In‐source decay of Boc‐carbo‐ β 3 ‐peptides in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (2005)

Over the past decade there has been growing interest in β-amino acids and their derivatives1 which are relevant intermediates for the syntheses of compounds of pharmaceutical potential and are important constituents of natural products such as alkaloids, peptides and beta-lactam antibiotics.2, 3 The presence of β-amino acids in place of α-amino acids in the naturally occurring pharmaceutically active peptides is known to increase the activity and the stability of the peptides.4-6 Synthetic oligomers of β-amino acids, i.e., β-peptides, are among the most studied class of molecules in foldamer chemistry.7, 8 Changes in their substitution pattern generate a variety of interesting structural features in these molecules,9-11 which have thus begun to prove immensely useful in bioactive peptide mimicry.12 In-source decay (ISD) combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry has been established as a useful method for sequencing peptides and proteins.13-22 One of the main advantages of ISD, compared with collision-induced dissociation (CID)23 and post-source decay (PSD),24 is that the amino acid sequences of proteins can be determined without predigestion.25 ISD is a convenient method for the assignment of product ions from proteins and α-peptides because ISD gives cn, bn, an, yn, and zn series ions16, 21, 22 (using the nomenclature of Roepstorff and Fohlman26 and Biemann27). The product ions produced by loss of NH3 or H2O from yn and/or bn series ions, as is observed with PSD,24 are absent from ISD spectra. While ISD is reported to be a better method for much larger peptides and even proteins,13 the yield of fragmentation in ISD is generally lower for smaller peptides22 because the low m/z region is obscured by matrix clusters. Takayama and Tsugita reported the influence of the acidic and basic character of amino acid residues on the peptide fragment ions produced by ISD under MALDI conditions.16 It was shown that peptides containing basic arginine and/or lysine residues near the N-terminus produce cn and an series ions in positive-ion mode, whereas a peptide that has an acidic amino acid cluster near the N-terminus and a basic residue near the C-terminus formed yn and zn series ions. There have been several reports on the mechanism of the ISD process.14, 17, 20, 21, 25 Recently, Kocher et al. have investigated the mechanism of ISD from the viewpoint of a comparison between electron-capture dissociation (ECD) and ISD.22 Based on their studies using H/D exchange experiments, they have shown that ISD involves radical reactions, and that cleavage in ISD is mediated by hydrogen radicals (H.) generated photochemically from the MALDI matrix (this work22 used 2,5-dihydroxybenzoic acid). Recently, we reported a mass spectral study of a series of new Boc-carbo-β3-peptides using liquid secondary ion mass spectrometry (LSI-MS), electrospray ionization (ESI) and tandem mass spectrometry (MS/MS).28, 29 In the present study, we report a mass spectral study of these peptides using ISD-MALDI. As there are no reports on metallation studies under ISD conditions, we have also studied the interactions of the peptides with alkali metal ions and selected transition-metal ions (Cu2+, Ag+, Fe2+ and Zn2+) of biological importance. The MALDI mass spectra were recorded using a Kompact MALDI SEQ laser desorption time-of-flight (TOF) mass spectrometer (Kratos Analytical, Manchester, UK) equipped with a pulsed nitrogen laser (λmax 337 nm, pulse width 3 ns). As per the manufacturer's specifications the maximum laser power is set at an arbitrary value of 180. The spectra were acquired in positive-ion mode. Ions were accelerated into the TOF analyzer at a voltage of 20 kV. The sample spots were irradiated just above the threshold, using linear mode with delayed extraction. For ISD experiments the laser fluence was raised to 10% above threshold. Thus, the laser power (irradiance) used to produce a good ISD mass spectrum was analyte-dependent. All the mass spectra shown in this paper were accumulated using 50 laser shots across the sample spot. The matrix materials, α-cyano-4-hydroxycinnamic acid (CHCA), 2,5-dihydroxybenzoic acid (2,5-DHB), and nor-harmane, and the trifluoroacetic acid (TFA) and metal salts (chlorides) used in this study were purchased from Sigma-Aldrich Chemie, GmbH & Co. (Steinheim, Germany). Matrix solutions of 10 mg/mL concentration were prepared in acetonitrile/0.1% TFA-water (70:30 v/v). Analyte solutions of 200 pmol/µL concentration were prepared in methanol/water (60:40 v/v). Methanol and water used were commercially available and of HPLC grade, and were obtained from SD Fine Chemicals (Mumbai, India). For cationization experiments, alkali metal and transition-metal salt solutions were prepared in deionized water (10 mg/mL). Equal volumes of matrix and salt solution were mixed in a separate vial. To make an analyte/matrix deposit, typically 0.5 µL of the analyte solution was placed on a stainless steel sample slide (part no. D 3665 TA, Kratos Instruments) and the solvent was removed by forced air evaporation at room temperature. Subsequently, 0.5 µL of the matrix solution was placed above the sample spot covering the analyte film, and again the solvent was removed by forced air evaporation at room temperature. In case of cationization studies, 0.5 µL of matrix/salt mixture was placed above the sample spot covering the analyte film. The syntheses of the peptides studied in this work have recently been reported by some of us.30, 31 All the peptides were prepared from corresponding monomers by conventional methods using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole hydrate (HOBt) in dichloromethane (dimethylformamide for penta- and hexa-peptides). The Boc-carbo-β3-peptide esters (1–6) and acids (7–12) contain30, 31 one to six C-linked carbo-β3-amino ester and acid residues with S-configuration at the amine center (Scheme 1). We used three different matrices, viz., CHCA, 2,5-DHB and nor-harmane, to study the ISD of these peptides. Of these only CHCA was found to yield reasonably good spectra even with low laser power, whereas the other two matrices did not give good spectra even at high laser power. The relatively low proton affinity (PA) of CHCA (PA = 201 kcal mol−1) seems to be one of the main reasons why it is a hot matrix for these peptides.32, 33 Boc-carbo-β3-peptide esters and acids 1–12. The ISD mass spectra of the monomeric amino ester and acid (1 and 7) show abundant [M+H-Boc+H]+, [M+Na]+ and [M+K]+ ions, with the [M+H]+ ion totally absent. The absence of the [M+H]+ ion may perhaps be due to its fast dissociation leading to elimination of the Boc group. The formation of the [M+H-Boc+H]+ ion can be rationalized in terms of a McLafferty rearrangement involving a hydrogen migration from one of methyl groups of the tert-butyl group to the carbonyl oxygen in the Boc moiety,34 followed by the loss of 2-methylprop-1-ene and subsequent loss of CO2. This is similar to the behavior observed in the LSI and ESI mass spectra of these peptides.28 The di-peptide ester exhibits a simple spectrum with the [M+H-Boc+H]+ ion (m/z 519) as the most intense peak. The spectrum also shows abundant [M+Na]+ and relatively less abundant [M+K]+, [M+Na-C(CH3)3+H]+ and [M+H-Boc+H-acetone]+ (m/z 461) ions. A low-abundance peak at m/z 276, corresponding to the y ion, is observed in the spectrum. The tri-peptide ester also yields an intense [M+H-Boc+H]+ ion at m/z 762 and abundant [M+Na]+ and [M+Na-Boc+H]+ ions and low-abundance [M+K]+, [M+Na-C(CH3)3+H]+ and [M+H-C(CH3)3+H]+ ions. The spectrum also displays low-abundance y, y (m/z 519) and b (m/z 487) ions. The ISD spectrum of the tetra-peptide ester exhibits abundant [M+Na]+, [M+Na-Boc+H]+ and [M+H-Boc+H]+ ions and low-abundance [M+K]+, [M+K-Boc+H]+ and [M+K-C(CH3)3+H]+ ions. The peptide backbone fragment ions that are present in the spectrum are y, y, y (m/z 762) and b (m/z 730) ions. The penta-peptide gives abundant [M+Na]+ and [M+H-Boc+H]+ (m/z 1248) ions, and the low-abundance ions are [M+K]+, [M+K-C(CH3)3+H]+, [M+Na-C(CH3)3+H]+, [M+K-Boc+H]+, [M+Na-Boc+H]+ and [M+H-Boc-acetone]+. The C-terminal ions observed are y (m/z 1005), y, y and y, and the only N-terminal ion is b at m/z 973. The hexa-peptide ester also shows abundant [M+K]+ (m/z 1630), [M+Na]+ (m/z 1614) and [M+H-Boc+H]+ (m/z 1492) ions; the relatively low-abundance ions are [M+K-C(CH3)3+H]+, [M+Na-C(CH3)3+H]+, [M+K-Boc+H]+, [M+Na-Boc+H]+ and [M+H-Boc+H-acetone]+. The spectrum also displays C-terminal (y to y) and N-terminal (b to b) ions (Fig. 1). Fragmentations characteristic of the side-chain carbohydrate are absent in the spectra of these peptides. Linear positive-ion MALDI-TOF-ISD spectrum of the hexa-peptide ester 6 obtained with CHCA as matrix, utilizing laser power = 60 (33% of maximum arbitrary units 180). The peptide acids 7–12 show similar ions, but low intensities as compared to those of the corresponding esters. This may perhaps be due to the formation of salts with metal ions which probably reduce the volatility of the peptide acids.35 We note that the results observed for the peptide esters and acids are similar to those obtained in the LSI-MS and ESI-MS/MS analyses of [M+H-Boc+H]+, which showed mainly y and b sequence ions. However, these ISD spectra are much weaker compared to LSI-MS and ESI-MS/MS spectra. We also note that the appearance of only y and b ions is in contrast with the formation of c and z ions from α-peptides under ISD-MALDI with 2,5-DHB as a matrix.16, 21, 22 The absence of c and z ions may perhaps be due to the presence of a carbohydrate moiety in these peptides at the carbon adjacent to the amine group which is the cleavage point leading to c and z ions. In order to examine the interactions of these peptides with alkali metal ions and a selected group of transition-metal ions, we examined the ISD-MALDI spectra of the peptides with added metal ions. All the alkali metal ions bind strongly with the peptides (1–12), which is reflected in the abundant [M+Cat]+ ions (Cat = Li, Na, K, Rb, Cs) in the spectra. Representative spectra of the alkali-cationized hexa-peptide are given in Fig. 2. The peptides also yield moderately abundant [M+Cat-C(CH3)3+H]+, [M+Cat-Boc+H]+ and [M+H-Boc+H]+ ions, except that the [M+H-Boc+H]+ ion is totally absent in case of cationization with Li. This indicates that the affinity of Li+ towards the peptides is much higher than that of the other alkali metal ions. Surprisingly, the spectra do not exhibit any metallated sequence ions, which is in contrast with the behavior of these peptides under LSI and ESI conditions.29 However, low-abundance free y and b ions are observed in the cases of tetra- to hexa-peptides. The lower abundance of y and b ions may be due to the dominance of [M+Cat]+ and [M+Cat-Boc+H]+ ions, which in turn can be ascribed to the presence of carbohydrate moieties in these peptides. Linear positive-ion MALDI-TOF-ISD spectra of the hexa-peptide ester 6 doped with (a) Li+, (b) Na+, (c) K+, (d) Rb+, and (e) Cs+. The formation of [M+X-Boc+H]+ (where X = H, Cat) y and b ions may be rationalized in two ways:16 (i) the fragment ions can originate from intact ionized molecules such as [M+X]+ and [M+X-Boc+H]+ within the residence time after the ionization event, and (ii) the intact ionized molecules and fragment ions can be formed independently, i.e., neutral fragments are produced directly from neutral analytes via energy transfer from matrix molecules activated by laser irradiation, and the neutral fragments can be ionized subsequently. Similarly to the alkali metal ions, the selected group of transition-metal ions, Cu2+, Fe2+ and Ag+, also form stable complexes with these peptides, which is again reflected in the abundant metallated molecules (Fig. 3). However, Zn2+ does not form a stable complex, which may be due to the absence of basic amino acid residues in these peptides.36 In the case of metallation with Ag+, the [M+H-Boc+H]+ ion is insignificant, suggesting that the complex formed by Ag+ may be more stable than those of Cu2+ and Fe2+. The low abundance of the [M-H+Fe]+ ion suggests the formation of a relatively weak complex between Fe2+ and peptides. This is evident from the presence of y and b ions which could arise from the [M+H-Boc+H]+ ions (Fig. 3(b)). It is also noteworthy that, when Cu2+ is used, the intensity of metallated molecule ions is enhanced; this is consistent with a recent report by Zenobi et al.37 that CuCl2 acts as an electron scavenger through its reduction from Cu2+ to Cu+, which leads to the enhancement of analyte signal in the ISD spectra. Absence of cationized y and b ions suggests that, under these ISD conditions, the complexes formed are highly stable and do not easily dissociate. Linear positive-ion MALDI-TOF-ISD spectra of the hexa-peptide ester 6 doped with (a) Cu2+, (b) Fe2+, and (c) Ag+. In conclusion, ISD-MALDI of Boc-carbo-β3-peptides produces abundant ionized molecules like [M+Cat]+, [M+Cat-Boc+H]+ and [M+H-Boc+H]+ and low-abundance y and b ions. Except for low ion yields, these results are similar to those obtained in the LSI-MS and ESI-MS/MS analyses of [M+H-Boc+H]+ ions. The absence of c and z ions, which is a common feature in ISD-MALDI spectra of normal peptides, may be due to the presence of a carbohydrate moiety adjacent to the amine carbon. Metallation studies with alkali and selected transition metals also produce only ionized molecules and low-abundance y and b ions. The absence of [M+H-Boc+H]+ ions with Li+ and Ag+ indicates that these metals form stable complexes with this class of peptides. From the present study we feel it is difficult to comment on the involvement of the H-atoms in the ISD process as was suggested previously.22 For such mechanistic studies we may require peptides with a minimal number of exchangeable hydrogen atoms. Also in the present study we did not observe cn and zn ions which are reported to be generated via H-atom-induced reactions. With 2,5-DHB (the matrix used previously22) we could not obtain meaningful spectra for the present compounds, and the CHCA matrix which was used in the present study is known to participate mainly in proton-transfer reactions. The authors thank Dr. J. S. Yadav, Director, IICT, Hyderabad, for facilities and Dr. M. Vairamani for cooperation. R.S., P.N.R. and V.S. are grateful to CSIR, New Delhi, for financial support. R. Srikanth*, N. Prasada Raju*, P. Nagi Reddy*, R. Srinivas [email protected]*, G. V. M. Sharma , V. Subash , Palakodety Radha Krishna , * National Centre for Mass Spectrometry, Indian Institute of Chemical Technology, Hyderabad – 500007, India, Discovery Laboratory, Organic Chemistry Division III, Indian Institute of Chemical Technology, Hyderabad – 500007, India.