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Membrane Protein Analyses Using Alkylated Trihydroxyacetophenone (ATHAP) as a MALDI Matrix Yuko Fukuyama,*,†,‡ Chihiro Nakajima,† Shunsuke Izumi,§ and Koichi Tanaka†,‡ †

Koichi Tanaka Laboratory of Advanced Science and Technology, Shimadzu Corporation, 1, Nishinokyo-Kuwabaracho, Nakagyo-ku, Kyoto 604-8511, Japan ‡ Koichi Tanaka Mass Spectrometry Research Laboratory, 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: Membrane proteins containing hydrophobic regions have been difficult to analyze using MALDI-MS, probably due to the use of conventional matrices with a low affinity for hydrophobic peptides. Recently, we reported 1(2,4,6-trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)) as a matrix for hydrophobic peptides. In this study, ATHAP was applied to analyze membrane proteins containing transmembrane domains. As a result, we detected intact molecular ions for bacteriorhodopsin (BR) containing seven transmembrane domains that are difficult to detect using 2,4,6-trihydroxyacetophenone or sinapinic acid, by using ATHAP. In addition, we detected digest ions containing all seven transmembrane domains that are difficult to detect using α-cyano-4-hydroxycinnamic acid (CHCA), by using ATHAP. Moreover, ions for hydrophobic digests containing a single transmembrane domain for cadherin 1 (CDH1), fibroblast growth factor receptor 4 (FGFR4), epithelial cell adhesion molecule (EPCAM) recombinant proteins, and human epidermal growth factor receptor type 2 (HER2) were detected with higher sensitivity using ATHAP than with CHCA, confirming that ATHAP improved the membrane protein analyses, especially for hydrophobic regions such as transmembrane domains.

A

Fourier-transform MS have been advanced to satisfy these purposes.3,10 However, despite significant progress in the technology, sequence coverage of membrane proteins remains patchy and there are some transmembrane regions that remain refractory to analyze.10 MALDI-MS has the potential to ionize hydrophobic peptides or proteins as singly ionized molecules if they are put on the sample plate, but they have been difficult to analyze. One of the difficulties is probably due to the use of conventional matrices with a lower affinity for hydrophobic peptides.33−35 Recently, we reported on a novel matrix additive, octyl 2,5-dihydroxybenzoate (alkylated DHB (ADHB)),33,34 and a novel matrix, 1-(2,4,6-trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone (ATHAP)),35 for hydrophobic peptides. They incorporate in their structures a hydrophobic alkyl chain, which has an affinity for hydrophobic peptides. Thus, the additive ADHB enriched hydrophobic peptides in the rim of a matrix− analyte dried spot, and the matrix ATHAP preferentially detected hydrophobic peptide ions by itself. Hydrophobic

bout 30% of proteins are membrane proteins, classified into membrane associated (mostly hydrophilic) or integral membrane proteins (mostly hydrophobic).1−5 Membrane proteins play a critical role in cellular processes.3 They are important for disease and drug discovery.6 Currently, more than 50% of drug targets act on membrane proteins.3,7−9 However, analysis of membrane proteins is challenging due to their hydrophobic nature and low abundance. Analytical technologies to improve sequence coverage of membrane proteins and their transmembrane domains are under development.10 Mass spectrometry (MS) has become a core tool in membrane proteomics7 because matrix-assisted laser desorption/ionization (MALDI)11,12 and electrospray ionization (ESI)13 were developed. The greatest difficulty with MS analyses of membrane proteins stems from their hydrophobic nature. To solubilize them, several additives such as chaotropes,14−16 detergents,17−23 organic solvents,24,25 organic acids,26−28 and ionic liquid17,29−31 have been reported. These additives need to be removed prior to MS, or minimize the negative effect on sensitivity, as reported by several reviewes.3,10 Shotgun approaches have been developed to improve the throughput of the process.3,10,32 Currently, ESI-MS and © 2016 American Chemical Society

Received: October 1, 2015 Accepted: January 10, 2016 Published: January 21, 2016 1688

DOI: 10.1021/acs.analchem.5b03700 Anal. Chem. 2016, 88, 1688−1695

Article

Analytical Chemistry

The washed gel piece was dehydrated with CH3CN and dried in a vacuum centrifuge. DTT (10 mM) in 100 mM ammonium bicarbonate was added, incubated for 1 h at 56 °C, and then IAA (50 mM) was added and incubated for 45 min at room temperature. The obtained solution was washed with ammonium bicarbonate (100 mM and 50 mM) and dehydrated with CH3CN, then dried in a vacuum centrifuge. Trypsin at an enzyme/substrate ratio of 1:10 (w/w) was added, and the obtained mixtures were then incubated at 37 °C overnight. The obtained CDH1, FGFR4, or EPCAM tryptic digestion solution was diluted with water for analysis using MALDI-TOFMS. The HER2 tryptic digestion solution was obtained by extraction with CH3CN/H2O/TFA (60:40:0.1 and 80:20:0.1, v/v); after the supernatant was dried in a vacuum centrifuge, CH3CN/ H2O/TFA (50:50:0.1, v/v) was added to dissolve or dilute it for analysis using MALDI-TOFMS. Matrix and Sample Preparation. The membrane proteins used in this study are listed in Table 1. Protein and tryptic

peptides were detected with higher sensitivity using ATHAP compared with 2,4,6-trihydroxyacetophenoene (THAP) or αcyano-4-hydroxycinnamic acid (CHCA) throughout an entire matrix−analyte dried spot, and detection of hydrophilic peptides was suppressed.35 As a result, hydrophobic peptides, which are difficult to analyze using CHCA, were detected using ATHAP, and thus sequence coverage was increased.35 These evaluations were carried out for standard peptides. However, a systematic study of disease-associated membrane proteins and their transmembrane domains using ATHAP has not been undertaken. Herein, ATHAP is used for analyzing intact molecules and tryptic digests for bacteriorhodopsin (BR) containing seven transmembrane domains. An evaluation is performed of detecting transmembrane regions or intact molecules through a comparison using ATHAP with conventional matrices such as CHCA, THAP, or sinapinic acid (SA). In addition, tryptic digests for human cadherin 1 (CDH1), human fibroblast growth factor receptor 4 (FGFR4), human epithelial cell adhesion molecule (EPCAM) recombinant proteins, and human epidermal growth factor receptor type 2 (HER2) containing a single transmembrane domain are analyzed using ATHAP to confirm the detection of their transmembrane domains.

Table 1. Membrane Proteins Used in This Study membrane protein analytes bacteriorhodopsin (BR)

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human cadhelin 1 (CDH1) human fibroblast growth factor receptor 4 (FGFR4) human epithelial cell adhesion molecule (EPCAM) human epidermal growth factor receptor type 2 (HER2)

EXPERIMENTAL SECTION Materials. 1-(2,4,6-Trihydroxyphenyl)octan-1-one (alkylated trihydroxyacetophenone, ATHAP) was purchased as a synthetic compound from Nard Institute, Ltd. (Osaka, Japan). α-Cyano-4-hydroxycinnamic acid (CHCA) and sinapinic acid (SA) were purchased from LaserBio Laboratories (SophiaAntipolis Technopole, France). Bacteriorhodopsin (BR) from Halobacterium salinarum, octyl-β-D-glucopyranoside (OGP), methylenediphosphonic acid (MDPNA), 1,4-dithiothreitol (DTT), iodoacetamide (IAA), and ammonium bicarbonate (NH4HCO3) were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Trypsin (mass spectrometry grade) was obtained from Promega Corp. (Madison, WI, USA). Human cadherin 1 (CDH1), human fibroblast growth factor receptor 4 (FGFR4), and human epithelial cell adhesion molecule (EPCAM) recombinant proteins were purchased from Abnova Corporation (Taipei, Taiwan). Human epidermal growth factor receptor type 2 (HER2) SK-BR-3 protein was prepared inhouse.36 Trifluoroacetic acid (TFA) (HPLC grade) and acetonitrile (CH3CN) (LC/MS grade) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The 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 of Membrane Proteins. BR (100 pmol, 0.025 mg) was dissolved in 20 μL of 25 mM ammonium bicarbonate buffer with 1% OGP. Trypsin at an enzyme/ substrate ratio of 1:27 (w/w) was added followed by 25 mM ammonium bicarbonate with 1% OGP. The obtained mixtures were incubated at 37 °C overnight. The tryptic digestion solution was diluted with water for analysis using MALDITOFMS. CDH1 (2 pmol, 0.2 μg per lane), FGFR4 (6 pmol, 0.5 μg per lane), EPCAM (13 pmol, 0.2 μg per lane), or HER2 (ca. 2 pmol, ca. 0.2 μg per lane) was separated by SDS-PAGE using mini-quick gel (Anatech Co., Ltd., Tokyo, Japan) at 25 mA for 90 min. The protein band stained with CBB Stain One (Nacalai Tesque, Inc., Kyoto, Japan) was excised and washed with water.

transmembrane domain position (a.a.) (I) 24−42, (II) 57−75, (III) 92−109, (IV) 121−140, (V) 148−167, (VI) 186−204, (VII) 217−236 710−730 370−390 266−288 653−675

digestions were prepared as described above, and were analyzed after dilution with water or CH3CN/H2O/TFA (50:50:0.1, v/ v) at appropriate concentrations, or without dilution. ATHAP solution was prepared in CH3CN/H2O/TFA (75:25:0.1, v/v) at 5 mg/mL. CHCA solution was prepared in CH3CN/H2O/ TFA (50:50:0.1, v/v) at 10 mg/mL. MDPNA (1%) solution was prepared in water. 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) for analysis using MALDI-TOFMS. Alternatively, the analyte solution (0.5 μL), the matrix solution (0.5 μL), and the MDPNA solution were mixed on a stainless-steel plate. MDPNA solution reportedly suppresses background peaks and improves sensitivity for crude, contaminated, or mixture analytes.37,38 MALDI-MS. MALDI-TOFMS measurement was performed using an AXIMA Performance (Shimadzu/Kratos, UK) 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 400 data points in square regions of 1000 μm × 1000 μm with a 53 μm measurement pitch (i.e., a 20 × 20 lattice) using ATHAP or CHCA. All MS images were constructed from raster-scanned data sets composed of two shots of laser irradiation at each of the 6561 data points in square regions of 4000 μm × 4000 μm with a 50 μm measurement pitch (i.e., a 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 at different concentrations (0.01 to 10000 fmol) made by 101689


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Analytical Chemistry fold serial dilutions. The excitation laser power was adjusted to slightly above the threshold of analyte-ion detection using ATHAP, CHCA, or SA. As a result, a 30−40 higher laser power was used with ATHAP compared with CHCA. This is assumed to be due to the difference between the absorption spectra of ATHAP and CHCA. ATHAP has an absorption maximum at ca. 290 nm (data not shown), and CHCA has one at ca. 340 nm that is close to the wavelength of the nitrogen laser (337 nm). Computational Calculation of Hydrophobicity for Tryptic Digestions. The Sequence-Specific Retention Calculator (SSRCalc) Hydrophobicity for peptides was calculated using SSRCalc software (copyrighted by the Manitoba Centre for Proteomics and Systems Biology, available at http://hs2. proteome.ca/SSRCalc/SSRCalcX.html).39,40 An SSRCalc algorism optimized for tryptic peptides identified by off-line RP HPLC MALDI-MS (MS/MS) has been reported by Krokhin et al.39,40 It takes into account the amino acid composition, position of the amino acid residues (N- and C-terminal), peptide length, overall hydrophobicity, pI, nearest-neighbor effect of charged side chains (K, R, H), and propensity to form helical structures.40 A correlation with R2 ∼ 0.98 has been reported for 2000 peptides and demonstrated for ∼2500 peptides.40 The most important aspect is that this algorism is based on actual experiment results with a number of real analytes using MALDI-MS. In previous papers, we compared SSRCalc Hydrophobicity with other hydrophobicity indices such as the BB index41 or HPLC index42−44 to explain the property of an alkylated matrix ATHAP or a matrix additive ADHB for hydrophobic peptides.33,35 As a result, SSRCalc Hydrophobicity most clearly indicated the property. In that case, we had selected “0.1% TFA” as the separation system in the calculator to approximate our real preparation conditions in MALDI. At least in this paper, SSRCalc Hydrophobicity was considered an appropriate index for discussing the properties of ATHAP and other matrices.

Figure 1. Mass spectra of BR intact molecule (1 pmol) using (a) ATHAP, (b) THAP, or (c) SA in linear, positive-ion mode.

Transmembrane Domain Analyses for BR Using ATHAP. BR has seven transmembrane domains consisting of many hydrophobic amino acids. Peptides containing transmembrane domains are hydrophobic and thus have been difficult to detect as ions. BR tryptic digestion analyses using ATHAP or CHCA were evaluated. CHCA is a common matrix for peptides. Mass spectra for BR tryptic digestion (100 fmol) are presented in Figure 2. Table 2 lists the detected peptide ions in order of SSRCalc Hydrophobicity. Ions for digests containing all transmembrane domains were detected using ATHAP (as drawn on Figure 2). In contrast, ions for digests containing partly six, completely three, out of all the seven transmembrane domains were detected using CHCA (as drawn in Figure 2). A digest containing three transmembrane domains with SSRCalc Hydrophobicity of 88.5 (digest 1 in Table 2) was analyzed using ATHAP. Ions for digests with SSRCalc Hydrophobicity of 23.5 or higher (digests 1−21 in Table 2) were detected using ATHAP, but those with SSRCalc Hydrophobicity of 23.0 or lower (digests 22 and 23 in Table 2) were not detected or were detected at a lower S/N ratio. However, about half of the ions for digests with SSRCalc Hydrophobicity of 75.1 or lower (digests 5−23 in Table 2) and the majority of ions for the digests (digests 17−23 in Table 2) were detected using CHCA, whereas those with SSRCalc Hydrophobicity of 76.7 or higher (digests 1−4 in Table 2) or the majority of them with SSRCalc Hydrophobicity of 57.5 or higher (digests 1−16 in Table 2) were not detected. Overall, ions for hydrophobic digests containing transmembrane

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RESULTS AND DISCUSSION Intact Molecular analysis of BR Using ATHAP. BR has seven transmembrane domains, and thus is more hydrophobic than other proteins. To confirm the utility of ATHAP for hydrophobic proteins, BR intact molecular analyses were evaluated using ATHAP, THAP, or SA. BR (1 pmol) intact molecular ions were not detected using THAP or SA, but were detected using ATHAP (Figure 1). The detection limit of the ions was 100 fmol using ATHAP. The ions were not detected at 10 pmol or less using THAP or SA. They were detected at 50 pmol using THAP but not using SA. Therefore, the sensitivity improvement rate for ATHAP compared with THAP or SA was 100-fold or more. In this case, the sensitivity using ATHAP, THAP, or SA indicated high reproducibility (almost 100%), at least in this study, although these results are usually influenced by the instrument or analyte status. SA has been known as a common matrix for proteins or high−mass molecules. THAP has almost the same absorption spectrum as ATHAP, whereas SA has a different spectrum. THAP and ATHAP have absorption maxima at ca. 290 nm, and SA has one at ca. 325 nm which closes to the wavelength of the nitrogen laser (337 nm). Thus, the above result detecting BR intact molecular ions with high sensitivity using ATHAP is probably an effect caused by the alkyl chain of ATHAP. It was confirmed that ATHAP improved the detection of hydrophobic membrane proteins. 1690


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Figure 2. Mass spectra of BR trypsin digests (100 fmol) using (a) ATHAP or (b) CHCA in linear, positive-ion mode. The ion peaks of the digests containing transmembrane domains are denoted with asterisks. The simplified structure of BR proteins is drawn on each spectrum to indicate the seven transmembrane helices (A−G).48 The extracellular surface is at the bottom. Shaded areas indicate the sequence covered by the analyzed BR tryptic peptides.

Table 2. Correlation of Ion Detection for BR Trypsin Digests and SSRCalc Hydrophobicity Using ATHAP or CHCAa BR trypsin digests

detection (±)

no.

TM domain

SSRCalc Hydrophobicity

m/z (av.)

position (a.a.)

number of missed cleavage sites

ATHAP

CHCA

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

★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★

88.5 77.4 77.2 76.7 75.1 74.4 70.4 70.2 70.2 69.5 66.9 65.3 61.5 61.2 59.7 57.5 41.6 39.1 34.6 23.8 23.5 23.0 11.2

8471.1 5754.0 4844.8 5997.3 5385.5 5628.8 4416.3 3383.1 2976.6 3645.4 4747.7 3475.3 3203.8 3332.0 4290.1 2844.4 1858.3 988.3 1822.1 1325.5 1453.7 1291.4 977.1

96−172 186−238 96−142 186−240 189−238 189−240 189−229 145−172 148−172 143−172 54−95 54−84 14−42 14−43 14−53 14−39 123−142 230−238 173−188 173−184 173−185 85−95 44−53

2 2 0 3 1 2 0 1 0 1 1 1 0 0 1 0 0 0 1 0 0 0 0

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

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

★

a

Ion detection for 100 fmol BR trypsin digests using ATHAP or CHCA in linear, positive-ion mode (see Experimental section). Up to three missed cleavages were considered. “++” indicates that the ions were detected with S/N > 5; “+” indicates that the ions were detected with S/N of 2 to 5; and “−” indicates that the ions were detected with S/N < 2 or no ions were detected. The digests containing transmembrane domain are denoted with asterisks that correspond to the asterisks in Figure 2.

domains were preferentially detected using ATHAP. One of them was detected at m/z 8471.1 in the high-mass area. In the

present study, we confirmed that superhydrophobic digests cover all seven transmembrane domains, with SSRCalc 1691


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Hydrophobicity of 88.5, and in the high-mass area at m/z 8471.1 were detected better using ATHAP compared with CHCA. These results indicate that ATHAP is useful for detecting all hydrophobic peptides, membrane proteins and their digests containing transmembrane domains. The detection limit for hydrophobic peptides, such as the ions for digest 1 at m/z 8471.1 (SSRCalc Hydrophobicity 88.5), was 10 fmol using ATHAP. The ions were not detected using CHCA even with 1000 fmol, the maximum concentration in this experiment. Therefore, the sensitivity improvement rate by ATHAP compared with CHCA was 100-fold or more. In this case, the sensitivity using ATHAP or CHCA indicated high reproducibility (almost 100% for this hydrophobic peptide), at least in this study, although these results are usually influenced by instrument or analyte status. In a previous paper, we reported that the sensitivity improvement rate by ATHAP for standard peptides with a range of SSRCalc Hydrophobicity of 42.4 to 54.8 was 10-fold.35 Therefore, the 100-fold or greater sensitivity improvement rate was considered appropriate for the peptides with SSRCalc Hydrophobicity 88.5 in this study. The sensitivity improvement rate by ATHAP as compared with CHCA is expected to increase for higher hydrophobic digests. In Table 2, digests 7 and 13 were highly hydrophobic, but the intensity of the ions was lower than other digests with similar hydrophobicity. These exceptions are explained by considering the following factors affecting sensitivity in MALDI: (i) hydrophobicity (hydrophobic molecules are difficult to ionize due to the hydrophilic property of common matrices),33−35 (ii)

Figure 3. (a) Photo of the matrix−analyte dried spot on a sample plate, (b) MS image, and (c) mass spectrum of digest 1 (SSRCalc Hydrophobicity 88.5, m/z 8471.1) in Table 2 for BR trypsin digests (100 fmol) using ATHAP.

Table 3. Correlation of Ion Detection for EPCAM Trypsin Digests and SSRCalc Hydrophobicity Using ATHAP or CHCAa EPCAM trypsin digests

detection (±)

no.

TM domain


m/z (Av.)

position (a.a.)


ATHAP

CHCA

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

★ ★ ★

63.3 61.8 61.2 47.5 42.0 42.0 41.4 35.6 30.9 30.7 27.2 24.9 23.6 23.6 23.5 23.0 22.1 21.6 17.0 15.7 15.6 12.8 12.5 12.0 11.7

2430.1 2842.7 2558.3 2641.0 2871.2 2999.4 1179.5 1892.0 2755.0 2911.2 2211.5 2002.3 1108.3 2357.7 2201.5 1517.8 1321.5 1247.4 1942.2 905.0 1033.2 1611.7 1223.4 763.9 1455.5

266−290 266−293 266−291 180−202 231−255 230−255 141−149 203-218 82−106 81−106 297−314 109−125 256−265 107−126 107−125 161−173 34−44 219−229 45−61 222−229 222−230 126−138 154−163 174−179 127−138

0 3 1 0 0 1 0 0 0 1 3 0 0 2 1 2 0 1 0 0 1 2 1 0 1

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

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

a

Ion detection for 250 fmol EPCAM trypsin digests using ATHAP or CHCA in refrectron, positive-ion mode (see Experimental Section). Up to three missed cleavages were considered. “++” indicates that the ions were detected with S/N > 5; “+” indicates that the ions were detected with S/N of 2 to 5; and “−” indicates that the ions were detected with S/N < 2 or no ions were detected. Digests containing transmembrane domain are denoted with asterisks. 1692


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Table 4. Correlation of Ion Detection for HER2 Trypsin Digests and SSRCalc Hydrophobicity Using ATHAP or CHCAa HER2 trypsin digests

detection (±)

no.

TM domain


m/z (Av.)

position (a.a.)


ATHAP

CHCA

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

★ ★

71.7 71.3 70.5 56.4 53.2 52.4 51.0 50.0 50.0 43.1 41.2 40.9 40.8 38.9 37.5 37.2 37.1 37.1 36.2 35.7 35.6 34.8 34.6 34.0

2892.7 3048.9 7030.9 3104.7 3561.0 3300.8 2383.8 4404.8 5275.7 2262.6 2057.3 1119.4 1778.1 2811.3 2671.1 1486.7 1850.2 4749.2 2827.3 4684.1 1879.3 1527.9 1750.1 1680.9

648−676 648−677 370−432 785−811 899−929 460−487 435−456 1007−1046 1183−1230 518−536 766−784 888−896 817−831 689−713 690−713 176−188 737−753 289−330 689−713 1112−1153 600−615 144−157 33−47 158−170

0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 1 0 0 1 1 0

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

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

a Ion detection for 3 pmol HER2 trypsin digests using ATHAP or CHCA with MDPNA, in linear, positive-ion mode (see Experimental Section). Up to one missed cleavage was considered. “++” indicates that the ions were detected with S/N > 5; “+” indicates that the ions were detected with S/N of 2 to 5; and “−” indicates that the ions were detected with S/N < 2 or no ions were detected. Digests containing transmembrane domain are denoted with asterisks.

m/z value (generally, high-mass molecules exhibit limited ionization compared with low-mass molecules), (iii) amino acid sequences (for example, peptides containing arginine tend to ionize with higher sensitivity than peptides without it), (iv) missed cleavage in digestion (plural peptides derived from a single sequence position tend to decrease the intensity of each peptide), and (v) post-translational modification (for example, phosphorylated peptides or glycopeptides easily dissociate carbohydrates or phosphoric acid respectively, thus they were difficult to detect as intact molecules). The exceptions of the detection intensity for digests 7 and 13 in Table 2 are probably caused by (iv) above, that is, missed cleavage. For example, the sequence for digest 7 (missed cleavage site 0) partially overlaps with digest 2 (missed cleavage site 2), digest 4 (missed cleavage site 3), digest 5 (missed cleavage site 1), and digest 6 (missed cleavage site 2). Thus, the intensities of the ion peaks were partitioned, decreasing intensity for digests 7. In addition, digest 7 at amino acid (aa) position 189−229 lacked aa position 230−240 (VGFGLILLRSR), containing two arginine at aa position 238 and 240, compared with digest 2 (aa position 186−238), digest 4 (aa position 186−240), digest 5 (aa position 189−238), and digest 6 (aa position 189−240), which probably led to the decrease in intensity for digest 7 compared with digests 2, 4, 5, and 6. In this way, even though some of the above factors influence sensitivity, ATHAP was basically useful for the detection of hydrophobic digests for BRs. Figure 3 presents a photo of the matrix−analyte dried spot on a sample plate, a MS image, and a mass spectrum for digest 1 (SSRCalc Hydrophobicity 88.5, m/z 8471.1) in Table 2 for BR trypsin digests (100 fmol) using ATHAP. As a result, the

digest peptide was detected over the entire area of the matrix− analyte spot. Other digests containing transmembrane domain in Table 2 resulted in similar MS images using ATHAP. This homogeneous property of ATHAP is expected to contribute to high-throughput analyses of membrane proteins in MALDI. Transmembrane Domains and Hydrophobic Peptide analyses of CDH1, FGFR4, EPCAM, and HER2 Using ATHAP. CDH1, FGFR4, EPCAM, and HER2 are membrane proteins containing a single transmembrane domain and are involved in diseases. Membrane protein analyses using ATHAP or CHCA were evaluated. In the same way as for BR, the detected tryptic digests were listed in order of SSRCalc Hydrophobicity. The results for CDH1 and FGFR4 are presented in the Supporting Information (Table S-1 and S2), and those for EPCAM and HER2 are presented in Tables 3 and 4. All of the ions of digests containing transmembrane domains or hydrophobic digests were detected with higher sensitivity using ATHAP than with CHCA. EPCAM and HER2 digests containing transmembrane domains were not detected using CHCA but were detected using ATHAP. For all of the proteins, hydrophobic digests with higher SSRCalc Hydrophobicity were preferentially detected using ATHAP, and hydrophilic digests with lower SSRCalc Hydrophobicity were preferentially detected using CHCA. These results indicate that the detection of hydrophobic digest ions of membrane proteins that are difficult to detect using conventional methods, especially those containing transmembrane domains, was improved with the use of ATHAP. Potential of ATHAP for Membrane Protein Analyses. ATHAP is a matrix for hydrophobic peptides.35 This property 1693


Analytical Chemistry is based on a structure containing a hydrophobic alkyl chain that has an affinity for hydrophobic peptides. The peptides were detected over the entire area of the matrix−analyte spot. This technique simplified membrane protein analyses by changing the matrix, unlike other techniques based on a hydrophobic sample plate,45 liquid−liquid extraction preparation,46 solvent-free methods,47 or concentration using polystylene beads.4 All digestion ions containing transmembrane domains for membrane proteins were detected using ATHAP. The intact molecule was also detected. Sensitivity was at the pmol or fmol level on a sample plate. Membrane protein digestion consists of super hydrophobic peptides containing transmembrane domains, hydrophobic peptides, and hydrophilic peptides. Hydrophilic peptides have been predominantly reported, probably due to the techniques used (e.g., matrices and solvent in MALDI or liquid chromatography column and solvent in ESI, optimized for hydrophilic peptides). However, in this case, hydrophobic regions remain as unmeasurable areas that are not considered. Rapid, highly sensitive, and accurate analyses of these regions have been a challenge in MS. The present study indicated that ATHAP enabled analysis of these regions in MALDI. In addition, the possibility of complete sequencing for membrane proteins was indicated. In fact, the entire sequence of BR was confirmed (data not shown). A combination of the results using ATHAP and CHCA demonstrate that both hydrophobic and hydrophilic peptides for membrane protein digestions were analyzed. In the future, we expect to apply and advance this technique.

ACKNOWLEDGMENTS

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CONCLUSION An ATHAP matrix for hydrophobic peptides was applied for membrane protein analyses. We used ATHAP to analyze the intact hydrophobic molecule, which is difficult to analyze using THAP or SA, as well as peptides containing transmembrane domains, which are difficult to analyze using CHCA. BR digests containing all seven transmembrane domains that include highmass superhydrophobic peptides with SSRCalc Hydrophobicity of 88.5 were analyzed. It was confirmed that ATHAP enabled rapid analyses of hydrophobic domains of membrane proteins using MALDI-MS. These results are expected to contribute to disease-related protein research and drug discovery through membrane protein research. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03700. Correlation of ion detection for CDH1 trypsin digests and SSRCalc Hydrophobicity using ATHAP or CHCA and correlation of ion detection for FGFR4 trypsin digests and SSRCalc Hydrophobicity using ATHAP or CHCA (PDF)

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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). The authors thank Ms. Kaoru Kaneshiro and Ms. Chikako Hamana for technical support with HER2, Mr. Shin-Ichirou Kawabata for technical comments on BR, and Dr. Naoki Kaneko for technical comments on CDH1.

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AUTHOR INFORMATION

Corresponding Author

*Yuko Fukuyama. Phone: +81-75-823-2897. Fax: +81-75-8232900. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1694


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