forms a 1,2,3-triazole with high efficiency and specificity.4, 15-17 However, the triazole click products themselves do not usually possess intrinsic properties that can be detected by mass spectrometry or fluorescence. With these properties, inherent limitations such as low sample abundance, unexpected chemistry18 or sample complexity can be better addressed.19 As for most analyses, detection can be divided into two stages: a discovery stage that detects their presence and an elucidation stage that reveals their site and chemical nature. Standard automated software searches of MS/MS-based data tie the two stages together and often lead to an incomplete set of all modified sites, as the searches are restricted by requiring good ionization and fragmentations. Moreover, many search algorithms generate both false negative and false positive hits.20

A previous investigation analyzing the effects of including different

modifications in a search on the false positive rate found that including multiple modifications increases the number of false positive results.21 To improve on the current technology and analytical workflows, as detailed below, we describe a new analytical approach using a novel tag that separates the two stages of discovery and elucidation to maximize the detection of protein modifications. We developed a workflow that incorporates a brominated coumarin azide tag (3-azido-6-bromo-7-hydroxy-chromen-2-one, see Figure 1) that is fluorogenic and contains a distinct isotopic pattern imparted by bromine.

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Figure 1. Schematic representation of the click reaction of an alkyne with the fluorogenic brominated coumarin azide and the subsequent detection by fluorescence and isotopic signature in mass spectrometry. This tag possesses a fluorescent property that is highly selective and sensitive for detection and quantitation.22-24 For species with poor ionization, fluorescent detection allows "universal" discovery of tagged species. Additionally, a comparison of the fluorescent trace to MS/MSbased search results quickly discriminates between real and false hits because only labeled species have a fluorescent signal. Previously, a MS-compatible coumarin fluorophore tag with photoaffinity labeling was used to identify the ligand-binding site of a protein by leveraging fluorescence specificity over non-labeled peaks.25 Fluorescent tags have also been used to label N-glycans to both identify and quantify the glycosylation profile in proteins26 and employed to characterize the surface susceptibility of a monoclonal antibody.27 However, distinguishing labeled from non-labeled envelopes in MS is difficult in a complex matrix without a unique MS signature. To facilitate differentiating between labeled and non-labeled envelopes, the tag includes a halogen. Bromine's two isotopes create a unique pattern in a mass spectrum: 79Br and 81Br in a 1:1 ratio (79Br, 51%; 81Br, 49%), and this specific isotopic signature readily distinguishes the modified species from unmodified species. Brominated tags were previously used in tandem MS analysis to identify phosphorylation sites28 and facilitate de novo sequencing29 and protein identification.30 Bertozzi et al. leveraged the ability to search for distinguishable isotopic signatures and developed a pattern search algorithm known as isotopic signature transfer and mass pattern prediction (IsoStamp) to screen for the precursor ions of modified species,31 but IsoStamp still faces the challenges of MS limitations and can be prone to false positives and negatives. To examine how successfully this approach could identify modified residues, a monoclonal antibody (mAb) was conjugated with an N-HydroxySuccinimide (NHS)-ester alkyne at amine groups and labeled with the azide tag..32

The mAb-alkyne was used to demonstrate this

analytical approach, illustrated in Figure 1. Lysine or primary amine conjugation in a mAb is highly heterogeneous due to the more than eighty lysine residues, making detection of the conjugation site quite challenging.33-34 Both modified and unmodified peptides appear on the 3 ACS Paragon Plus Environment

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MS total ion chromatogram (TIC) but only the click triazole product has a fluorescent and isotopic signature. When a standard MS/MS-based search fails, the fluorescent peak directs the analyst to the corresponding peak in the TIC, and the unique bromine isotopic signature quickly distinguishes the site of modification. RESULTS AND DISCUSSION Characterization of Triazole Click Products. To determine how well a combination of fluorescence and a unique bromine isotopic signature could be used to identify conjugations/modifications, we characterized the click-chemistry products of an acylated lysine peptide alkyne, three other terminal alkynes with different functional groups, one strained cyclic alkyne and an alkyne-modified monoclonal antibody (Figure 2). The acylated lysine peptide alkyne represented one of the tryptic peptides from the modified mAb and this synthetic version was used as a corresponding model peptide.



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Figure 2. Alkynes used for click-chemistry product characterization. A model acylated lysine peptide alkyne (1) and other alkynes with different functional groups, including a standard chain peptide alkyne (2), propargyl alcohol (3), propargyl amine (4), a strained alkyne (5) and an alkyne-modified mAb (6). The mAb was modified by an NHS ester alkyne to covalently link an alkyne group to the mAb molecule with expected chemistry of acylation of amines in the lysine side chain and the N-termini. The characterization results show that the click-chemistry reaction went nearly to completion as expected (Figure S4).35

The extinction coefficient of the click-chemistry product was

-1

determined to be 18,522 M *cm-1 using the model peptide alkyne and propargyl alcohol (Figure S5). Under typical reverse-phase liquid chromatography conditions, the maximum excitation and emission wavelengths of the triazole click-chemistry product were 370 nm and 470 nm, respectively, from evaluation of two peptide alkynes and the strained alkyne (Figure S6). The intensity of fluorescence varied less than two-fold between pH 2 and 4 (Figure S7). The isotopic patterns of brominated species could be distinguished from non-brominated species up to 4000 Da per available experimental data obtained from tryptic peptides of the mAb-alkyne (Figure 3) and at least 7000 Da per simulation (Figure S8). We also discovered that the MS/MS spectra of the click-chemistry product contained a specific pair of reporter ions m/z=333.97 and 335.97 (Figure S9-S10).36-38 The limit of detection (LOD) for MS and fluorescence was obtained using the click-chemistry product from the model peptide alkyne. The fluorescence LOD of 0.4 fmole (Figure S11-S12) was an order of magnitude better than the MS LOD of 6.6 fmole, (Figure S13S14). The high sensitivity of fluorescence supports that it is a powerful tool to detect tagged alkynes. Additionally, it is important to note that the tag had minimal effect on ionization efficiency (Figure S15), maximum charge state (Table S1), and MS/MS pattern (Figure S16).


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All of these characteristics of the triazole product facilitate its detection and justified examining the workflow performance in a complex system.

Figure 3. Isotopic Pattern of Brominated Coumarin Triazole Products Across a Wide Molecular Weight Range. The MS precursor ion spectra up to ~4000 Da were from experimental results. The left-hand column shows the native peptides and those in the right- hand column show the isotopic pattern of the brominated peptides. The mass spectra of mono-brominated peptides up to approximately 1300 Da showed the unique doublet peaks contributed by 79Br and

81

Br (top).

The doublet shape was less evident for larger peptides because the 13C was stronger. However, the modified and unmodified peptides could be readily distinguished based on the most intense peak seen in the envelope (as annotated with *) and/or the width of the peak cluster in the mass spectrum. Detection of Conjugation Sites in a mAb. To examine how successfully this approach could detect modified residues, a recombinant monoclonal antibody was treated with an NHS-ester 6 ACS Paragon Plus Environment


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containing an alkyne.32, 34..Amines in lysine residues and at the N-terminus are expected to be acylated.

The mAb-alkyne was reacted with the brominated coumarin azide in a copper

catalyzed click-chemistry reaction and analyzed by tryptic mapping on a UPLC with both fluorescence and mass spectrometry detectors. To detect the expected conjugation sites in a known mAb using this approach, the software General Protein/Mass Analysis for Windows (GPMAW) was used to generate a list of native tryptic peptides of the mAb including one missed-cleavage, as modified lysine is not cleaved by trypsin. Once the peptide alkyne was detected as labeled, then the mass of the native peptide was obtained by subtracting the added mass of the tag. By matching against the in-silico predicted peptide list, the modified peptide was deduced. Even without MS/MS data, which did not always exist, the confidence of peptide deduction was still high. The identification of conjugated lysine 103 in the light-chain peptide APYTFGQGTKVEIK is described in the supporting information as an example [Figure S18-19]. Using this workflow, we found that all 13 lysines and the N-terminus in the light chain and 18 out of 31 lysines and the N-terminus in the heavy chain were conjugated by an alkyne (Table S3S4).

Our result demonstrates that the brominated coumarin azide tag is efficient for the

identification of species containing an alkyne. Detection of Unexpected Conjugation Sites in a mAb. The workflow was also effective at detecting sites of unexpected alkyne conjugation, which is more important and challenging [Table S5]. There were several fluorescent peaks that did not match any potential lysine/amine conjugation of the antibody, with one example shown in Figure 4. A conjugated peptide was fluorescently detected at retention time of 100.59 minutes with an intensity of 8.60E+07 RFU (relative fluorescence unit, 2.59% of the highest intensity), and a reporter ion pair was found in a MS/MS scan. This peak corresponded to a low-abundant species in the MS TIC (peak intensity =1.28E4, 0.11% of the highest intensity). Numerous ions were co-eluting at this specific scan, but the doubly-charged precursor ion with the bromine isotopic signature and an intensity at 5% of the most intense peak was readily obvious. This peptide did not match any peptide modified at a lysine residue. However, it matched a peptide in the heavy chain spanning residues 126-137 GPSVFPLAPSS*K. MS/MS data analysis revealed that serine 136 was modified (Figure 5). Though acylation of serine hydroxyl groups has been previously reported,32, 39-40 its occurrence is less common and might not be included in the parameters for the standard MS/MS search,


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thereby highlighting the limitation of the current standard method that requires pre-defined chemistry of modifications.

Figure 4. LC/Fluorescence/MS analysis of tryptic peptides of a mAb triazole product. A): Fluorescence chromatogram and reporter ion pair leading to tagged peptides in MS; B): MS TIC containing numerous peaks; C): MS1 spectra at one scan containing multiple m/z; D): Unique isotopic pattern identifying the tagged peptide.



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Figure 5. CID MS/MS of HC (126-137) GPSVFPLAPSS*K Showing the Modification of Serine 136. Additionally, utilizing the novel detection characteristics of this probe, four fluorescent peaks were elucidated that did not exhibit a traditional tryptic cleavage pattern.

For example, a

fluorescent peak at 91.06 minutes corresponded to a peak in the MS spectra with the bromine isotopic pattern and a doubly-charged precursor ion matching the m/z of DNAK*NSLYLQMN (Figure S20). This peptide resulted from a cleavage between asparagine and the following serine in the heavy chain spanning the residues 73-87 DNAK*NSLYLQMN\SLR. MS/MS analysis confirmed that lysine 76 was conjugated (Figure S21). Non-specific cleavage by trypsin can occur and has been previously reported;41-42 however, including non-specific cleavage as a search parameter requires more computational power and therefore may not be included in the search, thus leading to false negatives. Comparison of Workflows. We compared our approach with other analytical methods from the aspects of discovery and elucidation, and the results are illustrated in Figure 6. A standard automated MS/MS search using Byonic from ProteinMetrics43 discovered and elucidated only 33 species because good MS/MS is required for detection. A Matlab script used to search for the isotopic reporter ion pair discovered 36 species.

Alternatively, 93 labeled species were 9

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discovered using a combination of IsoStamp and a manual fluorescence-directed search. This approach combining the fluorescence and bromine isotopic signatures allowed for the discovery of 112 conjugation sites including 19 fluorescent species that had no MS signature [Figure S22, Table S6]. The 19 fluorescent species found by this method would not have been discovered using the isotopic signature only, highlighting the high sensitivity of fluorescence and the power of the combined approach. A control experiment was performed to analyze the product of the click-chemistry reaction between the azide tag and the antibody without alkyne conjugation to evaluate whether fluorescent peaks appeared without a true click-chemistry reaction.

This

product showed no fluorescent peaks, indicating that there are no false positives using fluorescence, and follow-up experiments using more sensitive MS techniques or enrichment can be performed to elucidate those 19 peaks.

Figure 6. Illustration comparing detection of labeled species by this workflow and other analytical workflows. Quantification of the Level of Protein Modification. Fluorescence detection was used for relative quantification of the level of conjugated sites. The most abundant modification LC (184-190) with lysine 188 modified was used as the normalization factor to report percent modified relative to that residue (Table S7). The modification level at a particular residue could be due to a combination of various factors, such as solvent accessibility and local interactions. Lysine 149 displayed a relatively low level of modification. As shown in the molecular model in Figure 7, lysine 149 (panel A) is solvent accessible but likely forms an electrostatic interaction with glutamate 195 due to its close proximity to the epsilon amine (2.8 Å), resulting in only 5% conjugation at that site. Conversely, lysine 188 (panel B) is solvent exposed but is nearly 6.0 Å 10 ACS Paragon Plus Environment


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away from aspartate 185. This distance makes an interaction unlikely and allowed a high level of conjugation, demonstrating the critical role the local environment plays. Overall, the levels of modification varied significantly, indicating the importance of quantifying levels of conjugation and highlighting the utility of the fluorescence moiety in the brominated coumarin azide tag for quantification.

Figure 7. Expanded view of molecular structure of antibody. A): Lysine 149 with 5% relative modification is solvent exposed, but likely to form a salt-bridge with nearby residue glutamate 195. B): Lysine188 with high degree of modification is solvent exposed but less likely to directly interact with aspartate185. CONCLUSIONS All together, we have demonstrated the capability of a brominated coumarin azide tag for its facile detection and quantification of protein conjugation/modifications. To the best of our knowledge, this is the first report of combining sensitive fluorescence and a unique bromine isotopic signature to detect alkynes. This workflow provided great benefits for detection of species with known chemistry and sites, and also significantly improved detection of undefined and low-abundance species that did not ionize or fragment well. In samples with few labeled species, the fluorescence immediately identifies which peak in the TIC the analyst should focus on. In the other extreme with many labeled species requiring an automated search, utilizing fluorescent peak retention time information allows for a quick way of removing false positives 11 ACS Paragon Plus Environment

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and identifying false negatives. Once the modification was detected, relative quantification was achieved using the fluorescent signal. This could be particularly useful in the biotechnology industry, where the drug to antibody ratio in antibody-drug conjugates is usually one of the critical quality attributes. We foresee that the brominated coumarin azide tag will have benefits in more challenging and impactful areas due to its inherent properties (fluorescence, isotopic signature) and elucidation options (MS/MS, reporter ions, sequence-based search) that maximize detection and quantification. We foresee that this tag and workflow will be particularly powerful for more complex systems including detecting protein modifications and drug targets in the cellular milieu44-45 and systems with undefined chemistry and/or sites for bioconjugation or degradation, and moreover, novel chemistry46 and biology. EXPERIMENTAL SECTION Reagents. Reagents of ACS grade or better were obtained from commercial sources without further purification. The recombinant monoclonal antibody (MW=148 kDa) was produced by stably-transfected Chinese hamster ovary (CHO) cells cultured in a bioreactor and purified at AbbVie Bioresearch Center (Worcester, MA). Peptide alkynes were custom synthesized by AnaSpec Corporate (Fremont, CA). Pentynoic acid, 2,5-dioxo-1-pyrrolidinyl ester (NHS-ester alkyne), was purchased from, Annova Chem Inc. (San Diego, CA). 3-azido-6-bromo-7-hydroxychromen-2-one (brominated coumrain azide) was purchased from Princeton BioMolecular Research, Inc. (Princeton, NJ). UPLC/Fluorescence/MS Analysis. Analysis of the tryptic peptides was performed on a Waters UPLC system using a reverse-phase column (Waters, Peptide BEH C18, 1x150 mm) coupled to an Acquity fluorescence detector (Waters, Milford, MA) and an electrospray source of a LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Waltham, MA) in tandem. The UPLC gradient was a linear gradient containing 98% of 0.08% formic acid plus 0.02% trifluoroacetic acid in water and shifting from 2 to 65% of 0.08% formic acid plus 0.02% trifluoroacetic acid in acetonitrile over 135 minutes. The fluorescence detector was operated at an excitation wavelength of 370 nm and emission wavelength of 470 nm.

The mass

spectrometer was operated in positive-ion mode with a scan range from m/z 200 to 2000 with 12 ACS Paragon Plus Environment


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alternating CID and HCD of the three most intense parent masses. Data was processed using the Xcalibur software system 2.6 (Thermo Fisher Scientific, Waltham, MA). NMR analysis. NMR spectra of the brominated coumarin azide were acquired on an Agilent 400-MR DD2 with ONE_NMR probe actively shielded 400 MHz at 30 °C. The software VNMRJ 4.0 was used to process the data.

Proton NMR (399.929 MHz, DMSO-d6) was

acquired with carbon decoupling with 64 scans, an acquisition time of 3.0 s and relaxation delay of 1 s. The 90º pulse width was 11.50 µs but the pulse was set to 5.75 µs. Line broadening of 0.2 Hz was added for smoothing purposes. Modification of mAb with Alkyne.47 The mAb was prepared at 15 mg/mL (100 µM) in PBS buffer (100 mM sodium chloride and 25 mM sodium phosphate, pH 7.4). The NHS-ester alkyne was dissolved in DMSO to produce a stock solution of 50 mM. The reaction was performed by mixing 120 µL of the NHS ester alkyne stock with 5.0 mL of the mAb solution, a mAb to alkyne molar ratio of 1:12. The reaction proceeded at ambient temperature for 2 hours. The modified mAb (referred as mAb-alkyne) was purified by a NAP-10 column (GE Healthcare, Marlborough, MA) into 20 mM Hepes pH 7.8. The concentration of the mAb-alkyne was measured to be 11.6 mg/mL using UV absorbance at 280 nm with an extinction coefficient of 205,720 M-1 cm-1. Formation of Brominated Coumarin Triazole Click Product.48 Alkynes (mAb-alkyne and five other alkynes) were reacted with brominated coumarin azide through a copper-catalyzed azide-alkyne click reaction.

The reaction conditions are described using a mAb-alkyne

generated in the previous step as an example. The mAb-alkyne was diluted to 2.5 mg/mL (16.67 µM). Brominated coumarin azide was dissolved in DMSO to produce an 8.0 mM stock solution. Fifty microliters of 40 mM CuSO4 with 100 µL of 100 mM ligand THPTA (tris(3ydroxypropyltriazolylmethyl)amine) was mixed to generate a Cu/THPTA solution. To 100 µL of 2.5 mg/mL mAb-alkyne (final 12.93 µM), 5 µL of brominated coumarin azide stock was added and mixed (final 310 µM) , followed by addition of 4 µL of Cu/THPTA solution (final 0.4 mM/1.0 mM) and 5 µL of 100 mM sodium ascorbate (final 4 mM). Finally, 15 µL of DMSO was added to prevent precipitation (final 12%). The reaction proceeded at ambient temperature for 1 hour. The brominated coumarin triazole click product from the mAb-alkyne (referred to as mAb-triazole) was purified using a Zebra desalting column (ThermoFisher Scientific, Waltham


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MA, Catalog number 89882) into 20 mM hepes, pH 7.8 buffer. The strained alkyne did not require the addition of CuSO4/sodium ascorbate to catalyze the reaction. MS/MS Search Using Byonic. Byonic (ProteinMetrics v 2.9.30) was used to search the data for modifications. An error of 20 ppm was allowed for both the precursor ion and both HCD and CID fragment ions. A maximum of two missed cleavages by trypsin were allowed, and enzyme specificity was set for cleavage C-terminal to both lysine and arginine residues but allowing nonspecific ("ragged") cleavage from both termini.

Cysteine modifications were fixed for

carboxymethylation, and custom modifications for the tag of +360.97 and +362.97 were allowed at residues K. False postive results were manually filtered out after the search completed. Isotopic Precursor Ion Search Using IsoStamp. IsoStamp (Isotopic Signature Transfer and Mass Pattern Prediction) software developed by the Bertozzi group was used to search for brominated precusor ions in mass spectrometry (MS). http://mass-spec-169.herokuapp.com.

The software can be accessed via

The mzXML-converted raw Xcaliber MS file was

processed by comparing grouped spectra to the predicted pattern as a function of mass and charge, then scored on the difference between the predicted and observed peak distribution for tagged vs. untagged species. The signal/noise ratio was set at equal to or greater than 3. Matches with an assigned score below 20 and with linear error greater than 0.80 were discarded. False positives were manually filtered and false negatives were retrieved by the fluorescencedirected approach after the search was completed. Reporter Ion Search. Matlab v 2015b (Mathworks) supplemented with the Bioinformatics toolbox was used to search the mzXML-converted raw mass spectrometry file. All of the MS/MS scans were searched for the presence of the reporter ions 333.97 and 335.97. If the ratio of the intensity of each reporter was less than 4, the scan number and retention time were recorded for the analyst for further analysis.

ASSOCIATE CONTENTS. Supporting Information Supporting Information is available free of charge on the ACS Publications website



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AUTHOR INFORMATION Corresponding Author *

Email: [email protected]

*

Email: [email protected]

Present Addresses ∥BioAnalytix,

790 Memorial Drive, Cambridge, MA 02139, United States

§

Mersana Therapeutics, Department of Analytical Chemistry, 840 Memorial Drive, Cambridge, MA 02139, United States Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

The authors thank Shanshan Liu for technical discussion on calculation of UV extinction coefficient; Kimberly Yach for characterizing the brominated coumarine azide. This activity is partially supported by a grant from NIH NIGMS (Grant 1R01GM101396 to Z.S.Z.). ABBREVIATIONS TIC, total ion chromatogram; NHS, N-HydroxySuccinimide; RT, Retention time.


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