trypsin non-covalent complex using covalent modification and mass spectrometry.

Research Article Received: 15 August 2013

Revised: 21 November 2013

Accepted: 1 December 2013

Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2014, 28, 413–429 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6797

Structural studies of the sBBI/trypsin non-covalent complex using covalent modification and mass spectrometry Ekaterina Darii1,2*, Guanalini Saravanamuthu1, Ivo G. Gut1,3 and Jean-Claude Tabet4 1

CEA/Institut de Génomique/Centre National de Génotypage, Evry, France CEA/Institut de Génomique/Centre National de Séquençage, Evry, France 3 Centro Nacional de Análisis Genómico, PCB, Barcelona, Spain 4 UPMC Paris Universitas, Paris, France 2

RATIONALE: The study of protein recognition sites is crucial for understanding the mechanisms of protein interaction. Mass spectrometry can be a method of choice for the investigation of the contact surface within the protein non-covalent complexes. METHODS: Probing the reactivity of essential amino acid residues of soybean Bowman-Birk inhibitor (sBBI) within the non-covalent sBBI/bovine trypsin complex was performed using covalent labeling by the BS3 cross-linker and charge tag with a quaternary ammonium group in combination with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. RESULTS: Significant modulation of the reactivity of essential K16 and S17 residues in the sBBI molecule upon binding to trypsin was established. The studies of sBBI proteolytic peptides with the same structure but carrying different labels using metastable dissociation in LIFT mode demonstrated that fragmentation pathways were oriented by used modification (BS3 cross-linker or charge tag). CONCLUSIONS: The effectiveness of the mass spectrometric approach including covalent modification for exploring protein-protein interaction sites has been demonstrated. The alteration of the reactivity of functionally important amino acid residues in the sBBI molecule is most likely related to changes in their microenvironment. It has been suggested that in the presence of charge tags fragmentation in LIFT mode proceeds through the formation of salt bridges between quaternary ammonium groups and acidic residues due to the occurrence of zwitterions (including basic and acidic residues). Despite the presence of one or several charge tags, fragmentation takes place yielding modulated bi/yj ion series depending on the positions of the tags. Copyright © 2014 John Wiley & Sons, Ltd.

Various biochemical processes are associated with proteinprotein recognition via non-covalent interaction under physiological conditions.[1,2] Such non-covalent binding often involves conformational rearrangements reinforced by formation of new salt bridges (SB) and hydrogen bonds between interacting partners.[3–7] One of the analytical approaches to investigate non-covalent protein complexes consists of the derivatization of proteins either alone or in the presence of interacting partners followed by comparative analysis of modified species using electrospray ionization mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization (MALDI)-MS.[8–10] The main factors responsible for the modulation of reactivity of amino acid residues upon protein interaction are steric hindrance and electrostatic effects, i.e., the influence of the microenvironment.[11–13] In addition, modifiable residues can be involved in new non-covalent bonds that maintain complex stability. Thus, the objective is to discriminate between the

Rapid Commun. Mass Spectrom. 2014, 28, 413–429

Copyright © 2014 John Wiley & Sons, Ltd.

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* Correspondence to: E. Darii, CEA/Institut de Génomique/ Centre National de Séquençage, 2 rue Gaston Crémieux, 91000 Evry, France. E-mail: [email protected]

residues that modulate the reactivity upon complex formation. Therefore, it is important to identify the accurate positions as well as the level of modification of derivatized residues within the proteins modified either alone or in the presence of interacting partners. Another strategy is to use cross-linking reagents for protein modification. These reagents are able to react with residues closely positioned in space. The analysis of intra- or intermolecular linked species provides helpful structural information.[14–19] NHS ester derivatives that specifically react with primary amino groups (at the N-terminus and side chains of lysine residues) and hydroxyl groups (at S, T and Y residues) are largely employed for protein modification. Selective modification of N-terminal α-amino groups and hydroxyl functions has been demonstrated under neutral and slightly acidic pH conditions, while enhanced modification of lysine ε-amino groups has been observed at slightly alkaline pH.[16,20–22] The reactivity of modifiable residues is generally governed by their nucleophilicity towards labeling agents.[11] Due to relatively high pKa (about 10.5 ± 1.1), ε-amino groups of lysine residues are less reactive towards NHS esters than N-terminal amino groups (pKa about 7.7 ± 0.5) in proteins.[23–25] Serine and threonine hydroxyl

E. Darii et al. groups (pKa ~13) as well as that of tyrosine (pKa ~10) represent relatively poor nucleophiles at neutral pH. Nevertheless, their nucleophilicity may be significantly increased due to interaction with adjacent space residues.[21,26–29] On the other hand, O-acetyl derivatives obtained by modification of OH groups became less stable at slightly alkaline pH due to hydrolysis.[29] In order to localize modification sites with higher confidence by MALDI-tandem mass spectrometry (MS/MS), metastable dissociations of singly charged modified peptides using the LIFT mode can be a useful tool. Generally, metastable fragmentation pathways depend on the amino acid sequence of the precursor ion. They can be affected by the presence of chemical modification.[30] Charge-directed mechanisms yielding bi and yj ion series have been mostly demonstrated for mobile proton containing peptides.[31–35] For peptides with charge sequestered by strongly basic residues (R, H and K),[36–40] ’charge-remote,’[41–43] or, alternatively, ’proton driven’ [38] mechanisms have been proposed. Preferentially, non-selective cleavages can take place in the presence of a mobile proton except in certain particular cases.[32] The generation of bi ions generally proceeds through the formation of a protonated oxazolone derivative and involves at least two residues (at the Cterminus of the cleavage site and its N-terminal neighbour).[44] Consequently, bn–1 fragment ions (n = total number of residues) are often not abundant and, in addition, b1 ions are rarely detected.[45] In this work, a mass spectrometric approach including covalent modification by cross-linking agent BS3 and ’charge tag’ containing quaternary ammonium groups[46] with subsequent MALDI-MS and -MS/MS analysis were applied for elucidation of the reactivity of essential residues in the small size sBBI molecule. Such an approach may be helpful to probe the contact surface between interacting partners and provide information about the dynamic state of the complex existing in solution, whereas X-ray crystallographic studies are related to the solid state. The structural basis of the trypsin-inhibitor binding has been largely reported.[3,4,47,48] Crystallographic studies of Bowman-Birk type inhibitors and their complexes with trypsin revealed that K16 is the most important sBBI residue in the sBBI/trypsin contact region. A new salt bridge between K16 of sBBI and D189 of trypsin is formed upon proteinprotein interaction.[49,50] The essential S17 residue of sBBI is

implicated in the intramolecular hydrogen bond with the Q21 residue in the free inhibitor (Scheme 1(a)). The binding of the inhibitor to trypsin results in the creation of a new hydrogen bond between the S17 residue of sBBI and S195 of trypsin (Scheme 1(b)).[49–52] The maximum activity of trypsin is observed in the pH range of 7–9.[53] The formation of the non-covalent complex between small size sBBI and trypsin was initially studied by ESI-MS.[55] Multicharged ions corresponding to the cationized ternary complex sBBI/trypsin/Ca2+ were observed in positive and negative mode under non-denaturing conditions (Mw = 31186 Da). The narrow charge-state distribution independent of the ion polarity suggested the preservation of the native structure under these experimental conditions. The reactivity of amino acid residues in the sBBI molecule towards the BS3 and CT+0 reagents was explored in the experiments with the inhibitor alone and its mixture with trypsin under neutral and slightly alkaline reaction conditions. The labeled species were analyzed in LIFT mode in order to determine the position of modified amino acid residues. These experiments additionally confirmed that metastable fragmentation pathways of structurally identical but differentially labeled peptides are guided by the properties of the tags.

EXPERIMENTAL Reagents General use chemical reagents were obtained from SigmaAldrich. Bovine trypsin and trypsin-chymotrypsin inhibitor from soybean were purchased from Sigma (Steinheim, Germany). Homobifunctional NHS ester cross-linker bis [sulfosuccinimidyl] suberate (BS3, Scheme 2(a)) was from Thermo Scientific (Rockford, IL, USA). The mass tag with fixed positive charge (6-trimethylammoniumhexyryl-Nhydroxysuccinimidyl ester denoted as CT+0, Scheme 2(b)) was prepared similarly to the procedure of Bartlet-Jones et al.[46] 4-(2-Hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES) was from Fluka (Buchs, Switzerland) and endopeptidase porcine trypsin was from Promega (Madison, WI, USA). Water was purified using a Milli-Q® Ultrapure water purification system from Millipore.

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Scheme 1. Schematic presentation of the trypsin-binding loop in the sBBI molecule (amino acid residues 14–22 in the N-terminal domain; P1-P1’ – inhibitor primary active site, numeration according to Schechter and Berger:[54] (a) free sBBI and (b) trypsin-bound sBBI.

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Structural studies of the sBBI/trypsin non-covalent complex

RESULTS AND DISCUSSION

Scheme 2. Chemical structures of reagents for protein covalent labelling: (a) cross-linker bis[sulfosuccinimidyl] suberate (BS3) and (b) charge tag 6-trimethylammoniumhexyryl-N-hydroxysuccinimidyl ester (CT+0). Chemical modification Cross-linking experiments were performed using BS3 under non-denaturing conditions according to the supplier’s instructions with some modifications. In order to optimize the procedure of labeling compatible with MS analysis, a series of experiments was performed under different conditions, e.g., protein concentration, incubation time and temperature, protein-to-labeling agent ratio. For each set of parameters, the experiments were replicated at least 3 to 5 times. Finally, the derivatization of proteins alone or their equimolar mixture was carried out at neutral and slightly alkaline pH at a protein concentration of 50 μM. The mixture with 20-fold molar excess of cross-linker was incubated 2 h at 4 °C in 20 mM HEPES and 2 M triethylammonium bicarbonate was used to adjust pH. Covalent modification by CT+0 was performed using the same reaction conditions. The reaction was quenched by addition of 1 M Tris/HCl to a final concentration of 20 mM. After labeling the protein species were diluted with water to 10 μM and subjected to extensive denaturation using dithiothreitol (DTT) and urea treatment at 70 °C in order to prevent the autolysis of bovine trypsin. Then, the denatured samples were digested by porcine trypsin from Promega according to the supplier’s instructions. Mass spectrometry


Cross-linking by BS3 Modification at neutral pH First, the products of derivatization of sBBI alone and presumably formed non-covalent complex sBBI/bovine trypsin using cross-linker BS3 (Scheme 2(a)) at neutral pH were explored. The degree of modification of non-digested species in the experiments with sBBI modified alone and within a mixture with trypsin was relatively low. No species related to covalently linked partners were observed. According to MALDI-MS/MS data presented in the second part, preferentially the N-terminal amino group and serine hydroxyl groups were labeled at neutral pH. The abbreviations for cross-linked species were used according to Madler et al.[16] Thus, monolink (or ’deadend’) type modification with one hydrolyzed end was denoted as P-XLOH and looplink (or crosslink) was denoted as P=XL. Figures 1(a) and 1(b) represent MALDI mass spectra of digest products obtained from sBBI modified at neutral pH alone and within an equimolar mixture with bovine trypsin, respectively. The analysis of data using the Biotools software confirmed the presence of sBBI digest products with (or without) modification in the peptide mixture. Tryptic digestion of modified species yielded a set of peptides with lysine or arginine at their C-terminus and generally without missing cleavages. Therefore, lysine residues remained mostly unmodified under these experimental conditions. The mass spectra display a very weak signal at m/z 1732.6 attributed to the non-modified N-terminal peptide D1-K16 (MW = 1731.6 Da) and a slightly more intense signal at m/z 1888.7 probably related to the same peptide with a monolink type modification resulting in a mass increment of 156 Da (for more details, see Table 1 with semi-quantitative estimation of modification degree of three representative peptides from sBBI at different pH and with or without interacting partner). It has to be noted that a missed cleavage at K6 is always impeded due to the presence of a P7 residue (Scheme 1).



415

MALDI-MS and -MS/MS experiments were performed on a MALDI-TOF/TOF mass spectrometer (Ultraflex II; BrukerDaltonics, Bremen, Germany) in positive mode. Freshly prepared saturated solutions of sinapinic acid (SA) and αcyano-4-hydroxycinnamic acid (HCCA) from Bruker Daltonics in 30% acetonitrile with 0.1% trifluoroacetic acid (TFA) were used as matrix for protein and peptide analysis, respectively. For MS experiments, the mixture of sample and matrix solutions was prepared at a 1:5 ratio (v/v) and deposited onto a stainless steel MALDI target plate. The peptide analysis was performed in reflectron mode. For MS and MS/MS experiments, the laser fluence was adjusted for the studied specimen without the use of delay extraction. The data were processed using the Biotools (BrukerDaltonics, Bremen, Germany) software. Semi-quantitative measurements of the degree of peptide modification were performed using the peak intensity ratios of representative modified/unmodified peptides. For each pair of ion species 17–23 spectra were used for calculations.

Preliminary MALDI experiments with intact sBBI/trypsin mixture did not yield enough evidence for complex formation. Indeed, a very poor signal (m/z about 31200) corresponding to the non-covalent complex was observed in MALDI mass spectra of the protein mixture under various experimental conditions. Although ESI-MS experiments confirmed the protein binding, no specific data about contact surface within the complex were obtained. In order to achieve more detailed information about protein interaction sites, chemical modification by NHS ester derivatives, especially by cross-linker BS3 and charge tag CT+0, followed by MALDI-MS analysis was employed. The difference between the MALDI mass spectra of proteins modified alone and within their equimolar mixture has been established, especially at slightly alkaline pH, suggesting the protective effect related to protein interaction. This can be an indirect indication of non-covalent complex formation prior to chemical modification. Indeed, the missed modified site in the complex may be caused by steric hindrance in this particular region due to non-covalent interaction.

E. Darii et al.

Figure 1. Representative zones of MALDI mass spectra of sBBI-modified (a) alone and (b) within the non-covalent complex using BS3 at neutral pH with subsequent denaturation and digestion. MALDI-MS analysis was performed using a mixture of analyte and saturated solution of matrix HCCA (see Experimental section).

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The peak observed at m/z 801.3 (Fig. 1(a)) can be attributed to the peptide S17–R23 (MW = 800.4 Da), and that at m/z 957.5 (Fig. 1(b)) was most likely related to the same peptide with a monolink type modification. Surprisingly, the signal at m/z 801.3 from the unmodified peptide was predominant in the experiment with sBBI alone, while the signal from the modified species was low (Fig. 1(a), Table 1: intensity ratio for modified/unmodified ion species = 0.59 ± 0.27). In contrast, in the experiment with the sBBI/trypsin mixture (Fig. 1(b)), the signal at m/z 957.5 from the modified peptide was significantly more intense than that at m/z 801.4 related to the non-modified species (Table 1: intensity ratio for modified/unmodified ion species = 4.5 ± 2.2). Therefore, in spite of possible steric hindrance upon complex formation, the reactivity of residues in the contact region can be even reinforced. Next, the peak at m/z 2809.0 was observed in both mass spectra, but with different intensity. It could be attributed to the peptide D1-R23 (Mw = 2514.0 Da for the unmodified peptide) resulting from a missed cleavage at K16 and carrying one ’loop-link’ and one monolink type modification. Indeed, the experimental m/z value of the singly charged peptide resulted from a mass increment of 294 Da, i. e., the sum of the mass increments for monolink (156 Da) and ’loop-link’ (138 Da) type modifications. In this part, we only present our final conclusions about tag positions, i.e., modified amino acid residues based on the LIFT experiments. The results and the discussion concerning metastable fragmentation patterns of unmodified and differently modified peptides are presented in the second part of this work for the reason of more comprehensive presentation. According to the MS/MS data, the precursor ion at m/z 1888.7 (shifted by 156 Da from the theoretical value) is generated by the N-terminal peptide D1-K16 with a monolink type modification at D1. The precursor ion at m/z 957.5 displaying the same mass shift (156 Da) corresponds to the charged peptide S17-R23 modified at S17. Furthermore, the m/z 2809.1 ion is most likely related to the peptide D1-R23 carrying a monolink modification at K16 and a loop link


between D1 and K6. The peak of the peptide with a missed cleavage at K16 was relatively more intense in the experiment with sBBI alone (Table 1) suggesting the higher reactivity of K16 in this case. The decrease in peak intensity for this peptide in the experiment with the sBBI/trypsin mixture (approximately one order of magnitude, see Table 1) confirms the protective effect related to complex formation. In addition, the minor peak at m/z 2827.1 (monoisotopic), most probably corresponding to the peptide D1-R23 with modifications at the D1 and K16 residues, was also present in both mass spectra, but to a lesser extent (Fig. 1). The presence of unmodified peptides D1-K16 and S17-R23 as well as species modified at D1 and S17 within the digested products gives evidence of the enzymatic cleavage after K16 (Scheme 3(a)). Most likely, this residue remained preferentially unmodified at neutral pH. Therefore, under these experimental conditions, not only the N-terminal amino group but also the hydroxyl group of serine residues can be labeled. Interestingly, S17 was less efficiently modified in the experiment with sBBI alone than in the experiment with the sBBI/trypsin mixture (about 8 times lower, Table 1). Probably, the protective effect in this case was less important than the change in reactivity towards the NHS ester. Similar behavior was observed for the DII-type inhibitor from the same family. In fact, the most intense signals were observed at m/z 1365.5 (Fig. 1(a)) and 1521.7 (Fig. 1(b)). The sequences of these precursor ions were analyzed in LIFT mode using the RapiDeNovo software module from Biotools (Fig. S1, Supporting Information). The provided data were compared to the sequences of other inhibitors which are structurally similar to sBBI.[56] It has been established that the corresponding unmodified (Mw = 1364.6 Da) and modified (Mw = 1520.6 Da) peptides were originated from the DII-type inhibitor. Most likely, this species is always present in the sample of sBBI from the supplier. In addition, the peak at about m/z 8500 corresponding to this inhibitor[56,57] was found in the control mass spectrum of



h.m. – highly modified; m.m. – moderately modified; l.m. – low modified ion species *the corresponding unmodified peptide was not observed due to enzymatic cleavage after unmodified K16; therefore, the signal of the m/z 801.3 ion (peptide S17-R23) that displayed less variation in abundance than other analyzed ions was used as reference for peptide D1-R23 with one mono- and one cross-link

1.4 ± 0.5 l.m. 0.007 ± 0.003

Note: m/z 1732.6 – protonated unmodified peptide D1-K16; m/z 1888.7 – the same peptide with monolink BS3 modification; m/z 1870.8 – the same peptide with internal cross-link; m/z 801.3 – protonated unmodified peptide S17-R23; m/z 957.5 – the same peptide with monolink BS3 modification; m/z 2809.1 – protonated peptide D1-R23 with one monolink and one internal crosslink; m/z 1365.5 – protonated unmodified peptide from DII-type inhibitor; m/z 1521.7 – the same peptide with monolink BS3 modification

0.22 ± 0.14 l.m. 4.3 ± 1.8 m.m. 0.045 ± 0.023 l.m.

0.59 ± 0.27 l.m. 4.5 ± 2.2 m.m. 0.66 ± 0.34 (low abundant ions) l.m. 2.3 ± 1.4 m.m. 3.7 ± 2.2 m.m. 0.34 ± 0.15 l.m. 830 ± 360 h.m.

Free sBBI at neutral pH sBBI/trypsin at neutral pH Free sBBI at slightly alkaline pH sBBI/trypsin at slightly alkaline pH

4.7 ±0.4 m.m. 3.7 ± 1.3 m.m. 6.9 ± 3.7 (low abundant ions) h.m. 120 ± 45 h.m.

3.2 ± 1.7 3.8 ± 2.0 0.57 ± 0.38

8.9 ± 3.5 h.m.

I (m/z 1521.7)/I (m/z 1365.5) I (m/z 957.5)/I (m/z 801.3) Modification conditions

(I (m/z 1888.7) + I (m/z 1870.8))/I (m/z 1732.6)

I (m/z 1888.7)/I (m/z 1870.8)

I (m/z 2809.1)/I (m/z 801.3)

Peptide S25-R36 from DII type inhibitor Peptide S17-R23 from sBBI Peptide D1-R23 with a missed cleavage at K16* N-terminal peptide D1-K16 from sBBI


intact sBBI. The main difference between the two inhibitors is the occurrence of an arginine residue at the P1 position (numerotation of Schechter and Berger),[54] namely, R24, instead of a lysine residue (K16) in the N-terminal active centre. The following serine residue (S25 or S17, respectively) is highly conserved in the sequences of this family of inhibitors.[57,58] MALDI-MS analysis evidenced a remarkable increase in abundance of the 1521.7 m/z ion corresponding to the peptide with a modified S25 residue in the experiments with the sBBI/trypsin mixture (approximately 30 times higher, Table 1). Furthermore, the modification of the S25 residue in the DII-type inhibitor was significantly reinforced compared to that of S17 of the main sBBI species. Modification at slightly alkaline pH The MALDI mass spectrum of the non-digested species from the sBBI/trypsin mixture subjected to modification by BS3 displayed a broad peak at about m/z 33500 (Fig. 2). This could be attributed to covalently linked partners. It has to be noted that after denaturation and digestion, no species corresponding to intermolecular cross-linking between sBBI and trypsin was detected. This could be explained by the lower stability of esters formed by BS3 acylation of the serine hydroxyl group. Probably, their hydrolysis can occur under denaturing conditions, i.e., heating at slightly alkaline pH.[29,59] The MALDI mass spectra of sBBI modified either alone or within an equimolar mixture with trypsin under slightly alkaline pH conditions are characterized by a common peak at m/z 2809.1 (Figs. 3(a) and 3(b)). This could be attributed to the peptide D1-R23 with a missed cleavage at the K16 residue. Fragmentation patterns of the m/z 2809.1 ion in LIFT mode (data not shown) were similar to those observed after modification under neutral conditions. Presumably, this peptide carried a monolink type modification at K16 and a loop link (cross-link) between D1 and K6 (Scheme 3(b)). On the other hand, the abundance of the m/z 2809.2 ion was reduced in the experiment with the sBBI/trypsin mixture (Table 1). Furthermore, the modification of sBBI in the presence of trypsin resulted in the generation of peptides produced by cleavage at K16. The respective peaks newly appeared, namely, (i) at m/z 801.4 and 957.5 assigned to the unmodified and modified peptide S17-R23, and (ii) at m/z 1870.8 (Fig. 3(b)). The latter corresponds to the N-terminal peptide D1-K16 carrying an intrapeptide cross-link with the mass increment of 138 Da. According to MS/MS data presented later, the m/z 1870.8 ion is probably generated by the peptide D1-K16 with a loop link between D1 and K6. Thus, K16 was modified in the experiment with sBBI alone but remained partially unmodified in the experiment with the protein mixture likely due to complex formation (Scheme 3). Interestingly, the Nterminal peptide carrying the internal cross link between D1 and K6 was preferentially formed in the experiments with a protein mixture under slightly alkaline conditions (Fig. 3(b), for more details, see peak intensity ratios I (1888.7)/I (1870.8), i.e., monolink versus loop-link formation from Table 1). The higher reactivity may be due to enhanced nucleophilicity of ε-NH2 of the K6 residues at slightly alkaline pH.



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Table 1. Semi-quantitative estimation of changes in reactivity of sBBI amino acid residues using the peak intensity ratios of BS3-modified and unmodified protonated peptides


E. Darii et al.

Scheme 3. Modification of sBBI alone and within the non-covalent complex using the BS3 reagent under (a) neutral and (b) slightly alkaline pH conditions with subsequent denaturation and digestion. The data concerning the change in reactivity of essential residues upon complex formation have been confirmed using covalent modification by a charge tag (CT+0).

Covalent labeling by a charge tag Modification at neutral pH

Figure 2. MALDI mass spectrum of equimolar sBBI/trypsin mixture after modification by BS3 under slightly alkaline conditions (pH 8.5). MALDI-MS analysis was performed using a mixture of analyte with SA as matrix at a ratio of 1:5 (v/v) (see Experimental section).

Covalent modification by CT+0 (Scheme 2(b))[46] was carried out under the same experimental conditions as that by BS3. The derivatization of one residue results in a mass increment of 155 Da. The presence of a fixed positive charge may enhance the signal in mass spectrometric detection. The MALDI mass spectrum of digestion products of sBBI modified by CT+0 at neutral pH, either alone or within the mixture with trypsin, displayed peaks at m/z 801.5, 956.4, 1732.5, and 1887.6 related to unmodified and modified peptides S17-R23 and D1-K16 (data not shown). Therefore, under these conditions, K16 remained at least in part unmodified giving rise to the formation of peptides D1-K16 and S17-R23 with or without modifications. Probably, the

418

Figure 3. Representative zones of MALDI mass spectra of sBBI-modified (a) alone and (b) within the non-covalent complex using BS3 at slightly alkaline pH with subsequent denaturation and digestion. MALDI-MS analysis was performed using a mixture of analyte and saturated solution of matrix HCCA (see Experimental section).




Structural studies of the sBBI/trypsin non-covalent complex D1 and the S17 residues underwent labeling by the charge tag. Thus, the results were similar to those obtained in the experiments carried out with BS3 at neutral pH. Indeed, the N-terminal residue, i.e., D1, and the S17 residue were partially modified, while the K16 residue remained preferentially unmodified (Scheme 4(a) and Table 2). The intensity of the peak at m/z 956.4 probably related to the modified S17-R23 peptide was too weak to perform its sequencing using the LIFT system. However, the peak at m/z 1887.6 attributed to the modified peptide D1-K16 was sufficiently intense to perform MS/MS experiments. It has been established that, as in the experiments with BS3, the mass tag was attached to the D1 residue of sBBI. This is in agreement with the higher reactivity of the N-terminal amino group compared to other modifiable groups at neutral pH. In addition, the m/z 1520.6 precursor ion corresponding to peptide S25-R36 from the DII-type inhibitor with CT+0 modification was analyzed under LIFT conditions. The respective metastable ion spectrum was compared with that of the unmodified product detected at m/z 1365.5. According to MS/MS data, the tag was attached to the essential S25 residue (Fig. S1, Supporting Information). Modification by CT+0 at slightly alkaline pH The results of experiments performed at slightly alkaline pH with sBBI alone or within a mixture with trypsin were compared. As in the case of BS3, a significant difference between the products was observed (Scheme 4(b), Table 2, Fig. 4). Presumably, the enhanced modification of the ε-amino group of lysine residues under slightly alkaline pH conditions compared to that at neutral pH highlights the protective effect related to enzyme–inhibitor interaction. The most intense signal in the mass spectrum of free sBBI after modification by CT+0 and subsequent digestion (Fig. 4(a)) was detected at m/z 2980.3 (monoisotopic peak). The respective ion can be attributed to the singly charged modified peptide D1-R23 with a missed cleavage at K16 (Mw = 2514.0 Da). The observed shifted value is consistent with the addition of three charge tags (+3*155 Da) giving the total mass increment of 465 Da. Clearly, the presence of three fixed positive charges requires the formation of doubly deprotonated species (the presence of two putative deprotonation sites) to provide the singly charged ion. The MS/MS data for this peptide are presented and discussed later. According to fragmentation patterns, modifications are most likely located at D1, K6 and K16.

Figure 4(b) illustrates the results of modification of the sBBI/ trypsin mixture using CT+0 under slightly alkaline conditions. The most intense peak at m/z 2042.8 (monoisotopic) is likely related to peptide D1-K16 (MW = 1731.6 Da) carrying two charge tags (mass increment of 210 Da). Its sequence was confirmed by metastable fragmentation. In conclusion, a similar reactivity of essential residues was observed in the experiments with CT+0 (Scheme 4) and BS3 (Scheme 3). In the case of the free inhibitor at slightly alkaline pH, the K16 residue was almost completely modified (peak intensity ratio of modified (3 CT +0)/unmodified ion species about 400 for free sBBI versus 2 for sBBI within the complex, Table 2). However, it became much less reactive in the presence of trypsin. In contrast, the reactivity of S17 was reinforced upon complex formation (peak intensity ratio of modified/unmodified ion species about 3.6 for sBBI within the complex vs 1 for free sBBI, Table 2). Effect of complex formation on the nucleophilic properties of sBBI essential residues According to the experimental results, the reactivity of essential K16 and S17 residues towards BS3 and CT+0 at neutral and slightly alkaline pH changes significantly during the complex formation. Particularly, the level of modification of K16 was reduced in the complex. This effect was more pronounced at basic pH due to the enhanced reactivity of lysine ε-amino groups under these conditions. Indeed, K16 was almost completely modified in the case of free sBBI, while it was at least partially preserved by the interacting partner upon complex formation. In contrast, the S17 residue in sBBI (as well as S25 in the DII-type inhibitor) demonstrated the enhanced reactivity within the complex (Tables 1 and 2). Such behavior of the serine residues S17 or S25 seems to be contradictory because they became buried within the trypsin pocket after complex formation (Scheme 1). Consequently, steric hindrance can take place. According to crystallographic studies, in the free sBBI molecule the S17 residue is involved in the intramolecular hydrogen bond with the Q21 residue, whereas a new hydrogen bond is likely formed between S17 (or S25 in the DII-type inhibitor) and S195 of trypsin upon interaction.[49,50] Thus, the reinforcement of the nucleophilicity of S17 (or S25) can be related to the changes in the microenvironment, especially to new hydrogen bond formation. Furthermore, it can be considered that the cooperativity effect appeared due to the presence of the neighboring SB between K16 (or R24 in the DII-type inhibitor)




419

Scheme 4. Modification of sBBI alone and sBBI within the non-covalent complex using CT+0 under (a) neutral and (b) slightly alkaline pH conditions with subsequent denaturation and digestion.

0.8 ± 0.2 (very low abundant ions) 1.7 ± 1.1 (very low abundant ions) Free sBBI at slightly alkaline pH sBBI/trypsin at slightly alkaline pH

h.m. – highly modified; m.m. – moderately modified; l.m. – low modified ion species *this table displays the peak intensity ratios for N-terminal peptide D1-K16 with one/two charge tag(s) and that of unmodified peptide; for homologous representation of data, the signal of the same peptide D1-K16 was used as reference for peptide D1-R23 with three charge tags

2.3 ± 1.1 m.m. sBBI/trypsin at neutral pH


Note: m/z 1732.6 – protonated unmodified peptide D1-K16; m/z 1887.6 – the same peptide with one CT+0; m/z 2042.8 – the same peptide with two CT+0; m/z 801.3 – protonated unmodified peptide S17-R23; m/z 956.4 – the same peptide with one CT+0; m/z 2980.3 – monocharged peptide D1-R23 with three CT+0; m/z 1365.5 – protonated unmodified peptide from DII type inhibitor; m/z 1520.6 – the same peptide with one CT+0

32 ± 13 h.m.

0.46 ± 0.28 l.m. 1.1 ± 0.3 l.m.

3.6 ± 1.3 m.m.

18.6 ± 1.7 h.m. 5.6 ± 2.5 m.m.

2.1 ± 0.9 (low abundant ions) m.m.

0.22 ± 0.9 l.m. 0.57 ± 0.22 l.m. 1.9 ± 0.8 m.m. Free sBBI at neutral pH

0.47 ± 0.12 (low abundant ions) l.m. 1.3 ± 0.5 (low abundant ions) l.m. 1.6 ± 0.4 (very low abundant ions) 90 ± 55 h.m.

0.22 ± 0.1 (low abundant ions) l.m. 0.29 ± 0.09 (low abundant ions) l.m. 380 ± 110 h.m.

I (m/z 1520.6)/I (m/z 1365.5) I (m/z 956.4)/I (m/z 801.3) I (m/z 1887.6)/I (m/z 1732.6)

I (m/z 2042.8)/I (m/z 1732.6)

I (m/z 2980.3)/I (m/z 1732.6)

Peptide S25-R36 from DII type inhibitor Peptide S17-R23 from sBBI Three charge tags * Two charge tags One charge tag

N-terminal peptide D1-K16 from sBBI

Modification conditions

420

Table 2. Semi-quantitative estimation of changes in reactivity of sBBI amino acid residues using the peak intensity ratios of CT+0-modified and unmodified monocharged peptides.

E. Darii et al. and D189 of trypsin. This effect can be enhanced in the case of a tighter SB, namely, if K is replaced by R. Consequently, this can explain why the modification level of the conserved serine residue was even higher in the case of the DII-type inhibitor (peak intensity ratio of modified/unmodified ion species approximately 75 for DII type inhibitor vs approximately 5 for sBBI, Table 2). Tight enzyme-inhibitor binding is accompanied by the changes in ionic and polar interactions and rearrangements of hydrogen bonds in the contact region. This results in remarkable enhancement of the reactivity of essential serine residues (particularly, for the DII-type inhibitor) even in the presence of steric hindrance. On the other hand, the degree of modification of K16 was lower within the complex compared to that for sBBI alone. The involvement of its ε-amino group in the SB with D189 of trypsin can affect the nucleophilicity of this residue. Therefore, its reactivity towards NHS ester derivatives can be decreased. Metastable dissociation studies In this part the results of metastable dissociation experiments are reported. The data were obtained using LIFT-TOF/TOF mode (i.e., PSD peptide sequencing).[60,61] The observed fragmentations are promoted by charge and take place due to residual internal energy distribution of precursor ion population. The positions of modified residues were explored by analyzing the fragmentation patterns of tagged and nontagged peptides. ’Sequence Editor’ software was employed to interpret metastable ion spectra. NHS ester reagents are commonly used for free amino group modification in solution. However, their reactivity towards protein hydroxyl groups has been also reported.[16,22,62] Thereby, the amino groups at the N-terminus and side chain of K residues as well as hydroxyl groups of S (and perhaps T and Y) residues can react with BS3 reagent and charge tag. Consequently, different possibilities should be explored to localize the introduced tag(s) and thus to obtain information on the interactive area. For this purpose, the simulated fragmentation patterns for peptides with the same structure but different modified residues were compared with experimental data. Then, the tag location giving the largest number of peaks assigned to PSD product ions (LNAP) was considered as the most probable. It should be noted that the concept of proton-driven fragmentation for peptides without a mobile proton reported by Bythell and al.[38,39] in detailed computational studies was particularly important for the following discussion. According to the authors, fragmentation pathways for these peptides still require the mobilization of protons which can be transferred to the amide nitrogen of the cleavage site. They involve the formation of SB, anhydride or imine-enol intermediates prior to dissociation and provide the same fragment ions as the classical oxazolone mechanism. The proton can be mobilized from the guanidinium group of the R residue, from the carboxylic group or from the amide bond. The proton transfer from the C-terminal carboxyl group to the R residue results in the SB formation and cyclization including the neighboring amide carbon atom. Consecutive fragmentation can proceed through a SB transition structure




Figure 4. Representative zones of MALDI mass spectra of sBBI-modified (a) alone and (b) within the non-covalent complex using CT+0 at slightly alkaline pH with subsequent denaturation and digestion. MALDI-MS analysis was performed using a mixture of analyte and saturated solution of matrix HCCA (see Experimental section). or an anhydride intermediate. The aspartic and glutamic acid residues[63] can be involved in the fragmentation processes via anhydride formation. In the case of amidic proton mobilization, the imine-enol intermediate is formed likely for the peptides without a free carboxyl group at the C-terminus. The following interpretation of experimental results is based on these considerations. Proline-containing peptide with lysine at the C-terminal position The precursor m/z 1732.6, 1888.7 and 1870.8 ions generated from the unmodified peptide D1-K16, the same peptide with a monolink BS3 modification and the same peptide probably carrying a loop link (mass increment of 138 Da), respectively, were analyzed in LIFT mode. The metastable ion spectrum of the precursor m/z 1732.6 ion matched well to the sequence of peptide D1-K16 from sBBI (Fig. 5(a), for more details, see Table S1, Supporting information). It was dominated by the fragment y10 ion (m/z 1071.1) generated by cleavage at the P7 residue due to currently observed ‘proline effect’[64,65] and displayed almost the complete yj ion series as expected by the presence of the C-terminal lysine residue, and the bi ion series (except for i = 1, 5, 8, 9) (Fig. 5(a)). The absence of b1 is in agreement with a classical oxazolone mechanism of bi ion formation.[45] Effect of the non-charged tag introduced by the BS3 reagent: one monolink or loop-link type BS3 modification on the peptide with K at the C-terminal position


One monolink type BS3 modification on the peptide with R at the C-terminal position The metastable ion spectrum of the m/z 801.3 ion of the unmodified peptide S17-R23 is shown in Fig. 6(a). The m/z 957.5 ion related to the modified peptide most likely carried



421

Different peak annotations of the metastable ion spectrum of the m/z 1888.7 ion corresponding to positions of the tag at the C-terminus, S4, S5, K6 and K16, were compared. The best correlation between experimental and theoretical data, i.e., LNAP, was found for modification at the N-terminus (annotation in Fig. 5(b)). This metastable ion spectrum was characterized by the same base peak (cleavage at P7 residue)

as that of the unmodified peptide and the similar yj ion series. The bi ion series was shifted by +156 Da (for more details, see Table S1, Supporting Information) as expected for modification at the N-terminus, and some bi fragment ions disappeared, namely, from b11 to b16. The peak at m/z 272 was annotated to the b1 product ion (Fig. 5(b)), while the same ion was missing in the case of the unmodified peptide. In fact, acylation of the N-terminal amino group can favor the formation of the b1 ions carrying modification[30] but hinders the other bi ion production. This is consistent with the higher reactivity of the N-terminal amino group towards acylation compared to other possible reactive sites.[16,22,29] Taken together, the metastable ion spectrum annotation based on the hypothesis of the tag position at D1 is consistent with the generally observed charge-directed fragmentation pathways of protonated peptides.[34,35,65] For the m/z 1870.8 ion different possibilities for link position have been considered. Most likely, an intralink was formed between D1 and K6. In fact, the high probability of loop-link formation between N-terminal amino groups and the nearest reactive residues has recently been reported.[16] The respective metastable ion spectrum with LNAP is shown in Fig. 5(c). It displays the yj ion series from y1 to y10 at the positions corresponding to theoretical values for unmodified species (Fig. 5(a)) and is dominated by the y10 fragment ion (cleavage at P7) as in the case of the unmodified peptide (Table S1, Supporting Information). On the other hand, fragment ions from y11 to y15 were absent and only weak signals attributed to the fragment b6 and b7 ions with a mass shift of 138 Da from theoretical values were detected. This confirms the presence of a cyclic crosslink between D1 and K6.

E. Darii et al.

Figure 5. Metastable ion spectra of ions related to unmodified N-terminal peptide D1-K16 and its BS3-modified forms with the annotation giving the LNAP: (a) protonated unmodified peptide (m/z 1732.6), (b) protonated peptide with one monolink type BS3 modification considered at D1 (m/z 1888.7), and (c) protonated peptide carrying intrapeptide cross-link considered between D1 and K6 (m/z 1870.8).

422

Figure 6. Metastable ion spectra of ions related to peptide S17-R23: (a) protonated unmodified peptide (m/z 801.3) and (b) protonated peptide with one monolink type BS3 modification considered at S17 (m/z 957.5).




Structural studies of the sBBI/trypsin non-covalent complex a monolink at S17. Actually, S17 is the only possible residue able to be acylated in the short peptide S17-R23. The corresponding metastable ion spectrum of the m/z 957 precursor ion with the peak annotation based on this hypothesis is shown in Fig. 6(b). The peaks of the fragment y1–y3 and y5 ions were not shifted, while the fragment b2 and b3 ions were shifted by 156 Da. The fragment b1 ion could be observed after modification with the same mass increment. The most intense peak (y5–17 ion) was generated by cleavage at the P19 residue. Fragmentation patterns for this charged peptide with the C-terminal R23 residue were characterized by the presence of the (yj–17) ion series which was not detected for peptide D1-K16. This suggests the specific ammonia loss from the guanidine moiety of the C-terminal R residue due to sequestered proton at this position.[34,39] One monolink type and one loop-link BS3 modification on the peptide with R at the C-terminal position Similar fragmentation patterns for the precursor m/z 2809.1 ion were observed in the experiments at neutral and slightly alkaline pH suggesting that the same residues were labeled in both cases. This ion probably corresponded to the peptide D1-R23 (Mw = 2514 Da) with a missed cleavage at K16 and carrying one monolink (mass shift of +156 Da) and one ’looplink’ (mass shift of +138 Da). Fragmentation patterns expected for the identical peptide sequence with different combinations of mono- and loop-link positions (at the N-terminal, K and S residues) were compared to experimental data. Finally, the best match between theoretical and experimental data, i.e., LNAP, was found in the case of monolink type modification at K16 and a loop-link between the N-terminal and K6 amino groups (Scheme 3). This is consistent with the enhanced reactivity of the N-terminal residue compared to other modifiable groups,

the preferential formation of a loop-link between the N-terminal amino group and the nearest modifiable residue,[16,22,29] and the inhibition of the cleavage after modified lysine residues.[66] Figure 7(a) shows the metastable ion spectrum of the m/z 2809.1 ion with the respective annotation (for more details, see Table S3, Supporting Information). The m/z values of the fragment yj ions from y1 to y7 were the same as for unmodified fragments, while the signals of yj ions from y8 to y17 were shifted by +156 Da. The fragment yj ions from y18 to y22 were absent as well as most of the bi ions except a very weak signal which could be attributed to the fragment b6 ion (Fig. 7(a)) with a mass shift of +138 Da from the theoretical value. This result is consistent with an internal cross-link between D1 and K6 and a monolink modification at K16 (Scheme 3). The y1 to y6 ion series was accompanied by the additional (yj–17) ion series suggesting the diagnostic loss of ammonia from the C-terminal R residue.[34] The y5 fragment ion (cleavage at the P19 residue) and y13 ion (cleavage between D10 and Q11) were the most abundant. Thus, in contrast to the shorter peptide D1-K16, the cleavage at the P7 residue was much less favored than after the D10 residue. This can be explained by the structural features of this internally cross-linked peptide. The charge is preferentially localized at the C-terminal R23 residue. This could be the reason for enhanced fragmentation before P19 and after D10 compared to fragmentation at P7. Effect of the fixed positive charge related to CT+0 modifications One charge tag on the peptide with K at the C-terminal position The most relevant peak annotation for the m/z 1887.6 ion corresponding to the modified N-terminal peptide is consistent with CT+0 at the D1 residue, as in the experiments




423

Figure 7. Metastable ion spectra of ions related to peptide D1-R23: (a) protonated peptide with intrapeptide BS3 cross-link considered between D1 and K6 and monolink modification considered at K16 (m/z 2809.1) and (b) protonated peptide D1-R23 with three CT+0 considered at D1, K6 and K16 (m/z 2980.3).

E. Darii et al. with BS3 (Fig. 8(a)). The m/z values of fragment ions from y1 to y6, and y10 were the same as for the unmodified peptide (Fig. 5(a), for more details, see Table S1, Supporting Information), while the signals of the bi fragment ions from b1 to b6, b9 and b10 were shifted by +155 Da from the theoretical values. Particularly, the peak annotated to the b1 fragment ion was detected, whereas the corresponding ion was absent in the case of the unmodified peptide. Fragmentation patterns of thw CT+0 modified peptide displayed an enhanced bi ion series, while some yj fragment ions were disfavored or disappeared. Especially, the b2 and b10 fragment ions generated by cleavage after D2 and D10 become the most abundant, while the formation of their complementary ions was disfavored. This could be due to the presence of a fixed positive charge at the N-terminus. In addition, the fragment y10 ion produced by cleavage at the P7 residue became less abundant than in the case of the unmodified and BS3modified peptides, whereas the formation of its complementary b6 ion was enhanced. Alternatively, for peptides carrying one charge tag the cleavage at D10 was preferentially observed. Finally, the comparison of metastable ion spectra (Figs. 5(a), 5(b) and 8(a)) indicates that fragmentation pathways are directed by the properties of the attached tag (i.e., charged or not). It has to be noted that, in addition, the loss of the 59 Da neutral related to trimethylamine from the end of the charge tag was observed under LIFT conditions. Two charge tags on the peptide with K at the C-terminal position The positions of charge tags in the twice modified monocharged peptide D1-K16 (m/z 2042.8) were explored in LIFT mode. The peak annotation that best fits the experimental data was obtained in the case of modification at the D1 and K6 residues (Fig. 8(b)). The m/z values of

fragment y1–y10 ions were the same as those of the unmodified peptide. The peaks annotated to fragment ions b1–b5 and y11–y15 were shifted by +155 Da relative to the theoretical values, whereas those of the b6 and b10 ions were shifted by +310 Da. As in the previous experiment, the b1 fragment ion appeared after modification with a mass increment of 155 Da. The majority of fragment ions showed a significant decrease in abundance due to the presence of two fixed positive charges. Only b1, b2 and b10 fragment ions emerged in this metastable ion spectrum, and the most abundant fragment ion was related to the loss of N(CH3)3 from CT+0. Three charge tags on the peptide with R at the C-terminal position Next, the metastable fragmentation in LIFT mode was employed to characterize the precursor ion at m/z 2980.3. In fact, a missed cleavage after K16 suggests the derivatization of this residue by CT+0 under slightly alkaline conditions. Two other possibilities for the location of modification were investigated. The annotation with LNAP was obtained in the case of the modification position on the D1 and the K6 residues. The respective metastable ion spectrum is illustrated in the Fig. 7b (for more details, see Table S3, Supporting Information). The presence of three charge tags caused a dramatic decrease in ion abundances compared to those displayed in the metastable spectrum of the same peptide modified by BS3 (Fig. 7(a)). However, despite the absolute lowering, the relative increase in abundance of the bi ion series over the yi ion series was observed. The fragment y1–y4 ions as well as their complementary b19–b22 ions were absent. The peaks annotated to fragment y5–y7 ion series matched the m/z values for unmodified peptide, while the peaks attributed to fragment y8–y10 ions were shifted by +155 Da as expected for the K16 residue modification. Furthermore, the peaks of fragment ions

424

Figure 8. Metastable ion spectra of ions related to CT+0 labeled peptide D1-K16: (a) monocharged peptide with one CT+0 considered at D1 (m/z 1887.6) and (b) monocharged peptide carrying two CT+0 considered at D1 and K6 (m/z 2042.8).




Structural studies of the sBBI/trypsin non-covalent complex from b1 to b3 and b5 with a mass shift of +155 Da and those attributed to b6 and b10 ions with a mass increment of 310 Da were present. This result supports a hypothesis that the modifications occurred at D1 and K6. Interestingly, in the case of the peptide D1-R23 with three charge tags (Fig. 7(b)), the b1 fragment ion was much more abundant than the b2 ion, whereas, for the shorter peptide D1-K16 with one charge tag at D1 (Fig. 8(a)), the b2 fragment ion was more abundant. In the case of the peptide with two charge tags (Fig. 8(b)) the abundances of these fragment ions were comparable. Apparently, the presence of three fixed charges hindered the fragmentation after D2. The peak intensities of the b10 and y13 fragment ions produced by cleavage after D10 were similar (Fig. 7(b)), while, in the case of the peptide D1-K16 with one charge tag, the b10 fragment ion was much more abundant than its complementary y6 ion (Fig. 8(a)). Indeed, a higher PA of the neutral fragment with R at the C-terminus compared to that for a fragment with K at this position resulted in enhanced formation of y13 from the b10/y13 ion pair (peptide D1-R23) compared to that of y6 from the b10/y6 ion pair (peptide D1-K16). Furthermore, the signals of the fragment b6 and b18/y5 ions formed by cleavage at P7 and P19, respectively, were low. Apparently, in the presence of fixed positive charges the fragmentation of peptide bonds at the N-terminal side of the P residues was less favored than those at the C-terminus to aspartic acid residues. This is in agreement with the commonly observed data[32,37,63] and can validate the chosen spectrum annotation. It has to be noted that some yi–17 fragment ions were detected in the metastable spectrum of the m/z 2980.3 ion, as in the case of the same peptide modified by BS3. The base peak corresponded to the loss of N(CH3)3 (59 Da) neutral fragment from the charge tag. According to experimental data, the following conclusions can be made: 1. Fragmentation patterns of unmodified tryptic peptides D1-K16 and S17-R23 display both bi and yj ion series and agree with the commonly accepted charge-directed fragmentation mechanism.[31,34–36] 2. In the case of tryptic peptides modified by BS3, the reinforced yj ion series was observed in the metastable spectrum due to the presence of basic residues (K and R) at their C-termini. In addition, the basicity of the N-terminal amide obtained by modification of the N-terminal amino group is decreased compared to amine. Consequently, the formation of bi ions was disfavored. The preferred cleavage at the N-terminus to proline residues is consistent with a charge-directed mechanism.[32,39,40,64,65] 3. The peptides modified by CT+0, namely, the peptide D1K16 with one tag (at D1) or two tags (at D1 and K6), and the peptide D1-R23 with three tags (at D1, K6 and K16), represent a reinforced b ion series due to the presence of a fixed positive charge at their N-termini.[38,39]


Monocharged peptide carrying one CT+0 In order to explain the generation of both bi and yj ion series, different structures for singly charged ions with one fixed positive charge have to be considered. First, it can be suggested that the monocharged ion exists in a canonical form with one fixed positive charge. In this case the experimentally observed fragmentation is difficult to rationalize in terms of a mobile proton model. However, the formation of bi/yj product ion series requires the free proton generation in an appropriate site despite the permanent charge of the quaternary ammonium group. It can be suggested that the charge tag assists the polarization of a spatially close amide bond due to a particular folded peptide conformation in the gas phase (Scheme 5). The resonance delocalization of a lone electron pair from nitrogen to the oxygen amide atom yields a protonated imine-enolate that is able to form a very stable SB with a permanent charge of the spatially neighboring charge tag. The mobilized amidic proton can migrate to another peptide bond and initiate its cleavage. In this way, the formed ion-dipole complex (Scheme 5(b)) dissociates giving the bi ion series (Scheme 5(c)). In addition, competitive intra-complex proton transfer can occur resulting in the generation of the yj ion series (Scheme 5(d)). The formation of the yj ions occurs via release of a neutral species carrying a SB formed between the quaternary ammonium group and the imine-enol moiety. In contrast, the bi product ions are generated with the neutral (yj–H) fragments in a canonical structure. Furthermore, the SB is maintained, and the positive charge remains at the oxazolone (or acylium) end-group (Scheme 5(c)). Alternatively, if the quaternary ammonium group is involved in a SB with a neighboring amide group, the possibility of zwitterion (ZW) formation at different positions

Scheme 5. Schematic presentation of (a) the amidic proton mobilization from the amide bond polarized by CT, (b) peptide bond cleavage via generation of an ion-dipole complex, (c) direct dissociation resulting in the bi ion formation, and (d) internal proton transfer giving rise to the yj ion series.



425

It should be noted that all these conclusions are based on the assumption that the spectrum annotation giving LNAP is the most relevant.

The role of salt bridges stabilizing tagged ions in the orientation of fragmentation

E. Darii et al. can be considered. The formation of zwitterions in the gas phase is more favored when one group is strongly acidic and the other is strongly basic. For example, for the D1(CT +0)D2E3SSK6PCCD10QCACTK16-tagged peptide, zwitterion (s) can appear through the protonation of the lysine (K6 or K16) residue and competitive deprotonation of any acidic residue (D1, D2, E3 or D10). It has been reported that peptide bond cleavage can be induced by a positively charged site as well as a deprotonated acidic residue.[38] In the case of the K6 residue protonation at its ε-amino group the proton can migrate to a proline amide bond and promote the K6-P7 bond cleavage yielding b6 and y10 complementary product ions. b6 was much more abundant due to the presence of a fixed positive charge at the N-terminus. In contrast, for the unmodified and the BS3-modified peptide the abundance ratio b6/y10 was reversed (Figs. 5(a), 5(b) and 8(a)). On the other hand, the negatively charged residue(s), namely, D1, D2, E3 and D10, induce the nearest amide bond cleavage yielding the competitive b1, b2, b3 and b10 (base peak) product ions, respectively (Scheme 6). This process involves the formation of the five- or six-membered cyclic anhydride and the complementary peptide from the ion-dipole intermediate system. The latter peptide can be protonated via intra-complex proton transfer. Therefore, the neutral ZW (bi–H) constituted by the deprotonated anhydride and quaternary ammonium group from CT+0 is formed. Interestingly, b10 is one of the most abundant products. This can be explained by the high probability of ZW formation with the participation of D10 and Q11 residues, because these residues are distant from the charge tag. The proximity to a permanent positive charge likely decreases the basicity of a protonation site. As a result, the ZW formation is disfavored. Consequently, the peptide bond cleavages assisted by proton mobilization via the formation of an SB involving a charge tag (at the N-terminus) are limited compared to those promoted by preformed zwitterions. Thus, the b10 ion must be generated from the zwitterion, whereas the b2 ion is produced by a classical process (Scheme 7). If both protonated and deprotonated sites of the ZW are not distant from the charge tag, strong Coulomb attraction between the quaternary amine at the N-terminus and the deprotonated carboxyl group takes place. This stable salt bridge ’clips’ the conformation as a non-covalent ring. The protonated site of the ZW can then induce the peptide fragmentation. However, after the opening of the ring at the position of the cleaved peptide bond, the two parts of the ring

remain strongly bound due to the presence of an SB. Thus, despite the metastable process, the system can survive unless consecutive dissociations occur resulting in generation of internal products. Finally, in all cases, the sequestered charge of the quaternary ammonium moiety is a spectator to the cleavages. This fixed charge assists a proton migration via SB formation and orients the cleavage towards the formation of bi fragment ions. In addition, if zwitterions pre-exist, the peptide bond cleavage is favored. However, zwitterions are more favored at a position distant from the charge tag. The bi ion series is reinforced when acidic residues are vicinal to the breaking bond due to the assistance of the negatively charged carboxylate group. Fragmentation of peptides with two or three charge tags The presence of two (or three) charge tags in singly charged peptides indicates that one (or more) negative charge(s) must be introduced, and the system is not really in a canonical form. Thus, in the case of one charge tag, the conformation might not be very rigid. However, in the presence of two charge tags, the conformation could be relatively frozen. Consequently, the probability of several ZW formations is reduced. Indeed, the involvement of two tags in salt bridges results in a steric hindrance for any proton transfer and hinders fragmentation. Therefore, the fragmentation patterns in Fig. 8(b) (peptide with two charge tags) were characterized by lowering of the ion abundance, in particular for b2 and b10 ions compared to the metastable spectrum with one charge tag (Fig. 8(a)). Furthermore, the b6 ion abundance (30% of the base peak [M–H–59]+) decreased to a few percent. Finally, in the presence of charge tags, fragmentation pathways are similar to those that can occur under non-mobile proton conditions and may be directed by the charge of the remote group (quaternary ammonium) through a SB formation due to the possible peptide chain bending. In the case of a peptide with one CT+0 at the N-terminus the peak of the b10 fragment ion was the most intense. It can be suggested that the competition between the formation and disruption of an SB (including different acidic residues and quaternary ammonium group) can take place. However, in the case of a peptide with two (or three) CT+0, at least one SB must be formed. This leads to an increase in the intensity of the b1 ion produced from the modified by charge tag D1 residue.

CONCLUSIONS Covalent modification using a BS3 cross-linker and a charge tag under different experimental conditions was applied to probe contact region within the non-covalent sBBI/trypsin

426

Scheme 6. Schematic presentation of (a) peptide bond cleavage assisted by nucleophilic attack of the carboxylate on the nitrogen atom of the protonated amide bond and (b) ion-dipole complex yielding competitive b10 (one of the most intense signals) and y6 (minor peak) product ions.


Scheme 7. Generation of the b2 fragment ion assisted by salt bridge and anhydride formation.



Structural studies of the sBBI/trypsin non-covalent complex complex. Comparative analysis of data using MALDI-MS and -MS/MS revealed significant changes in the reactivity of functionally important K16 and S17 amino acid residues in the sBBI molecule after the interaction with the enzyme. Particularly, the K16 residue remained partially unmodified within the complex. In contrast, the modification level of its neighboring S17 residue was reinforced, especially in the case of the DII-type inhibitor. It should be noted that no intermolecular linked species were observed in experiments with the BS3 cross-linker. Only peptides with a monolinktype modification and an internal cross-link were detected. The altered reactivity of amino acid residues involved in protein-protein interaction may reflect not only the decrease in their accessibility, but also the modulation of their nucleophilic properties due to changes in their microenvironment. This is likely related to the formation of new salt bridges and hydrogen bond systems. The metastable decomposition experiments performed under LIFT conditions were applied to locate the modification sites in labeled peptides. Clearly, the fragmentation patterns of differently modified peptides revealed the impact of modifications with or without fixed charge on the dissociation pathways. The abundances of bi/yj product ions were modulated. In particular, the effect of the C-terminal residue, namely, K or R, was counterbalanced by that of the charge tag at the N-terminus. Fragmentation yielding numerous bi/yj ions is promoted by proton transfer favored by preformed zwitterions. In the case of the modified peptide carrying only one positive charge the occurrence of zwitterions is not absolutely required for fragmentation due to relatively flexible conformation. The presence of n charge tags (n = 2 or 3) implies the appearance of (n–1) negative charges and the formation of (n–1) betain-like structures, i.e., salt bridges between the quaternary ammonium group of CT+0 and a negatively charged group. This prevents the further formation of zwitterions (including adjacent ionizable residues) important for fragmentation. Therefore, the fragmentation level became significantly altered when more than one charge tag was introduced.

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