3-Hydroxy-4-nitrobenzoic Acid as a MALDI Matrix for In-Source Decay

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3‑Hydroxy-4-nitrobenzoic Acid as a MALDI Matrix for In-Source Decay Yuko Fukuyama,*,† Shunsuke Izumi,‡ and Koichi Tanaka† †

Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu Corporation, 1, Nishinokyo Kuwabara-cho, Nakagyo-ku, Kyoto 604-8511, Japan ‡ Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8526, Japan S Supporting Information *

ABSTRACT: In-source decay (ISD) in matrix-assisted laser desorption/ionization (MALDI) is a rapid sequencing method for peptides. 1,5-Diaminonaphthalene (1,5-DAN) is a most frequently used matrix for ISD. However, using 1,5-DAN generates mainly c- and z-series ions by N−Cα bond cleavage, which makes it difficult to distinguish leucine (Leu) and isoleucine (Ile), and frequently lacks c(n−1)-series ions owing to proline (Pro) at residues n. Several oxidizing matrices generating aand x-series ions accompanied by d-series ions by Cα−C bond cleavage have been reported, but an issue remained concerning their sensitivity. 3-Hydroxy-4-nitrobenzoic acid (3H4NBA) has been reported as a matrix for 2-nitrobenzenesulfenyl-labeled peptides by Matsuo et al. (Proteomics 2006, 6, 2042−2049). Here, we used 3H4NBA as an oxidizing matrix for ISD. As a result, numerous a- and d-series ions for amyloid β 1−40 were generated with high sensitivity using 3H4NBA. Each of the two Leu and two Ile was identified by the d-series ions. The sensitivity of the a-series ions using 3H4NBA was a little lower than that of c-series ions using 1,5-DAN. The same tendency was observed for N-acetyl renin substrate and ACTH 18−39. The a-series ions were detected, even at the left side of Pro. The sensitivity of the a-series ions using 3H4NBA was higher than with other existing oxidizing matrices, such as 5-nitrosalicylic acid and 5-formyl salycilic acid. The ions were detected over the entire area of the matrix-analyte spot using 3H4NBA. 3H4NBA was confirmed to be a useful oxidizing matrix for ISD, leading to higher sequence coverage of peptides.

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FSA),19,20 4-nitrosalicylic acid (4-NSA),20 3-nitrosalicylic acid (3-NSA),20 or 2,5-bis(2-hydroxyethoxy)-7,7,8,8-tetracyanoquinodimethane (bisHE-TCNQ)21), which preferentially yields aand x-series ions accompanied by d-series ions by Cα-C bond cleavage. 5-FSA,9,10,19 2,5-DHB22 and 5-ASA22 were reported as a matrix yielding both c- and z-series ions, and a- and x-series ions. Reducing matrices 1,5-DAN and 2,5-DHB and oxidizing matrices 5-NSA and bisHE-TCNQ have been reported to yield ISD fragment ions with the highest sensitivity in each matrix group.9−11,21,23 Overall, 1,5-DAN has been reported as the most effective matrix for ISD,9,11,23 probably due to its exceptionally sensitive detection of fragment ions. However, there are some issues for 1,5-DAN as a reducing matrix for ISD. For example, leuicine (Leu) and isoleuicine (Ile) cannot be distinguished just by detecting c- and z-series ions. Leu/Ile differentiation using 1,5-DAN has been reported by the detection of d- or w-series ions using MALDI orthogonal TOFMS employed collisional cooling at elevated laser fluences24 and MALDI at low collision rate,25 but peak

atrix-assisted laser desorption/ionization (MALDI)1,2 and electrospray ionization (ESI)3 mass spectrometry (MS) have contributed to the analysis of peptides and proteins with advances in analytical techniques. In MALDI, collisioninduced dissociation (CID),4 postsource decay (PSD),5 and insource decay (ISD)6,7 have been used for fragmentation. PSD is not often used because of its lower intensity and it is timeconsuming for spectra acquisition.8 CID can efficiently cleave peptide bonds and give sequence information mainly based on b- and y-series ions. However, it is strongly affected by the peptide sequence and thus often yields incomplete sequence information.9 ISD enables rapid sequencing by detecting mainly c- and z-series ions, or a- and x-series ions with d-series ions, wherein the effect depends on the matrices.9,10 Matrices for ISD are mainly classified into two groups.9 One is a reducing matrix (e.g., 1,5-diaminonaphthalene (1,5DAN),11 2,5-dihydroxybenzoic acid (2,5-DHB),8,12−15 5aminosalicylic acid (5-ASA),16 5-amino-1-naphthol (5,1ANL),17 1,5-dihydroxylnaphthalene (1,5-DHN),17 2-aminobenzoic acid (2-AA),18 or 2-aminobenzamide (2-AB)18), which preferentially yields c- and z-series ions by N−Cα bond cleavage. The other is an oxidizing matrix (e.g., 5nitrosalicylic acid (5-NSA),19,20 5-formylsalicylic acid (5© XXXX American Chemical Society

Received: April 15, 2016 Accepted: July 17, 2016

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× 384 well, Shimadzu/Kratos, U.K.) for analysis using a MALDI time-of-flight mass spectrometer (TOFMS). MALDI-MS. MALDI TOFMS measurement was performed using an AXIMA Performance (Shimadzu/Kratos, U.K.) mass spectrometer equipped with a nitrogen UV laser (337 nm) in linear or reflectron positive-ion mode. Data was obtained using raster scanning with five shots of laser irradiation at each of the 1600 data points (at 40 × 40 lattice) in square regions of 1200 μm × 1200 μm ∼ 1700 μm × 1700 μm inside a matrix/analyte crystal.

intensity of the ions was not so high and the mass spectra consisting of plural ion species remain complex to analyze. In addition, the c(n−1)-series ions frequently are lacking owing to proline (Pro) at residues n.15 Oxidizing matrices such as 5-NSA solve the problems, but the sensitivity of ISD fragment ions are lower compared with reducing matrices such as 1,5-DAN. Sublimation of 1,5-DAN causes lower reproducibility and limits long-time measurement. Inhomogeneous matrix/analyte crystallization using 1,5-DAN results in lower reproducibility. Moreover, 1,5-DAN has been reported for carcinogenic property.26 Matsuo et al. reported 3-hydroxy-4-nitrobenzoic acid (3H4NBA) to be a matrix for selectively ionizing 2-nitrobenzenesulfenyl-labeled peptides.27 In this study, we use 3H4NBA as an oxidizing matrix for the ISD analyses. 3H4NBA is evaluated by comparison with 1,5-DAN and other matrices in MALDI-ISD analyses.

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RESULTS AND DISCUSSION ISD Analyses Using 3H4NBA. Figure 3 presents ISD spectra of amyloid β 1−40 using 3H4NBA or 1,5-DAN. Mainly a- and d-series ions were detected using 3H4NBA (Figure 3a), and c-series ions were detected using 1,5-DAN (Figure 3b). As shown in Figure 3a, c15-, c18-, and c27-ions were also detected using 3H4NBA. The mechanism for the formation of d-series ions from a-series ions has been reported by Asakawa et al.9,19,21 Generally, d-series ions provide structure information about side chains for amino acids, and thus d-series ions effectively distinguish Leu from Ile, since Leu and Ile have the same mass but a different structure. As a result, the two Leu and two Ile of amyloid β 1−40 were identified by the d-series ions (d17, d31, d32, and d34 ions) using 3H4NBA (Figure 3a). Differentiation between Leu and Ile was difficult using 1,5DAN (Figure 3b) because mainly c-series ions were detected. That is, the sequence coverage of amyloid β 1−40 was increased using 3H4NBA compared with 1,5-DAN. The sensitivity of a-series ions using 3H4NBA was a little lower than that of c-series ions using 1,5-DAN. Figure 4 presents ISD spectra of N-acetyl renin substrate using 3H4NBA or 1,5-DAN. As described above for amyloid β 1−40, a- and d-series ions were detected using 3H4NBA, and two Leu and one Ile were distinguished by the d-series ions (d5, d10, and d11 ions; Figure 4a,c). In the spectra, the c6 ion was absent using 1,5-DAN (Figure 4b), which corresponds to the report that c(n−1) ions are frequently lacking owing to Pro at residues n.15 On the other hand, a6 ions were detected using 3H4NBA (Figure 4a) to provide sequence information at residue 6. Thus, the a-series ions were more useful for obtaining peptide sequence information than the c-series ions. As a result, sequence coverage of an N-acetyl renin substrate was improved using 3H4NBA compared with 1,5-DAN. The influence of matrix clusters in low-mass areas was less using 3H4NBA than 1,5-DAN (Figure 4a,b). This improves sequence coverage close to the N-terminal. The resolution of the fragment ion peaks was higher using 3H4NBA than with 1,5DAN (Figure 4c,d). Leu/Ile differentiation has been carried out with other instrumental or analytical methods. Detection of w-series ions by multistage electron transfer dissociation (ETD) and higher energy collision activated dissociation (HCD) using Orbitrap fusion MS spectrometer28 resulted in high accuracy differentiation, but it needs time to perform HCD for each targeted z-series ion. Detection of d- or w-series ions using MALDI Fourier transform-ion cyclotron resonance (FTICR) mass spectrometer at intermediate source pressure,29 MALDI orthogonal TOFMS employed collisional cooling at elevated laser fluences using 2,5-DHB or 1,5-DAN,24 and MALDI at low collision rate using 1,5-DAN25 has been reported, but the mass spectra consisting of plural ion species remain complexity to analyze. An ISD analyses to reach is a rapid and simple peptides

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EXPERIMENTAL SECTION Materials. We synthesized 3H4NBA (Figure 1) by ourselves. 1,5-DAN (Figure 2), 5-NSA (Figure 2), and 5-FSA

Figure 1. 3-Hydroxy-4-nitrobenzoic acid (3H4NBA) as a novel matrix for ISD.

Figure 2. 1,5-Diaminonaphtalene (1,5-DAN), 5-nitrosalycilic acid (5NSA), and 5-formyl salycilic acid (5-FSA) as conventional matrices for ISD used in this study.

(Figure 2) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Amyloid β 1−40 was purchased from Peptide Institute, Inc. (Osaka, Japan). N-Acetyl renin substrate, ACTH 18−39, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, U.S.A.). Acetonitrile (CH3CN; LC/MS grade) was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Water used in all experiments was deionized using a Milli-Q ultrapure water purification system (Merck Ltd., Tokyo, Japan). All other chemicals were of analytical reagent grade. Preparation. Peptides were dissolved in water or CH3CN/ H2O/TFA (50:50:0.1, v/v) at 20 pmol/μL. The matrix solution was prepared in CH3CN/H2O/TFA (75:25:0.1, v/v) or CH3CN/H2O/TFA (50:50:0.1, v/v) at 10 mg/mL. The analyte solution (0.5 μL) and the matrix solution (0.5 μL) were mixed on a stainless-steel plate (sample plate 2.8 mm ring B

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Figure 3. ISD spectra of amyloid β 1−40 using (a) 3H4NBA and (b) 1,5-DAN in linear positive-ion mode.

neous, not a thin layer. Generally, thin homogeneous crystal formation results in high-resolution analyses with TOFMS. The dried spot using 3H4NBA was a relatively homogeneous minute thin granular crystal. In fact, ions were detected from almost the entire area of the matrix/analyte spot with comparatively higher peak resolution using 3H4NBA, whereas ions were detected from a partial “sweet-spot” of the matrix/ analyte spot with comparatively lower peak resolution using 1,5-DAN. Sublimation of 1,5-DAN is faster in a vacuum. This causes lower reproducibility and limits long-time measurement. However, sublimation for 3H4NBA is slower. Thus, 3H4NBA is effective for reproducibility or long-time measurement. The stability of 1,5-DAN in solution is lower due to oxidation. During oxidation, the color of the solution gradually changes from colorless or pale purple to black, and the sensitivity of the analyte ions is simultaneously reduced. In contract, the stability

sequencing including Leu/Ile differentiation, which is achieved by rapid measurement and simple ISD spectra. ISD analyses using 3H4NBA enable to detect sequential a- and d-series ions at once, leading to one of the ideal ISD analyses. 3H4NBA was also applied to top-down protein analyses. ISD of bovine serum albumin (BSA, 66 kDa) was performed using 3H4NBA in positive-ion mode. As a result, a-series ions were sequentially detected at m/z 5942.8 or less (Figure S-1 in Supporting Information). About 40 a-series ions (a9∼a51 ions) were detected. This result is comparable to the report in which only c-series ions (c9∼c49 ions) were detected using 1,5-DAN.11 Improvement of the ISD method for proteins remains to be solved. Figure 5 presents photo images of a matrix/analyte dried spot on a MALDI sample plate using 3H4NBA and 1,5-DAN. The dried spot using 1,5-DAN was a densely distributed, large needle crystal that was comparatively thick and inhomogeC

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Figure 4. ISD spectra of N-acetyl renin substrate at (a, b) m/z 600−950 and (c, d) m/z 1200−1530 using 3H4NBA (top) and 1,5-DAN (bottom) as matrices in reflectron positive-ion mode.

with about the same intensity using both 3H4NBA solutions. Although there are no cases actually using the matrix solutions prepared four months ago, higher stability of 3H4NBA leading to high reproducibility maximizes advantage of MALDI MS and ISD, that is, rapid and reproducible analyses. Toxicity of 3H4NBA has not been reported, but 1,5-DAN has been reported to contain carcinogens.26 A National Cancer Institute carcinogenesis technical report indicates that 1,5-DAN was carcinogenic in female Fischer 344 rats.26 The low toxicity of 3H4NBA enable it to be usable as a matrix for ISD. Figure 6 presents ISD spectra of ACTH 18−39 using 3H4NBA, 5-NSA, or 5-FSA as oxidizing matrices. As a result, numerous a- and d-series ions were detected with higher sensitivity using 3H4NBA than with other oxidizing matrices. Additionally, matrix cluster ion peaks in low-mass areas were suppressed using 3H4NBA. Chemical Structure of 3H4NBA for Cα−C Bond Cleavage. The structure of 3H4NBA (Figure 1) is similar to that of 5-NSA (Figure 2) as an oxidizing matrix. According to Asakawa et al., the NO2 group on 5-NSA has high affinity for NH groups from the amide portion of peptide backbone and accepts hydrogen from the NH group, providing a- and x-series ions.9 In this case, the NO2 groups work as hydrogen acceptors.9,10,19,30 Both 3H4NBA and 5-NSA possess NO2 groups, but their positional relation is different. OH groups on a matrix have been reported to work as hydrogen donors,10,15,16,31 while NO2 groups have been reported as hydrogen acceptors.10,19,30 A comparison between the positional relations of the OH group and the NO2 group of 3H4NBA or 5-NSA indicates that the relation of 3H4NBA, that is, the “3,4-relation” more effectively promoted highly sensitive detection of a- and d-series ions compared with that of 5NSA, that is, the “2,5-relation.” This fact is similar to the report

Figure 5. Photo of a matrix/analyte dried spot on a MALDI sample plate using (a) 3H4NBA and (b) 1,5-DAN.

of 3H4NBA in solution was comparatively high. The solution is a faint yellow color which is nearly unchanged, and the sensitivity of the analyte ions is retained for several weeks or more. In fact, we demonstrated comparison of MS and ISD spectra of amyloid β 1−40 between using 3H4NBA and 1,5DAN solutions prepared on the day or four months ago, that is, refrigerated for four months (Figure S-2 in Supporting Information). In MS spectra using 1,5-DAN, [M + H]+ was detected with about fiftieth sensitivity using the 1,5-DAN solution prepared four months ago (black color) compared with using the 1,5-DAN solution prepared on the day (colorless). Any ISD fragments were not detected using the 1,5-DAN prepared four months ago, although a lot of c-series ions were detected using the 1,5-DAN prepared on the day. On the other hand, in MS spectra using 3H4NBA, [M + H]+ was detected with approximately the same intensity using both 3H4NBA solutions (colorless) prepared on the day and four months ago. In ISD spectra, a lot of a-series ions were detected D

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Figure 6. ISD spectra of ACTH 18−39 using (a) 3H4NBA, (b) 5-NSA, or (c) 5-FSA in reflectron positive-ion mode.

considered essential to improve the sensitivity of ISD fragment ions.

by Nonami et al. in which the positional relation of an NH group and an N group of β-carboline, that is, the “1,4-relation” of the hydrogen donor and hydrogen acceptor, functions as a highly sensitive matrix for proteins, carbohydrates, and synthetic polymers.32 The “3,4-relation” (proximity) of the hydrogen donor and hydrogen acceptor in 3H4NBA is considered to improve the sensitivity of the ISD fragment ions. Moreover, according to Brown et al., OH groups on a matrix form hydrogen bonds with basic sites of the analytes and then contribute to an exothermic proton transfer.12−15 This report leads to the hypothesis that OH groups on 3H4NBA form hydrogen bonds with basic sites of the peptides and then contribute to an proton transfer between NO2 groups and the peptides to promote generation of a- and x-series ions accompanied by d-series ions using 3H4NBA. MS spectra for matrix ion peaks in low mass area were confirmed using 3H4NBA (Figure S-3 in Supporting Information). Molecular related ions of 3H4NBA were not clearly detected (too small to be detected) in positive-ion mode, and [M − H]− was detected in negative-ion mode. In this case, there is almost no difference between MS spectra of matrix with and without analytes, or in MS and ISD condition. As a result, it was difficult to obtain information about ISD mechanism using 3H4NBA from the matrix ion peaks. From the reports of 1,5-DAN, 2,5-DHB, 5-FSA, 5-ASA, and 5-NSA,9−15,19−25 the order of ion yield (sensitivity) of a-series ions is 5-NSA > 5-FSA > 5-ASA, 2,5-DHB > 1,5-DAN. 5-NSA, that is, a benzoic acid containing OH group and NO2 group in the “2,5-relation”, tends to provide a-series ions with higher sensitivity. In this study, comparison of 3H4NBA with 5-NSA demonstrated that a-series ions were detected with higher sensitivity in the “3,4-relation” of the OH group and the NO2 group than the “2,5-relation”. The positional relation is

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CONCLUSION 3H4NBA was reported as a novel ISD matrix. It provides a- and d-series ions by Cα−C bond cleavage with high sensitivity. The d-series ion production enables us to distinguish Leu and Ile, and a-series ion production, even at the left side of Pro, enables us to improve the sequence coverage of peptides. Having fewer matrix cluster ions improves the sequence coverage close to the N-terminal. The matrix/analyte crystal is comparatively thin and homogeneous, leading to rapid analysis and high mass resolution using a TOF mass spectrometer. Toxicity of 3H4NBA has not been reported. The slow sublimation and high stability in the solution for 3H4NBA were also confirmed. 3H4NBA thus solved almost all of the problems with 1,5-DAN and is expected to contribute to rapid and simple ISD analyses in MALDI.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01471.

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Supporting Figures S-1, S-2, and S-3 (PDF).

AUTHOR INFORMATION

Corresponding Author

*Phone: +81-75-823-2897. Fax: +81-75-823-2900. E-mail: [email protected]. Notes

The authors declare no competing financial interest. E

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