Leveraging Gas-Phase Fragmentation Pathways for Improved

Nov 30, 2016 - The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As inter...
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Leveraging Gas-Phase Fragmentation Pathways for Improved Identification and Selective Detection of Targets Modified by Covalent Probes Scott B. Ficarro,†,‡,§ Christopher M. Browne,†,§ Joseph D. Card,†,‡ William M. Alexander,†,‡,§ Tinghu Zhang,†,§ Eunyoung Park,†,§ Randall McNally,†,§ Sirano Dhe-Paganon,†,§ Hyuk-Soo Seo,†,§ Ilaria Lamberto,†,§ Michael J. Eck,†,§ Sara J. Buhrlage,†,§ Nathanael S. Gray,†,§ and Jarrod A. Marto*,†,‡,∥ †

Department of Cancer Biology and ‡Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts, 02115, United States § Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, United States ∥ Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115, United States S Supporting Information *

ABSTRACT: The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays.

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tandem mass spectrometry (LC−MS/MS) whereby lowexpression targets or those labeled at low-stoichiometry are not reproducibly detected or quantified. In previous work, we utilized in vitro labeling assays followed by nanoflow LC−MS/MS22 to characterize the binding of acrylamide-based kinase inhibitors to their target proteins.23−28 A detailed and comprehensive reanalysis of these data suggested that cysteine side chains covalently modified via a thioether linkage exhibited common gas-phase fragmentation pathways. Here, we leverage these predictable, inhibitor-specific fragment ions to (i) significantly improve identification of modified peptides via commercial search algorithms and (ii) explore the potential for use of targeted mass spectrometry assays as a foundation for rapid, selective detection of inhibitormodified peptides in complex mixtures.

he past decade has witnessed a resurgence of interest in the pursuit of drugs which exert therapeutic effect through covalent modification of cellular targets.1−3 Selective covalent inhibitors which utilize diverse reactive “warheads” have been developed for numerous enzyme families.4−9 A subset of these has been successfully developed to yield clinical-grade probes targeting kinases,10,11 deubiquitinating enzymes,12 and other catalytically active proteins.1 Examples include Neratinib (Her2/EGFR, breast cancer),13 Afatinib (EGFR, non-small cell lung cancer),14 Ibrutinib (BTK/ITK, chronic lymphocytic leukemia),15 and VLX1570 (USP14, multiple myeloma).12 Despite these promising results the characterization of on-/offtarget molecules for lead compounds, in addition to subsequent medicinal chemistry optimization, remains a significant challenge.16 Mass spectrometry is an integral component of analytical platforms used to characterize covalent probes. Several approaches have been developed that rely on direct detection of targets based on affinity-tagged probes or the use of broad-reactivity reagents in a competition format with native inhibitors to provide an indirect readout of targets.17−21 Although informative, these approaches may be limited by (i) the use of tagged analogues which may not faithfully reproduce the physicochemical properties of the native probe or (ii) stochastic properties of shotgun liquid chromatography− © XXXX American Chemical Society

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MATERIALS AND METHODS Materials. Synthetic peptides were produced by use of Fmoc chemistry and purified by reversed phase HPLC.

Received: August 29, 2016 Accepted: November 7, 2016

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DOI: 10.1021/acs.analchem.6b03394 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry Inhibitors were synthesized as described23,25,27,29,30 or obtained from commercial sources. All other compounds, unless noted otherwise, were obtained from Sigma-Aldrich. Preparation of Cellular-Derived Tryptic Peptides and Covalently Modified Synthetic Peptide Standards. K562 tryptic peptide aliquots were prepared as described.31 Peptides modified with inhibitors were produced by incubating a 10-fold molar excess of inhibitor with synthetic peptides (FGLCSGPADTGR or YMANGCLsLNYLR, sL = 15N-1, 13 C-6 leucine) or reduced, desalted tryptic bovine serum albumin (BSA) peptides in 1:1 DMSO/100 mM triethylammonium bicarbonate, pH 8.5 at 37 °C overnight. Carbamidomethylated BSA peptides were prepared by incubation of desalted, reduced BSA with 20 mM iodoacetamide for 30 min in the dark at room temperature. Collision Energy Profiling. Covalently modified synthetic peptides were diluted 1:200 with 50% acetonitrile/water with 1% acetic acid and directly infused into a QExactive HF mass spectrometer at a flow rate of 3 μL/min using the Ion Max source with HESI-II probe (spray voltage = 4 kV, sheath gas = 1). Spectra at a range of collision energies (typically 10−100 eV) were acquired in tune mode from m/z 100−1500 at a resolution of 15 000. Intensities of ions were extracted and exported to.csv using a multiplierz script32 and plotted using R (version 3.0.2). Nanoflow LC−MS/MS Analysis of Inhibitor Conjugated BSA Peptides. BSA peptides conjugated to THZ1 or THZ531 (5 pmol) were loaded onto a self-packed precolumn (4 cm POROS 10R2 packed into 100 μm fused silica) using a NanoAcquity UPLC system. Peptides were resolved on an analytical column (12 cm 5 μm Monitor C18, Orochem, Ontario, CA, packed into 30 μm fused silica; emitter tip was ∼1 μm in diameter22) by gradient elution (5−60% B in 40 min, A = 0.2 M acetic acid in water, B = 0.2 M acetic acid in acetonitrile, flow rate ∼30 nL/min) and introduced to an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific, San Jose, CA) by electrospray ionization (spray voltage = 2.2 kV). The instrument was operated in data dependent mode and performed MS/MS (HCD, 30 and 40% CE, quadrupole isolation, 2 Da isolation width, image current detection with 15K resolution, target = 5 × 104, max fill time = 100 ms) on the 10 most abundant ions in each MS scan (image current detection with 120 K resolution, target = 5 × 105, max fill time = 500 ms). We used multiplierz scripts32 to deisotope fragment ions as well as normalize multiply charged species to z = 1 if one or more 13C isotopes were present. When applicable, .mgf files were filtered to remove inhibitor related ions. Processed peak lists were searched using MASCOT version 2.2.1 against a forward reverse human NCBI refseq database appended with the sequence of bovine serum albumin. Search parameters specified variable inhibitor modification (with or without a scored neutral loss corresponding to retro-Michael addition) and variable oxidation of methionine, a precursor tolerance of 10 ppm, and a product ion tolerance of 25 mmu. Search results were converted to .xls and filtered to 1% FDR using multiplierz scripts.32 To assess the impact of inhibitor modification on peptide charge state, a separate LC−MS/MS analysis of a mixture of THZ1, THZ531, and carbamidomethylated BSA peptides was performed. The net increase in charge state was determined by evaluating the most intense charge state for inhibitor modified vs carbamidomethylated peptides using multiplierz scripts.32

Precursor Ion Scanning. Synthetic peptide FGLCSGPADTGR (∼30 fmol) conjugated to Ibrutinib was spiked into 1 μg of K562 tryptic peptides and analyzed by nanoflow LC−MS/MS as described above. Peptides were introduced to a QTRAP 5500 mass spectrometer (ABSciex, Framingham, MA) by electrospray ionization (spray voltage = 2.2 kV). The mass spectrometer conducted cycles of Q3MS followed by precursors of 475.19 (CE = 35) or 304.12 (CE = 55). Precursor scans were acquired over a mass range of 400− 900 at 1000 amu/s, summing 2 scans with a threshold of 2 cps and a step size of 1 Da. Q1 and Q3 were operated at unit and high resolution, respectively. Precursor triggered MS/MS data were acquired using the same mixture described above. The mass spectrometer was programmed to perform precursor scans for 475.19 at a collision energy of 35 eV using above parameters. ER (enhanced resolution) scans were acquired for the five most abundant precursor peaks; peptides charge 2+ to 5+ were subjected to MS/MS using a rolling collision energy (Q1 resolution = low). Data files were converted to .mgf with the ABSciexMSDataConverter version 1.3 beta and Ibrutinib related ions removed using a multiplierz script.32 The preprocessed .mgf was searched using MASCOT version 2.2.1 against a forward reverse human NCBI refseq database appended with the sequence of the synthetic peptide. Search parameters specified variable Ibrutinib modification (with a scored neutral loss corresponding to retro-Michael addition) and variable oxidation of methionine, a precursor tolerance of 0.5 Da, and a product ion tolerance of 0.5 Da. Search results were converted to .xls and filtered to 1% FDR using multiplierz scripts.32 Additional experiments aimed at detecting a QL47 conjugated peptide (50 fmol FGLCSGPADTGR) in 1 μg of K562 tryptic digest were performed in a similar fashion, scanning for precursors of 482.16 (CE = 35) or 394.17 (CE = 55) after obtaining a full-scan mass spectrum (Q3MS).

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RESULTS AND DISCUSSION We chose five acrylamide-warhead kinase probes which we had previously characterized along with two approved covalent kinase inhibitor drugs (Table 1). Each probe was incubated Table 1. List of Inhibitors Used in This Study and Their Protein Targetsa

a

probe

primary target(s)

reactive group

ref

JNK-IN-2 THZ1 THZ531 TL10−201 (T50) Ibrutinib Neratinib QL47 HBX-19818 MI-2

JNK1 CDK7 CDK12/13 JAK3 BTK/ITK HER2/EGFR BTK USP7 MALT-1

acrylamide* acrylamide* acrylamide* acrylamide acrylamide acrylamide* acrylamide C−Cl chloroacetamide

27 23 29 25 15 13 30 35 36

* denotes a dimethylamino group installed near the warhead.

separately with its recombinantly produced target, after which we verified covalent modification of each intact protein by mass spectrometry as previously described23,25,27,29 (data not shown). To determine the site of labeling, digested proteins were desalted and analyzed by nanoflow LC−MS/MS. Figure 1A shows a JNK1-derived peptide (LMDANLCQVIQME) containing Cys116 modified by JNK-IN-2. We noted that several ions detected in the MS/MS spectrum could not be B

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THZ1, Neratinib, Table S1) These data suggest the presence of inhibitor class-specific dissociation pathways. To explore these fragmentation pathways in the context of alternative warheads and target families, we interrogated a deubiquitinase (DUB) inhibitor, HBX-1981835 as well as the paracaspase inhibitor MI-236 (Table 1 and Table S1), both of which modify cysteine residues through different electrophilic groups. We observed thiolated ions in the MS/MS spectra of peptides labeled with each probe, confirming this as a common dissociation pathway shared across covalent inhibitors which modify their targets through a thioether bond (Figure S1D,F,H). Consistent with the data above, we observed amide bond cleavage within each inhibitor (Figure S1G “1” and H “1”). Interestingly peptides covalently modified with HBX-19818 or MI-2 did not undergo retro-Michael addition but rather exhibited a low-yield elimination reaction to produce a series of dehydroalanine-containing b- and y-type fragment ions (Figure S1F). Importantly, the dissociation pathways described above are agnostic with respect to peptide sequence, charge state, and proteolytic enzyme. Collectively these results suggest that probes which form covalent adducts through a thioether linkage dissociate under MS/MS conditions to yield predictable, structurally specific fragment ions. Despite our ability to identify covalently modified peptides, we noticed that in most cases the associated fragment ions provided relatively low spectral match scores when using the commercial MASCOT algorithm37 for database search and sequence assignment. As one example, the high-resolution MS/ MS spectrum in Figure 2A (corresponding to THZ531 modified CDK12 target peptide) yielded a low-confidence peptide score of only 15.7. A majority of kinase inhibitors contain one or more heterocyclic rings that impart significant gas-phase basicity, leading to increased peptide charge state (typically ≥3+, Figure S2). Dissociation of higher charge state peptides can yield complicated product ion spectra containing multiply charged fragment ions which can diminish the performance of search algorithms. To account for these effects we modified our spectral processing scripts to normalize all fragment ions to the 1+ charge state. This step alone increased the MASCOT score for the MS/MS spectrum to 40. In addition, we surmised that the myriad of MS/MS ions derived from fragmentation of the inhibitor (Figure 2B peaks labeled 1−9; see Table S1, “CDK12” for proposed structures) further diminished the quality of spectral matches. To test this hypothesis we preprocessed MS/MS spectra (e.g., prior to submission for MASCOT database search) based on the fragmentation pathways described above. First, we defined neutral loss of the inhibitor from the peptide backbone (retroMichael addition) as part of the variable modification in MASCOT; this step increased the peptide score to 50. Second, we removed the predictable inhibitor-related ions described in Table S1, “CDK12” (see also Figure 2B), which further improved the MASCOT score to 66 (Figure 2C). To assess these improvements across a larger and more diverse population of modified cysteine-containing peptides, we incubated THZ531 and THZ1, which target CDK12 and CDK7, respectively, with reduced bovine serum albumin (BSA) and compared MASCOT peptide scores for spectra which were subject to minimal (deisotope only) or extensive preprocessing. We observed that use of the full preprocessing scheme as described above led to consistent and significant improvements in MASCOT scores for >90% of MS/MS spectra (Figure 2D). These results demonstrate that the predictable, inhibitor-

Figure 1. Acrylamide kinase inhibitor/target conjugates generate predictable thiolated ions. MS/MS spectra for (A) JNK-IN-2 labeled JNK1 peptide; (B) Ibrutinib labeled ITK peptide; (C) Neratinib labeled EGFR peptide. Fragment ions containing the peptide N- (btype) or C- (y-type) termini are indicated with blue and red glyphs above and below the peptide sequence. Modified cysteine residues are shown in bold, italic font. Thiolated ions are highlighted in purple and denoted with “*”. Structures for thiolated ions are shown adjacent to each mass spectrum.

assigned to canonical b- or y-type fragments which result from gas-phase cleavage of peptide amide bonds.33 Further investigation revealed that these and other fragments were derived from the inhibitor (Table S1, JNK). In particular, one fragment (Figure 1A, “∗” and Table S1, JNK, “thiolated ion”) corresponded to cleavage of the peptide−probe adduct, yielding an ion containing the intact inhibitor in addition to the target cysteine thiol. These data further confirm Cys116 on JNK1 as the site of covalent modification. Other fragments resulted from various elimination reactions, for example, loss of the dimethylamino group or the inhibitor itself (retro-Michael addition) or cleavage of amide bonds within the inhibitor. To more easily reference the latter fragments, we propose a nomenclature whereby each amide linkage in an inhibitor is numbered based on proximity to the cysteine thiol (iy1 and ib1, iy2 and ib2, etc.). In addition, we observed neutral loss of the inhibitor from canonical b- and y-type ions via retro-Michael addition (Table S1). Intrigued by these results, we went on to validate the same fragmentation pathways in several wellcharacterized acrylamide probes, including the covalent drugs Ibrutinib and Neratinib, which target BTK and HER2/EGFR, respectively (Figure 1B,C, Figure S1A−C,E, and Table S1).10,23−27,29,30,34 In addition we observed intramolecular dissociation at specific linkages within the probes, including fragmentation adjacent to alkylated amines (relevant to THZ531, Ibrutinib, and TL10-201, see Table S1) as well as cleavage at dimethylamino moieties (JNK-IN-2, THZ531, C

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Figure 3. Normalized fragment ion intensity vs collision energy (CE) for inhibitor specific (thiolated and iy1) ions produced by MS/MS of a triply charged synthetic cysteine-containing peptide (FGLCSGPADTGR) labeled with (A) Ibrutinib, (B) Neratinib, and (C) QL47. For comparison, each plot includes profiles for b-/y-type ions produced by MS/MS of the unlabeled triply charged peptide.

Figure 2. Preprocessing peak lists from fragment ion spectra to account for inhibitor-related dissocation pathways significantly increases MASCOT peptide scores. (A) The deisotoped MS/MS spectrum for a THZ531 labeled CDK12 peptide yields a relatively lowconfidence MASCOT score, largely due to an abundance of inhibitor related fragment ions (B, no. 1−9; see Table S1 for proposed structures). Additional steps, comprising normalization of all fragment ions to z = 1, including neutral loss of inhibitor as part of the MASCOT variable-mod definition and subtraction of peaks corresponding to internal fragmentation of the inhibitor yields a high-confidence MASCOT peptide score (C). Ions of type b and y are shown in blue and red. Neutral loss ions are shown in green. ++ indicates a doubly charged ion. (D) Scatter plots of MASCOT scores for BSA peptides modified by (left) THZ531 or (right) THZ1 for MS/MS data subject to deisotoping (y-axis) or full spectral preprocessing (x-axis). Dotted green lines represent MASCOT score cutoffs for a 1% FDR.

and Figure S3A−D). In addition, for specific ion types (e.g., JNK thiolated ion) and charge states there exists sufficient overlap in CE profiles such that a single collision energy generated significant signal intensity (>50% max) for peptides of different sequence (Figure S3E,F). Finally, inhibitors incorporating a dimethylamino group produced a characteristic fragment at m/z = 112.07 which was observed across a broad range of collision energies (Figure S3D, red) suggesting its potential use as a class-specific diagnostic ion. We next wanted to leverage our understanding of the energetics associated with inhibitor-specific ions to explore the potential application of precursor ion scanning mass spectrometry to detect these fragments. As a proof of principle, we labeled a synthetic peptide with Ibrutinib and spiked it into a complex mixture of tryptic peptides derived from human myeloid K562 cells. We then acquired precursor ion scan data on a triple quadrupole mass spectrometer using optimized collision energies as described above. Figure 4 and Figure S4 show that selective detection of the thiolated (Figure 4F) or alkylated amine cleavage product (m/z 304.12) (Figure S4F) ions provide more than an order of magnitude improvement in selectivity as compared to standard full mass range data acquisition (Figure 4C, Figure S4C). As a further test of selectivity we next triggered full-scan MS/MS acquisition based on precursor ion signals corresponding to Ibrutinib thiolated ions (m/z 475.19; Figure 4D). After preprocessing of the resulting peak lists, a MASCOT search yielded 22 peptidespectral matches (PSMs), corresponding to 16 unique peptide sequences. Notably, the spiked-in synthetic was the only Ibrutinib-modified peptide detected (Figure 4G, MASCOT score ∼49), even though it was among the lowest intensity precursor ions in the base peak chromatogram (BPC). We observed similar gas phase enrichment for precursor ion data

associated fragmentation pathways can be leveraged to improve our ability to identify covalently modified peptides. On the basis of the results above, we speculated that inhibitor-associated fragmentation pathways may be used as the basis for highly selective precursor scanning or other targeted mass spectrometry assays.38 As a prerequisite to these experiments, we sought to understand the yield of inhibitorspecific fragment ions as a function of kinetic energy during MS/MS. Toward this end, we conjugated synthetic cysteinecontaining peptides with seven different acrylamide inhibitors. Next we used direct infusion of the mixture to acquire MS/MS spectra for each modified peptide across a wide range of collision energies (CE). Plots of ion yield as a function of CE revealed several interesting trends. For example, we observed that use of higher CE resulted in gas phase enrichment of structure-specific fragment ions from each inhibitor relative to canonical peptide b- and y-type ions. In fact, the iyn ions typically reached a maximum yield at ≥40 eV, a CE where peptide fragment ion intensities were greatly reduced (Figure 3 D

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down approaches to investigate antibody−drug conjugates,43,44 metabolite-modified proteins,45,46 and drug binding to serum albumin.47,48 However, the potential for idiosyncratic toxicity resulting from drugs with a covalent mechanism-of-action continues to motivate the development of analytical platforms for rapid characterization of on- and off-target proteins. These assays would be implemented early in the drug discovery pipeline to assist in prioritization and triage of lead compounds. Pursuant to this goal, we systematically interrogated gas phase fragmentation behavior for a small cadre of cysteine-directed covalent probes. We identified several novel dissociation pathways that yield fragment ions which can be predicted based on probe structure and reactive warhead. Our results provide evidence for several informative trends: (i) Probes covalently bound by a thioether linkage produce a characteristic thiolated ion independent of peptide sequence and charge state; (ii) certain dissociation pathways appear to be classspecific, for example, acrylamide warheads undergo a retro Michael addition to regenerate the intact probe, while inhibitors with dimethylamino groups yield a low-mass fragment at m/z = 112.07; (iii) information for these novel fragmentation pathways can be used to markedly improve peptide sequence identification scores for shotgun proteomic methods; (iv) use of higher CE provides gas phase enrichment of predictable, inhibitor-specific fragments relative to canonical peptide b- and y-type ions. Our data suggest that inhibitor-specific dissociation pathways may be leveraged to develop selective mass spectrometry assays to characterize covalently modified targets (Figure 4). In this initial report we focused on three predictable fragment ions (thiolated, iy1, and alkylated amine cleavage), but others may provide higher selectively, either individually or in combination (Figure S3D). While our data suggest that a single collision energy can generate sufficient yield for multiple inhibitorspecific fragments and different target peptide sequences, it would be interesting to explore whether the use of ramped CE would improve signal-to-noise for simultaneous detection of multiple diagnostic ions. Similarly, it may be possible to use customized acquisition APIs49−51 to build scan functions which trigger MS/MS based on simultaneous detection of multiple inhibitor-specific fragments. Finally, it will be interesting to interrogate a wider range of warheads5 to build a more complete library of inhibitor-/class-specific dissociation pathways. Overall these methods will comprise a valuable addition to the proteomic toolbox for characterization of covalent inhibitor binding sites in mixtures ranging from simple in vitro reactions to complex cellular lysates.

Figure 4. Novel dissociation pathways associated with thioether linked covalent probes provide for significant gas-phase enrichment during precursor ion scanning mass spectrometry. (A) Base-peak chromatogram (BPC) and (B) extracted ion chromatogram (XIC; precursor ±0.5 Da) from Q3MS scans and (C) individual Q3 full-scan mass spectrum recorded during analysis of a synthetic cysteine-containing peptide (FGLCSGPADTGR; indicated by “CYS”) labeled with Ibrutinib and spiked into a mix of tryptic peptides derived from human myeloid K562 cells. Red arrow indicates the elution time (A and B) or m/z (C) for the labeled peptide. (D) Base-peak chromatogram, and (E) XIC (precursor ±0.5 Da) from precursor ion spectra (precursors of 475.19, corresponding to the thiolated ion of Ibrutinib; abbreviated as “Prec”), and (F) individual precursor ion mass spectrum recorded during the same LC−MS/MS analysis. Red arrow indicates the elution time (D and E) or precursor ion signal (F) for the Ibrutinib labeled peptide. (C, F) The % values in each panel represent the gas phase enrichment, calculated as the relative contribution of each ion (red arrow) as compared to the total ion current in that spectrum. (G) MS/MS spectrum of Ibrutinib labeled peptide triggered by precursor scans for m/z = 475.19, corresponding to the thiolated ion of Ibrutinib. Fragment ions containing the peptide N- (b-type) or C- (y-type) termini are indicated with blue and red glyphs above and below the peptide sequence. Inhibitor-specific ions are labeled with green glyphs (see Table S1 for proposed structures of ions labeled nos. 1−5).

obtained for QL47 (targeting thiolated ion and iy1; Figure S5). These data suggest that predictable inhibitor-associated fragmentation may be used as a basis to develop selective mass spectrometry assays for detection of proteins targeted by cysteine-directed covalent probes.

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ASSOCIATED CONTENT

S Supporting Information *

CONCLUDING REMARKS Recent clinical progress demonstrated by various inhibitors including Neratinib,13 Afatinib,14 Ibrutinib,15 and VLX157012 has rekindled enthusiasm for covalent drugs. Similarly, data from the human genome project along with large-scale functional genomic studies have highlighted the need for selective probes which will enable biochemical analysis of enzyme function.39,40 Mass spectrometry is well-suited to interrogate covalent protein modifications; in fact, several studies demonstrated the utility of mass spectrometry approaches to characterize small molecule−protein adducts, these include profiling of intact proteins41,42 as well as top-

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03394. Common fragmentation pathways are observed in MS/ MS spectra of inhibitor conjugated peptides; bar graph illustrating shift in charge state distribution observed after conjugation of reduced BSA peptides; normalized fragment ion intensity vs collision energy plots; selective detection of an Ibrutinib labeled peptide by precursor ion mass spectrometry; selective detection of a QL47modified peptide using precursor ion scanning mass E

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spectrometry; and inhibitor related ions produced by MS/MS of modified peptides (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: (617) 632-3150 (office). Fax: (617) 582-4471. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Strategic Research Initiative at the Dana-Farber Cancer Institute and the National Cancer Institute (Grant CA188881, to J.A.M) in addition to the National Institutes of Health Grants CA182736-03 and CA179483-03 (to N.S.G.) and Grants GM110352 and CA201049 (to M.J.E.).

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