Chromogenic Chemical Probe for Protein Structural Characterization

Jul 15, 2013 - Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry. John P. O'Brien...
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Chromogenic Chemical Probe for Protein Structural Characterization via Ultraviolet Photodissociation Mass Spectrometry John P. O’Brien, Jeff M. Pruet, and Jennifer S. Brodbelt* Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station A5300 Austin, TX 78712, United States S Supporting Information *

ABSTRACT: A chemical probe/ultraviolet photodissociation (UVPD) mass spectrometry strategy for evaluating structures of proteins and protein complexes is reported, as demonstrated for lysozyme and beta-lactoglobulin with and without bound ligands. The chemical probe, NN, incorporates a UV chromophore that endows peptides with high cross sections at 351 nm, a wavelength not absorbed by unmodified peptides. Thus, NN-modified peptides can readily be differentiated from nonmodified peptides in complex tryptic digests created upon proteolysis of proteins after their exposure to the NN chemical probe. The NN chemical probe also affords two diagnostic reporter ions detected upon UVPD of the NN-modified peptide that provides a facile method for the identification of NN peptides within complex mixtures. Quantitation of the modified and unmodified peptides allows estimation of the surface accessibilities of lysine residues based on their relative reactivities with the NN chemical probe.

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frequencies of modified peptides among a vast pool of more abundant unmodified peptides. The low abundances of modified peptides arise because chemical probe reactions with proteins are typically limited to reduce excessive modification that might lead to denaturation of the proteins and sampling of non-native conformations. This deficiency can be addressed by employing selective enrichment methods or via more selective MS/MS methods to target the modified peptides. Enrichment methods typically require the incorporation of an affinity tag into the chemical probe, thus assuring enhanced extraction of the labeled peptides after digestion of the tagged proteins.38−40 This strategy comes at the expense of requiring synthesis of more elaborate chemical probes for which the affinity tags may add considerable bulk, thus reducing reagent accessibility to the protein. An alternative approach to facilitating the differentiation of probe-modified peptides from unmodified ones relies on selective MS/MS methods. Collision-induced dissociation (CID) is the most widely used MS/MS method in the bottom-up workflow, and thus efforts to increase its selectivity for targeted approaches have focused on incorporation of CIDcleavable bonds to capitalize on the enhancement of specific neutral losses or production of characteristic ions due to cleavage of a labile bond.37−39,41−54 For example, selectively cleavable, labile bonds have been incorporated into a number of

he integration of chemical labeling methods with tandem mass spectrometry (MS/MS) has become an increasingly popular strategy for structural biology studies, providing information about higher-order protein structure and revealing insight into protein−ligand interactions and structure/function relationships.1−7 These labeling methods include photoinduced oxidation,8−16 hydrogen−deuterium exchange (HDX),16−22 noncovalent labeling,23−25 cross-linking, and use of site-specific chemical probes to evaluate the local reactivity of key amino acid residues.26−37 Such methods can be used to identify interacting proteins, to define spatial constraints within proteins, and to reveal the surface accessibilities and reactivities of specific regions or residues of proteins. Two of the major benefits of using probe-based mass spectrometric methods compared to traditional methods like X-ray crystallography and NMR spectroscopy for structural biology studies are the low amount of protein needed and the potential for adaptation to higher throughput workflows. Chemical probe-based methods typically involve incubation of a native protein or protein complex with a reagent whose reactivity reflects the degree of exposure and relative reactivity of specific sites of the protein. The proteins are most often analyzed using a bottom-up LC−MS/MS approach to track the modified sites in the resulting proteolytic peptides. The surface accessibilities/reactivities of various regions of the protein are estimated by quantifying the resulting modified peptides relative to unmodified ones based on the assumption that the more exposed sites will show a higher degree of modification. This strategy is conceptually straightforward, but data analysis is considerably more challenging due to the low abundances and © 2013 American Chemical Society

Received: April 30, 2013 Accepted: July 15, 2013 Published: July 15, 2013 7391

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cross-linking or surface accessibility agents.37,38,44,45,47−49 Alternatively, cross-linking agents that release reporter ions have proven to be a successful approach for identification of cross-linked peptides.50,51 In addition, the identification of cross-linked peptides has been enhanced by inclusion of isotopically labeled probes39,52,53 or by the incorporation of a UV photocleavable moiety that allows release of the crosslinked peptides upon MALDI53 or UV irradiation prior to mass spectrometric analysis.54 We and others have been exploring electron- and photonbased methods to enhance the selectivity of MS/MS schemes.26,55−57 For example, we reported a surface accessibility probe that incorporated a labile N−N bond that preferentially cleaved upon ETD, resulting in a characteristic neutral loss that was used to pinpoint modified peptides.26,55 We have also designed chromogenic cross-linking agents that endowed cross-linked peptides with high photoabsorption cross sections at 10.6 μm or 355 nm.56,57 We exploit the latter concept to create the UV chromogenic probe described in the present report. As illustrated in Figure 1 the amine-reactive chemical probe, termed “NN”, incorporates a hydrazone moiety with high

from the full mass spectrum. UVPD mass spectra were acquired using fifteen 3 mJ pulses. NN and Acetylation Derivatization. The surface accessibility reagent, NN, was synthesized in-house as previously reported.26 Reactions of NN with each protein were undertaken using a variety of NN:protein molar ratios in order to maximize the total number of probed amine sites while avoiding denaturation of the protein. Feedback about the number of reacted amine sites was obtained by examining the ESI mass spectrum of each protein prior to proteolysis and by circular dichroism measurements to assess denaturation. Additional details are provided as Supporting Information. Data Interpretation and Analysis. CID mass spectra were searched using MassMatrix, a free online database search algorithm for peptide MS/MS data (www.massmatrix.net).58−60 The reactivities of each lysine ε-amine and the N-terminus were calculated by dividing the sum of the areas of all peptides containing each modified residue by the sum of the areas of all the peptides (both modified and unmodified) containing the same residue as summarized in eq 1: Residue Percent Reactivity =

∑ Area of all peptides containing modified residue N ∑ Area of all peptides containing residue N × 100

(1)

Predicting Surface Accessibility. The surface accessibilities of lysine and N-termini residues were calculated from the PDB structures using the online software program GETAREA.61 The agreement between NN reactivity results and the calculated solvent accessibilities is not expected to be quantitative, and some of the issues that modulate NN reactivity are described in the Supporting Information. The pKa values of the amine sites of each protein were estimated using pKa prediction online software, PROPKA.62

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RESULTS AND DISCUSSION Properties of the Chromogenic Chemical Probe. An overview of the chemical probe/UVPD strategy is shown in Figure 2. Via the well-known N-hydroxysuccinimide coupling reaction, chemical probe NN was designed to react with primary amines (i.e., epsilon-amine group on the side-chain of lysine and N-terminus) (Figure 1). Lysines occur in relatively high frequency in proteins, and they are often key highly reactive residues found on the more exposed surfaces of proteins, thus making them one of the most popular targets for chemical modification-based structural methods. As exploited in this study, the NN reagent incorporates a UV chromophore that endows the peptides with high cross sections for absorption of 351 nm photons. Only those peptides that contain the NN-tag undergo photodissociation upon exposure to 351 nm irradiation. The NN-modified peptides produce diagnostic b and y ions upon 351 nm UVPD, thus allowing the peptides to be sequenced and readily differentiated from nonmodified peptides (which do not absorb 351 nm photons and thus do not dissociate). Moreover, the NN moiety also produces two characteristic reporter ions of m/z 130 and 224 upon UVPD. Structures for these reporter ions are proposed in Figure 1B, in which the ion of m/z 224 arises from cleavage of the amide bond between NN and the peptide and the ion of m/z 130 is produced upon cleavage of the same amide bond plus the N−N hydrazine bond. These characteristic reporter

Figure 1. Structure of (A) NN and (B) NN reporter ions.

photoabsorptivity at 351 nm, thus allowing facile differentiation of modified and unmodified peptides. The NN-modified peptides give readily interpretable b/y fragmentation patterns and also yield unique reporter ions upon UVPD, thus allowing the peptides to be pinpointed in complex mixtures upon LC− UVPD analysis. The NN chemical probe is characterized and evaluated for the determination of relative reactivities of primary amine sites of two proteins in the present study, including mapping changes in primary amine reactivity induced by ligand binding.

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EXPERIMENTAL SECTION Liquid Chromatography, Mass Spectrometry, and Ultraviolet Photodissociation. All experiments were performed on a Velos Pro Elite mass spectrometer (San Jose, CA) equipped with an UltiMate 3000 RSLC nano liquid chromatography system. The mass spectrometer was modified to allow photodissociation in a manner similar to that described previously.56 A Coherent ExciStar-XS excimer laser (Santa Clara, CA) operated at 351 nm at 500 Hz was used for UV photodissociation. For all LC/UVPD runs, the first event was a full mass scan (m/z range of 350−1800) followed by consecutive UVPD events on the ten most abundant ions 7392

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Figure 2. Overview of the NN chemical probe/351 nm UVPD strategy. (1) A protein is incubated with NN. NN is denoted by a purple ⧫. (2) The protein is enzymatically digested. (3) The mixture of modified and unmodified peptides is analyzed by LC−MS/MS. (4a) NN-modified peptides absorb 351 nm photons, undergo selective fragmentation, and are easily pinpointed by the EIC of the reporter ions. (4b) NN peptides are sequenced using their UVPD diagnostic fragmentation patterns. (5) Unmodified peptides do not absorb 351 nm photons and are sequenced by CID. An apostrophe (’) signifies the neutral loss of water or ammonia from a fragment ion.

ions upon UVPD provide a key means to pinpoint the elution of the low abundance NN-modified peptides amidst a large number of higher abundant unmodified peptides, as illustrated more explicitly later. Evidence for the striking change in UV absorption cross sections upon incorporation of the NN group is reflected in the UV absorbance profiles for glutathione (a model tripeptide) before and after attachment of the NN moiety. (See Figure 1 of the Supporting Information). Unmodified glutathione exhibits no absorbance at 351 nm, but the absorbance of NN-modified glutathione is substantial. Cross sections in the gas phase do not quantitatively parallel cross sections in aqueous solution, but there is often good general agreement although the absorption maxima may shift. Upon UV irradiation in the gas phase, unmodified peptides do not absorb and thus do not dissociate (Figure 3A for tryptic peptide IDALNENKVLVLDTDYK). However NN-modified peptides absorb UV photons and dissociate into diagnostic b and y ions upon 351 nm UVPD, as illustrated for peptide IDALNENKNNVLVLDTDYK in Figure 3B (in which the subscript NN in the peptide sequence is used to indicate the location of the modification at the lysine residue). UVPD can thus be used to readily differentiate the NN-modified peptides from unmodified ones as well as provide extensive sequence coverage of the modified peptides. Probing the NN-Reactivity of Lysozyme. The entire strategy is first demonstrated for lysozyme, a 14.3 kDa protein containing six lysine residues. Lysozyme was modified using a 7:1 NN:protein molar ratio. Figure 2A of the Supporting Information shows the 11+ charge state of lysozyme after reaction with NN, with the most abundant product having a single NN modification and other products having up to a

Figure 3. UVPD-MS spectra of (A) unmodified IDALNENKNNVLVLDTDYK (3+) and (B) NN-modified IDALNENKVLVLDTDYK (3+) beta-lactoglobulin tryptic peptide. An asterisk indicates the precursor ion, and a “Δ” indicates that the fragment ion contains the NN chromophore. Reporter ions of m/z 130 and 224 are indicated.

maximum of four NN modifications. The companion CD spectra of NN-modified lysozyme and unmodified lysozyme are shown in Figure 2B of the Supporting Information, and the lack of change in the CD profiles implies little disruption of the secondary structure of lysozyme after NN-modification (although CD monitors the average global structure of proteins rather than specific local changes). The base peak LC−MS chromatogram for the chymotryptic digest of NN-modified 7393

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accessibility values determined from GETAREA. Variations in the percent reactivity values among the three digests are not unexpected because each protease generates a different set of peptides, some of which are more or less likely to be detected by virtue of their size, number of basic sites, and hydrophobicity, all factors which modulate the elution order, ionizability, and the MS/MS pattern of each peptide. In fact, the overall average sequence coverages obtained for lysozyme based on each digest were 98% for trypsin, 94% for chymotrypsin, and 92% for Glu-C, thus reflecting the variability in the numbers of identified peptides irrespective of the NN reaction. Lys96 exhibits the lowest reactivity among all the primary amine sites, a finding that is consistent with the lowest surface accessibility of this residue. Lys13 likewise showed marginal reactivity, suggesting its low accessibility in lysozyme. Lys1 had the potential to react at its alpha and/or epsilon amine, but only a single addition of the NN probe was observed at this residue. The reactivity of the epsilon amine is presumed to be lower than the reactivity of the α-amine due to difference in the pKa values in solution: α-NH2, ∼9.0, and ε-NH2, 11.25, thus making the alpha amine more likely to be deprotonated and thus more nucleophilic. The reactivities of Lys1, Lys33, Lys97, and Lys116 are the highest, suggesting their greater accessibility and in agreement with the surface accessibilities predicted from the known crystal structure of lysozyme (Figure 4 of the Supporting Information). The NN reactivity values in Table 1 collectively suggest the trend Lys97 ≈ Lys33 ≈ Lys116 ≥ Lys1 > Lys13 > Lys96. Lys13 was expected to have moderate solvent exposure based on the crystal structure, but this was not mirrored in the NN reactivity results. Such discrepancies may arise from a number of factors, some arising from intrinsic differences between crystal structures and protein structures in solution, the impact of other intramolecular interactions that are not well modeled by the surface accessibility calculations of individual reactive groups but may have a large influence on nucleophilicity, and the sheer size and hydrophobicity of the NN chemical probe. Moreover, the pKa values of the primary amines influence the expected distribution of each amine among its free base and protonated forms in solution, thus influencing the nucleophilicity of the reactive amine group. The primary amine side-chain of Lys 96 displays no NN reactivity, and this parallels a previous study that attributed the low reactivity of Lys96 to the formation of a hydrogen bond between the amine and the carbonyl oxygen of His15.64 Lys33 and Lys1 were predicted to have similar accessibilities as Lys13, yet Lys33 and Lys1 exhibit significantly higher reactivities with the NN probe. The lower pKa values of Lys33 and Lys1 (αNH2) compared to Lys13 make them more nucleophilic and thus could contribute to their higher reactivities in solution.

lysozyme (Figure 4A) shows the elution profile of all peptides, both modified and unmodified. It is this indiscriminate

Figure 4. LC−MS results showing (A) base peak chromatogram and (B) extracted ion chromatogram of m/z 130 for the chymotrypsin digest of NN-modified lysozyme upon UVPD. The location of NN modification sites are indicated in bold font in the peptide sequences.

detection of all peptides that confounds the ability to pinpoint the key NN-modified peptides for the measurement of surface accessibility/relative reactivity. The identification of NNmodified peptides in the LC runs was facilitated by generating extracted ion chromatograms (EIC) based on the m/z value of the NN reporter ion (m/z 130) produced upon UVPD. The EIC shown in Figure 4B showcases the merits of the UVPD reporter ion for tracking the elution of the NN-modified peptides. Two representative UVPD mass spectra obtained for NN-modified chymotryptic peptides KNNIVSDGNGM and KNNVFGRCE are shown in Figure 3 of the Supporting Information. The b/y series confirm the peptide sequence, and all b ions retain the NN modification as expected due to its location on the N-terminal lysine for each peptide. It is the selectivity of the NN chromophore coupled with a 351 nm UVPD that simplifies the identification and analysis process by removing false positives common to identification in CID. The reactivity of each primary amine lysozyme was estimated from eq 1 by quantifying the abundances of the NN peptides and unmodified peptides for each potential reaction site. The unmodified and NN-modified peptides identified for each lysozyme digest and subsequently used in eq 1 are summarized in Table 1 of the Supporting Information. The experimentally measured percent reactivity results are summarized in Table 1 along with the comparative surface

Table 1. NN Percent Reactivities of Lysozyme in the Absence or Presence of N-Acetylglucosamine (NAG) Based on the Trypsin, Chymotrypsin and Glu-C Digests and Predicted Lysine Surface Accessibilities and pKa Valuesa Surface accessibility (%) residue Lys Lys Lys Lys Lys Lys a

1 13 33 96 97 116

PROPKA (pKa)

PDB:2LYZ 45% 42% 34% 27% 63% 52%

11.25 11.60 10.15 10.15 10.45 10.17

% reactivity (trypsin) native

with NAG

78+ 11 >0 39 ± 9 0±0 91 ± 2 92 ± 2

72 ± 10 0±0 62 ± 6 0±0 86 ± 1 89 + 3

% reactivity (chymotrypsin)

% reactivity (glu-C)

Native

with NAG

native

with NAG

± ± ± ± ± ±

0±0 >0 73 ± 3 0±0 19 ± 3 81 + 7

19 ± 6 3±1 88 ± 6 0±0 17 ± 3 11 + 2

11 ± 1 2±1 86 ± 6 0±0 5±2 8+1

0 1 68 0 26 84

0 0 2 0 3 0

Surface accessibilities and pKa values for pdb structure 2LYZ were calculated using GETAREA and PROPKA 3.0. 7394

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Table 2. NN Percent Reactivities of Beta-Lactoglobulin in the Absence and Presence of Palmitic Acid (pam) Based on the Trypsin, Chymotrypsin, and Glu-C Digests and the Surface Accessibilities (SA) and pKa Valuesa GETAREA SA (%)

a

PROPKA pKa

% reactivity (trypsin)

% reactivity (chymotrypsin)

% reactivity (glu-C)

residue

unbound

pam

unbound

pam

native

pam

native

pam

native

pam

LEU 1 LYS 8 LYS 14 LYS 47 LYS 60 LYS 69 LYS 70 LYS 75 LYS 77 LYS 83 LYS 91 LYS 100 LYS 101 LYS 135 LYS 138 LYS 141

− 100 60 45 21 51 53 61 100 54 35 73 45 39 71 77

− 100.0 59 49 14 40 51 60 100 61 46 69 60 46 64 80

− 10.46 10.61 10.46 9.71 10.95 10.95 10.41 10.47 10.53 10.29 10.99 11.51 10.91 10.34 10.08

− 10.46 10.63 10.42 9.14 12.04 10.97 10.62 10.45 10.50 10.22 11.20 10.11 10.45 11.19 10.38

0±0 1+0 0±0 >0 ± 0 6±1 83 + 6 0±0 0±0 91 ± 5 0+0 >0 ± 0 1±0 0±0 0±0 1±0 2+0

0±0 1±0 0±0 >0 ± 0 3±1 32 + 6 0±0 0±0 92 + 6 0±0 >0 ± 0 2±0 0±0 0±0 1±0 2+1

0±0 0+0 1±0 0+0 0+0 88 ± 8 0+0 0+0 51 ± 7 0±0 >0 ± 0 20 ± 2 0±0 0+0 0±0 45 + 5

0±0 0±0 1±0 0±0 0+0 59 ± 3 0±0 0±0 56 ± 2 0±0 >0 ± 0 18 ± 3 0±0 0±0 0±0 45 + 2

0±0 >0 ± 0 0±0 0±0 >0 ± 0 37 + 1 0±0 0±0 1+0 0±0 >0 ± 0 1+0 0±0 0+0 0±0 0+0

0±0 >0 ± 0 0±0 0±0 >0 ± 0 10 ± 1 0±0 0±0 2±0 0+0 >0 ± 0 >0 ± 0 0±0 0+0 0±0 0+0

Surface accessibilities and pKa values for pdb structures 1BSY and 3UEW (Pam bound) were calculated using GETAREA and PROPKA 3.0.

enzymatic activity.67−69 The NN probe reactions were undertaken at a 7:15:1 NN:NAG:lysozyme ratio prior to proteolytic digest and LC−UVPD analysis. The NN reactivities were calculated as described earlier and are summarized in Table 1. Lys13 maintained the same low reactivity in the presence of NAG, but some changes in NN reactivity were noted for the other primary amines. On the basis of the compilation of the results from the three different digests, the reactivities of Lys1, Lys 97, and Lys116 were found to be slightly lower, those of Lys13 and Lys96 displayed no change, and that of Lys33 showed the most significant increase upon NAG binding. The proximity of Lys1, Lys 97, and Lys 116 to the NAG binding region may lead to the decrease in surface accessibility upon ligand binding. Although Lys33 is not expected to lie within a known NAG binding region, the notable increase in its reactivity suggests a conformational change of lysozyme that leads to the greater exposure of Lys33. NN-Reactivity of β-Lactoglobulin. To further evaluate the NN probe/UVPD methodology, a second well-characterized protein was evaluated. β-lactoglobulin (18.3 kDa) contains 162 amino acids of which 15 are lysine sites. Although the details of the biological function of β-lactoglobulin remain poorly understood, it is known to bind both fatty acids and retinol, which suggests its possible role as a transporter protein.70 β-lactoglobulin was incubated with NN at a 1:1 protein:NN ratio, a ratio that yielded good NN reaction efficiency and did not appear to cause significant alterations in the native conformation of the protein based on circular dichroism analysis (Figure 2C of the Supporting Information). [Using a higher 5:1 NN:protein ratio led to subtle changes in the CD spectra (data not shown)]. Aliquots of the resulting NN-modified protein were digested using three different proteases prior to LC−MS/MS analysis per the workflow described earlier. The unmodified and NN-modified peptides identified for each digest and subsequently used in eq 1 are summarized in Table 3 of the Supporting Information. The protein was also incubated with palmitic acid, a known fatty acid binding partner,71 prior to the NN probe reactions at a net ratio of NN:palmitic acid:beta lactoglobulin of 1:1:1.

The results obtained from the NN probe/UVPD methodology are in good qualitative agreement with previous chemical probe-type studies.63−65 Suckau et al. reported the order of reactivity as K97 > K33 ≫ K1 (ε-NH2) > K13 > K116 > K96 > R−NH2 using an acetylation reagent and most notably observed the highest reactivity for Lys97 and Ly33 and lower reactivity of the other potential acetylation sites.63 Schniable and Przybylski found five reactive sites (α-NH2 of K1, K1, K33, K97, and K116) based on using a fluorescein isocyanate aminereactive probe.64 Using an estrone−glucuronide succinimide probe and tryptic digestion of lysozyme, Smales et al. noted the greatest reactivity of Lys33 and Lys97 as well as Lys116.65 To evaluate the reactivity of the NN probe relative to a smaller, more conventional chemical probe, comparative results were obtained using acetic anhydride, a well-known aminereactive acetylation reagent.63 Lysozyme was incubated with acetic anhydride (20:1 acetic anhydride:protein molar ratio) prior to tryptic digestion and LC−MS/MS analysis. Because acetylation does not attach a UV chromophore, all analysis was undertaken using CID alone. The acetylation reactivities are summarized in Table 2 of the Supporting Information, yielding the trend Lys97 ≈ Lys116 ≈ Lys1 > Lys 13 ≈ Lys 33 ≈ Lys96. For five of the sites (Lys1, Lys33, Lys 96, Lys 97, and Lys116), there is remarkable agreement between the NN and acetylation reactivities. However, there is a significant difference in reactivity for Lys13 for which lower NN-reactivity was found compared to the acetylation reactivity. We hypothesize that the low NN reactivity of Lys13 may arise from the larger size of the NN probe, a factor that contributes to its diminished accessibility to certain partially exposed sites. Additionally, Lys13 forms close polar contact with the C-terminus (Figure 5 of the Supporting Information), which may further suppress the reactivity of Lys13 toward a bulkier chemical probe. The ability of NN to reveal changes in accessibility upon protein−ligand binding was also evaluated, as demonstrated for lysozyme in the presence of the cognate ligand, N-acetylglucosamine (NAG). Lysozyme cleaves glycosidic bonds of bacterial cell walls, thus providing its well-known antimicrobial activity.66 NAG is known to interact with lysozyme to inhibit the hydrolysis of cell walls, thus significantly suppressing the 7395

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Lys135 ≈ Lys138 > Lys47 > Lys83 > K141 ≈ Lys14 ≈ Lys60 ≈ Lys101. The acetic anhydride probe exhibited broader reactivity over a greater array of the Lys amines. Similar low reactivities were observed for lysines 14, 60, 83, 101, and 141, suggesting that these residues are truly inaccessible regardless of the chemical probe.

All of the results are summarized in Table 2 along with the surface accessibility values obtained by GETAREA calculations based on the X-ray crystal structures.71,72 Modification of ten of the potential sixteen primary amine sites was discovered. The N-terminus (Leu1), Lys70, Lys75, Lys83, Lys101, and Lys135 sites exhibited no reactivity and Lys8, Lys14, Lys47, Lys91, and Lys138 showed extremely low but measurable reactivity toward the NN probe. The greatest NN reactivities were observed for Lys60, Lys69, Lys77, Lys 100, and Lys141. The overall order of reactivity based on the compilation from the three digests is Lys69 ≈ Lys77 > Lys 141 > Lys 100 > Lys60 > Lys14 ≈ Lys138 ≈ Lys91 ≈ Lys 8 > N-term Leu1 ≈ Lys70 ≈ Lys75 ≈ Lys83 ≈ Lys101 ≈ Lys135. The NN reactivity does not change appreciably in the presence of palmitic acid, except for amine sites Lys 60 and Lys69. The NN reactivity data showed partial agreement with the surface accessibilities predicted using GETAREA based on the reported structure of β-lactoglobulin (Supplemental Figure 6). Lys83, Lys47, Lys101, and Lys135 all had lower predicted solvent exposures compared to the other residues, and these exhibited no detectable reactivity with NN. Lys8 is known to bind a “lock and key” dimer with itself in solution, and the calculated surface accessibility of the monomer is not expected to depict the true solvent exposure.72 It is also known that the local environments can affect the reactivities of amine sites, and in particular the residues possessing acidic and basic side-chains may engage in hydrogen-bonding interactions that influence the nucleophilicity of particular amine sites. Lys141, Lys77, and Lys100 were anticipated to be among the most reactive sites of the protein as they all had predicted accessibilities above 70%, and this outcome is reflected in the NN reactivities across the series of digests. The unusually high reactivity of Lys69 deserves special consideration because it is not predicted to be among the seven most accessible sites. The significant modification of Lys69 as revealed by all three digests suggests that some factor other than surface accessibility and pKa affects its reactivity. βlactoglobulin is known to have a hydrophobic beta-barrel structure that can bind retinol, among other hydrophobic compounds.70 Binding ligands, such as palmitic acid, are known to interact with Lys69.71 Thus, the enhanced NN reactivity of Lys69 might arise from a unique hydrophobic−hydrophobic interaction between the NN probe and the retinol binding site of β-lactoglobulin. Overlaying the structures of β-lactoglobulin before and after palmitic acid binding (Figure 7 of the Supporting Information) suggests that the structures are nearly identical. With the exception of residues in the ligand binding site region (Lys60 and Lys69), one might expect that the surface accessibilities and reactivities of the probe toward the lysines would be similar in the presence or absence of the ligand based on the overlaid structures. After incubation of β-lactoglobulin with palmitic acid, the NN reactivities of Lys60 and Lys69 changed rather significantly, suggesting a conformational change induced by palmitic acid binding or shielding of these sites by palmitic acid. In a similar manner to that described above for lysozyme, the smaller acetylation reagent, acetic anhydride, was again employed for beta-lactoglobulin. The protein was reacted at a 20:1 acetic anhydride:protein molar ratio prior to tryptic digestion and LC−CID-MS/MS analysis. The acetylation reactivities are summarized in Table 4 of the Supporting Information, revealing the reactivity order of Lys70 ≈ Lys75 ≈ Lys77 > Lys 91 > Lys8 ≈ Lys69 ≈ K100 > N-term Leu1 >

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CONCLUSIONS The ability to pinpoint chemical probe-modified sites of proteins is showcased by a UVPD method that activates only those peptides that contain the chromogenic tag that exhibits high absorptivity at 351 nm. This method allows the lowabundance-modified peptides to be identified in complex proteolytic mixtures, as demonstrated for lysozyme and betalactoglobulin in the presence and absence of their cognate ligands. The NN probe-modified peptides undergo efficient photodissociation to produce diagnostic fragmentation patterns that contain b/y sequence ions as well as two characteristic reporter ions. The reporter ions generated by UVPD allow construction of extracted ion chromatograms that allow the elution of the modified peptides to be tracked. Peptides that do not contain modified lysines do not undergo UVPD, thus allowing facile differentiation of modified and unmodified peptides in complex mixtures.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding from the NSF (Grant CHE-1012622) and the Welch Foundation (Grant F1155) is acknowledged.

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Analytical Chemistry

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dx.doi.org/10.1021/ac401305f | Anal. Chem. 2013, 85, 7391−7397