Alkylated Trihydroxyacetophenone as a MALDI Matrix for Hydrophobic

Technical Note pubs.acs.org/ac

Alkylated Trihydroxyacetophenone as a MALDI Matrix for Hydrophobic Peptides Yuko Fukuyama,*,† Chihiro Nakajima,† Keiko Furuichi,‡ Kenichi Taniguchi,† Shin-ichirou Kawabata,† Shunsuke Izumi,‡ and Koichi Tanaka† †

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, 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: Hydrophobic peptides are difficult to detect in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), because of the hydrophilic properties of conventional matrices and the low affinity for hydrophobic peptides. Recently, we reported on alkylated dihydroxybenzoic acid (ADHB) as a matrix additive for hydrophobic peptides; however, the peptides were detected in the rim of the matrix-analyte dried spot. Here, we report on a novel matrix, alkylated trihydroxyacetophenone (ATHAP), which is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. ATHAP increased the sensitivity of hydrophobic peptides 10-fold compared with α-cyano-4-hydroxycinnamic acid (CHCA), in which the detection of hydrophilic peptides was suppressed. The peptides were detected throughout the entire matrix-analyte dried spot using ATHAP, overcoming the difficulty of finding a “sweet spot” when using ADHB. In addition, ATHAP functioned alone as a matrix, unlike ADHB as an additive. In phosphorylase b digests analysis, hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix, confirming that ATHAP led to increased sequence coverage and may extend the range of target analytes in MALDI-MS.

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atrix-assisted laser desorption/ionization (MALDI)1,2 and electrospray ionization3 mass spectrometry (MS) have progressed in sensitivity and accuracy for proteomics.4,5 However, MS is often problematic in the analysis of hydrophobic peptides.6−12 In general, the majority of cancer markers are glycoproteins including hydrophilic and hydrophobic regions.13−15 However, hydrophobic peptides are readily excluded from target analytes, because of difficulty in their detection. The difficulty in detecting hydrophobic peptides in MALDIMS is probably due to the feature of MALDI optimized for hydrophilic compounds.6 Conventional MALDI matrices have hydrophilic properties and, thus, a low affinity for hydrophobic peptides. Commonly, a matrix must have an affinity for the target analytes. Hence, we undertook the development of matrices that have an affinity for hydrophobic peptides. Recently, we reported on a novel matrix additive, O-alkylated dihydroxybenzoic acid (ADHB), for hydrophobic peptides.16 ADHB is a 2,5-dihydroxybenzoic acid derivative incorporating a hydrophobic alkyl chain on the hydroxyl group, which has an affinity for hydrophobic peptides. Adding ADHB to CHCA improved the sensitivity of hydrophobic peptides by 10-fold to 100-fold. This sensitivity improvement resulted from the enrichment of hydrophobic peptides in the rim of a matrixanalyte dried spot. © 2013 American Chemical Society

However, the following three issues remain with ADHB. (I) Hydrophobic peptides were detected in the rim of the matrix-analyte dried spot, and finding the “sweet spot” was difficult. (II) ADHB was an additive and, thus, was unavailable without conventional matrices. (III) Hydrophilic peptides ions were also detected; therefore, the detection of hydrophobic peptides ions may have been limited. To resolve these issues, we launched a study to develop a novel matrix for hydrophobic peptides. In this study, 1-(2,4,6trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) was synthesized to improve hydrophobic peptide analysis. ATHAP is a 2,4,6-trihydroxyacetophenone derivative incorporating a hydrophobic alkyl chain on the acetyl group and thus is expected to have an affinity for hydrophobic peptides. Distribution of the analytes on the matrix-analyte dried spot is confirmed using MS imaging. A systematic study on sensitivity for hydrophobic and hydrophilic peptides is Received: June 18, 2013 Accepted: September 5, 2013 Published: September 24, 2013 9444

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Analytical Chemistry

Technical Note

After 60 min incubation at room temperature in darkness, the gel piece was washed and dried in a vacuum centrifuge. The gel piece was then rehydrated with 50 μL of 50 mM NH4HCO3 containing 800 ng of Lys-C and incubated at 37 °C overnight. The obtained digest solution (2 pmol/μL) was used as an analyte solution for MALDI-MS. Sample Preparation for MALDI-MS. Peptides or protein digests were dissolved in 50% ACN/0.1% aqueous TFA (v/v) at appropriate concentrations. ATHAP solution was prepared in 75% ACN/0.1% aqueous TFA (v/v) at 5 mg/mL. CHCA or THAP solution was prepared in 50% ACN/0.1% aqueous TFA (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 × 384 well, Shimadzu/Kratos, UK) to be analyzed by matrix-assisted laser desorption ionization, coupled with time-of-flight mass spectroscopy (MALDITOFMS). 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 positive-ion mode (for peptides) or reflectron positive ion mode (for protein digests). All MS data was obtained by raster scanning with five shots of laser irradiation at each of the 400 data points in square regions of 1000 μm × 1000 μm with a 53 μm measurement pitch (i.e., 20 × 20 lattice), using ATHAP or CHCA. MS imaging was carried out by BioMap software (copyrighted by Novartis, available at http://maldi-msi.org/). All MS images were constructed from raster-scanned data sets comprised of two shots of laser irradiation at each of the 6561 data points in square regions of 4000 μm × 4000 μm with 50 μm measurement pitch (i.e., 81 × 81 lattice). The detection limit was defined as the lowest quantity of analyte detected as [M + H]+ with a signal-to-noise ratio (S/N) of ≥2 when each series of analyte solution at different concentrations (0.01 to 10000 fmol) made by 10-fold serial dilutions was evaluated. Sensitivity improvement was determined by dividing the detection limit using CHCA by that using ATHAP. SSRCalc Hydrophobicity for peptides was calculated by using SequenceSpecific Retention Calculator software (copyrighted by the Manitoba Centre for Proteomics and Systems Biology, available at http://hs2.proteome.ca/SSRCalc/SSRCalcX.html). The HPLC Index was calculated using the Sequence Calculator software functionality supplied by the MALDI-TOFMS instrument manufacturer.

performed using ATHAP compared with CHCA, and sensitivity improvement with ATHAP is discussed.

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EXPERIMENTAL SECTION Materials. 1-(2,4,6-Trihydroxyphenyl)octan-1-one (ATHAP) as a novel matrix was synthesized as described in the following section. α-Cyano-4-hydroxycinnamic acid (CHCA) was purchased from LaserBio Laboratories (SophiaAntipolis Technopole, France). 2,4,6-Trihydroxyacetophenone (THAP), ACTH 18-39, phosphorylase b from rabbit muscle, iodoacetamide (IAA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). NF-κB inhibitor SN50, OVA-BIP hybrid peptide, βamyloid 22-42, β-amyloid 1-11, and β-conglycinin hydrolysate 165-178 FAS inhibitor thioesterase antagonist soy were obtained from AnaSpec, Inc. (San Jose, CA). Humanin, catestatin, neuropeptide S, nocistatin, and β-amyloid 1-16 were purchased from Peptide Institute, Inc. (Osaka, Japan). Lysyl endopeptidase, (Lys-C, mass spectrometry grade), trifluoroacetic acid (TFA, HPLC grade), and acetonitrile (ACN) were 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. Synthesis of 1-(2,4,6-trihydroxyphenyl)octan-1-one (ATHAP, C8-ATHAP). ATHAP incorporating an octanoyl group (Figure 1) was synthesized as follows. 1,3,5-Trihydrox-

Figure 1. 1-(2,4,6-Trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone, ATHAP) as a novel matrix.

ybenzene (6.1 g, 0.048 mol) and anhydrous aluminum chloride (20 g) in a mixture of nitrobenzene (15 mL) and carbon disulfide (25 mL) were treated with octanoyl chloride (6.9 g, 0.042 mol) in nitrobenzene (7 mL). The reaction mixture was synthesized by Friedel−Crafts reaction in accordance with the previous report17 to obtain ATHAP (5.4 g, 50%) as pale yellow crystal powder: 1H NMR (400 MHz, CDCl3): δ = 5.83 (s, 2H), 1.61 (quint, J = 7.2 Hz, 2H), 1.60 (quint, J = 7.2 Hz, 2H), 1.27 (m, 4H), 0.86 (t, J = 7.2 Hz, 3H); 13C NMR (125 MHz, DMSO): δ = 205.2, 164.4, 164.1, 103.7, 94.6, 43.0, 31.1, 28.7, 28.6, 22.0, 13.9. High-resolution mass spectrometry: Calculated for C14H21O4+ [M + H]+ 253.144; Found. 253.144. Lys-C In-Gel Digestion of Phosphorylase b. Phosphorylase b (100 pmol) was separated by SDS-PAGE using miniquickgel (Anatech Co., Ltd., Tokyo, Japan) at 25 mA for 90 min. The coomassie-stained protein band with CBB Stain One (Nacalai Tesque, Inc., Kyoto, Japan) was excised and washed with water. The washed gel piece was dehydrated with ACN and dried in a vacuum centrifuge. Then 50 μL of 20 mM aqueous TCEP was added to the gel in a small plastic tube in order to reduce disulfide bonds, and the mixture was incubated at 37 °C for 60 min. S-Alkylation was performed by replacing the TCEP solution with 20 mM IAA in 100 mM NH4HCO3.

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RESULTS AND DISCUSSION Optimal Chain Length of the Acyl Group of ATHAP. Comparative evaluation of different acyl group chain lengths (C6, C8, C10, or C12 acyl group) in the ATHAP structure indicated that the octanoyl (C8) or decanoyl (C10) group was the most effective for sensitivity improvement for hydrophobic peptide humanin (HPLC Index 117.4, SSRCalc Hydrophobicity 50.0) (see Table S-1 in the Supporting Information). The peptide was detected at 1 fmol/well using ATHAP incorporating the octanoyl group (C8-ATHAP) or decanoyl group (C10-ATHAP), which corresponds to 10-fold sensitivity improvement over that using THAP or CHCA. C8-ATHAP was used in all experiments described below. Analysis of Hydrophobic and Hydrophilic Peptide Mixture Using ATHAP. An equimolar mixture of hydrophobic and hydrophilic peptides (humanin with HPLC Index 117.4, SSRCalc Hydrophobicity 50.0, and β-amyloid 1-11 with HPLC Index 1.4, SSRCalc Hydrophobicity 13.5) was analyzed using 9445

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Analytical Chemistry

Technical Note

enriched hydrophobic peptides in the rim of the matrix-analyte dried spot. Sensitivity improvement by ATHAP is from the matrix itself, unlike ADHB, which depends on the enrichment effect. Based on these results, raster scanning was used in all of the experiments in this study. Sensitivity Improvement by ATHAP and Peptide Hydrophobicity. Sensitivity for peptides with a range of Hydrophobicity (SSRCalc Hydrophobicity of 5.2 to 54.8, HPLC Index of −60.2 to 200.0) was evaluated using ATHAP or CHCA. The HPLC Index is a Hydrophobicity scale based on predictive retention coefficients of peptides by reverse-phase high-performance liquid chromatography (RP-HPLC), which takes into account amino acid composition.18−20 It has been reported to correlate with sensitivity improvement of hydrophobic peptides by ADHB.16 SSRCalc Hydrophobicity is based on a sequence-specific algorithm for peptide retention prediction in RP-HPLC, which takes into account amino acid composition, position of amino acid residues, peptide length, and three-dimensional (3D) conformation.21−23 Table 1 indicates the sensitivity improvement rate by ATHAP compared with that by CHCA, which was calculated by dividing the detection limit for peptides using CHCA by that using ATHAP. ATHAP improved the sensitivity of hydrophobic peptides with SSRCalc Hydrophobicity of 42.4 or higher (analytes 1−4 in Table 1) 10-fold (sensitivity improvement rates of 10). However, ATHAP decreased sensitivity (sensitivity improvement rates of THAP.24−26 CHCA coordinates metal ions by the carboxyl oxygens in addition to the hydroxyl oxygens. Although THAP does not possess a carboxylic acid group, it does form a chelate complex with metal ions by the carbonyl and hydroxyl oxygens. In contrast, ATHAP does not possess a carboxylic group and has difficulty forming a chelate complex with metal ions between hydroxyl oxygens and a hydrophobic alkyl group. The result may be inhibition of sodium or potassium ion transfer from matrix to analytes in a plume. In addition, ATHAP is hydrophobic, thus has a low affinity for hydrophilic metal ions. These ideas are hypotheses that must be clarified. Analysis of Phosphorylase b Lys-C Digests. Phosphorylase b Lys-C digests were analyzed using ATHAP or CHCA to confirm sensitivity improvement for hydrophobic peptides in digests. Figure 3 presents the mass spectra of 1 pmol

Table 2. Correlation of the Ion Detection for Phosphorylase B Lys-C Digests and SSRCalc Hydrophobicity Using ATHAP or CHCAa phosphorylase b Lys-C Digests

Detection (±)

no.

SSRCalc Hydrophobicity

HPLC Index

tn/z (Ave.)

ATHAP

CHCA

1 2 3 4 5 6b 7b 8b 9 10 11 12 13 14 15 16 17b 18 19 20 21 22 23 24b 25c

55.9 53.9 53.1 51.0 50.7 45.8 45.1 42.8 38.9 35.1 33.9 33.6 33.4 31.4 31.4 31.2 30.9 30.4 29.9 28.4 25.8 24.7 24.5 18.9 9.3

142.3 109.0 102.5 109.5 78.4 69.5 46.2 52.7 56.8 66.1 37.5 14.3 61.6 36.9 31.2 59.8 43.7 27.5 36.3 38.1 24.5 27.3 44.1 8.7 −0.7

3602.2 3823.5 3890.3 3823.5 2198.6 2155.6 2742.0 2969.5 3504.9 1855.1 1657.0 2130.5 2629.0 1610.9 1814.1 1526.8 2043.3 1942.3 2449.7 1304.7 1263.4 1178.3 1290.5 1254.5 1102.2

++ ++ ++ ++ ++ ++ + + ++ ++ + ++ ++ ++ ++ ++ ++ ++ − ++ − − + + −

− − − − + + + − − ++ + − ++ ++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++

a

The ion detection for 1 pmol phosphorylase b Lys-C in-gel digests using ATHAP or CHCA in positive-ion mode (see Experimental Section). “++” indicates that the ions were detected with S/N ≥ 5, “+” indicates that the ions were detected with a S/N of 2 to 5, and “−” indicates that no ions were detected. The ion peaks for digests 1 to 25 are annotated with asterisks in Figure 4; digests 2 and 4 were indistinguishable in the mass spectrum. b The peptides are carbamidomethylated and are not reflected in the SSRCalc Hydrophobicity or HPLC Index. cThe peptide is acetylated, which is not reflected in the SSRCalc Hydrophobicity or HPLC Index.

SSRCalc Hydrophobicity of 38.9 or higher (digests 1−9 in Table 2) were not detected or were detected at a lower S/N ratio. These results demonstrated that hydrophobic digests were detected with higher intensity using ATHAP than when using CHCA. In addition, hydrophobic peptides ions that could not be detected by CHCA (digests 1, 4, 8, 9, and 12 in Table 2) were detected using ATHAP. Several hydrophilic digests were not detected using ATHAP. Sequence coverage of phosphorylase b was improved from 30% using CHCA or 45% using ATHAP, to 51% using both CHCA and ATHAP. Coverage was calculated for the peptide ion at m/z 3823.5 as digests 2 or 4 (in Table 2); however, it might be increased (30% using CHCA, 49% using ATHAP, and 55% using both CHCA and ATHAP) when both digests 2 and 4 were detected. Thus, the combination of ATHAP and CHCA was expected to enable analysis of a wide variety of peptides, including both hydrophilic and hydrophobic peptides. Sensitivity Improvement for Hydrophobic Peptides by ATHAP. This study confirmed that sensitivity improvement by ATHAP correlated with the SSRCalc Hydrophobicity. Detection of hydrophobic peptides with higher SSRCalc

Figure 3. Positive-ion mass spectra of 1 pmol phosphorylase b Lys-C digests using (a) ATHAP and (b) CHCA in m/z 1000−3000 (top) and m/z 3000−5000 (bottom). The digest ion peaks are annotated with asterisks and listed in Table 2. The ion peaks observed at m/z 1500 or lower using ATHAP were assumed to be byproducts.

phosphorylase b digests. Ion peaks corresponding to digests are denoted with asterisks in Figure 3 and listed in Table 2 in order of SSRCalc Hydrophobicity. Ions for digests with an SSRCalc Hydrophobicity of 30.4 or higher (digests 1−18 in Table 2) were detected using ATHAP, but ions for digests with an SSRCalc Hydrophobicity of 25.8 or lower (digests 21−25 in Table 2) were not detected or were detected at a lower S/N ratio. Ions for digests with an SSRCalc Hydrophobicity of 33.4 or lower (digests 13−25 in Table 2) were detected using CHCA, but ions for digests with an 9447

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Analytical Chemistry

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Hydrophobicity (e.g., 42.4 or higher in Table 1) was improved using ATHAP. In fact, we experimentally observed that the order of SSRCalc Hydrophobicity for the peptides in Table 1 was the same as their retention time order using RP-HPLC (data not shown). The order for the matrices was CHCA < C6ATHAP < C8-ATHAP < C10-ATHAP < C12-ATHAP. These findings lead to the hypothesis that hydrophobic peptides were preferentially detected with hydrophobic C8-ATHAP, and that hydrophilic peptides were detected with hydrophilic CHCA. Comparison of C6-, C8-, C10-, or C12-ATHAP for 1 pmol phosphorylase b digests indicated that the ions for peptides with SSRCalc Hydrophobicity of 33.4 or higher were not detected or were detected at a lower S/N ratio using C6ATHAP as CHCA in Table 2, the ions for the digests with SSRCalc Hydrophobicity of 51.0 or higher, that could not be detected by CHCA, were detected using C10-ATHAP as C8ATHAP in Table 2, and a few ions were detected using C12ATHAP (see Table S-2 in the Supporting Information). These demonstrate the effect of acyl group chain length of ATHAP for various hydrophobic peptides in a mixture. As a result, hydrophobic peptides were preferentially detected using C8- or C10-ATHAP, corresponding to the result in Table S-1 in the Supporting Information (see the “Optimal chain length of the acyl group of ATHAP” section), and hydrophilic peptides were detected using C6-ATHAP. Hence, sensitivity was probably improved by ATHAP based on the Hydrophobicity of C8- or C10-ATHAP with an affinity for hydrophobic peptides.

REFERENCES

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CONCLUSION This study demonstrated that the novel matrix ATHAP increased sensitivity of hydrophobic peptides 10-fold, compared with CHCA. Peptide ions were detected throughout the entire matrix-analyte dried spot using ATHAP. However, the detection of hydrophilic peptides was suppressed. ATHAP functioned alone as a matrix, unlike ADHB as an additive. All three issues of ADHB were resolved using ATHAP. The sequence coverage for phosphorylase b digest analysis was increased using ATHAP, wherein hydrophobic peptides, which were not detected with CHCA for 1 pmol, were detected with this matrix. Consequently, ATHAP is expected to expand the range of target analytes in MALDI-MS.

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Technical Note

ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is granted by the Japan Society for the Promotion of Science (JSPS) through the “Funding Program for WorldLeading Innovative R&D on Science and Technology (FIRST Program),” initiated by the Council for Science and Technology Policy (CSTP). 9448

dx.doi.org/10.1021/ac4018378 | Anal. Chem. 2013, 85, 9444−9448