Technical Note pubs.acs.org/ac
Enhanced Visualization of Small Peptides Absorbed in Rat Small Intestine by Phytic-Acid-Aided Matrix-Assisted Laser Desorption/ Ionization-Imaging Mass Spectrometry Seong-Min Hong,† Mitsuru Tanaka,† Saori Yoshii,† Yoshinori Mine,‡ and Toshiro Matsui*,† †
Division of Bioresources and Bioenvironmental Sciences, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan ‡ Department of Food Science, University of Guelph, 435 Gordon Street, Guelph, Ontario, N1G 2W1, Canada S Supporting Information *
ABSTRACT: Enhanced visualization of small peptides absorbed through a rat intestinal membrane was achieved by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-IMS) with the aid of phytic acid as a matrix additive. Penetrants through intestinal peptide transporter 1, i.e., glycylsarcosine (Gly-Sar, 147.1 m/z) and antihypertensive dipeptide, Val-Tyr (281.2 m/ z), were chosen for MALDI-IMS. The signal-to-noise (S/N) ratios of dipeptides Gly-Sar and Val-Tyr were seen to increase by 2.4- and 8.0-fold, respectively, when using a 2′,4′,6′-trihydroxyacetophenone (THAP) matrix containing 5.0 mM phytic acid, instead of the THAP matrix alone. Owing to the phytic-acid-aided MALDIIMS method, Gly-Sar and Val-Tyr absorbed in the rat intestinal membrane were successfully visualized. The proposed imaging method also provided useful information on intestinal peptide absorption; to some extent, Val-Tyr was rapidly hydrolyzed to Tyr by peptidases located at the intestinal microvillus during the absorption process. In conclusion, the strongly acidic additive, phytic acid, is beneficial for enhancing the visualization of small peptides using MALDI-IMS, owing to the suppression of ionization-interfering salts in the tissue.
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solutions (e.g., solvent washing,10 chemical derivatization,11 nanomaterial-based platform,12 and organic matrixes13,14) have been proposed to improve the poor visualization of small analytes in the tissues. However, the solvent washing and chemical derivatization techniques still have the disadvantage of including the redistribution of analytes in tissues. Nanoparticleassisted laser desorption ionization imaging mass spectrometory12 and newly developed matrixes such as 9-aminoacridine13 and metal-phthalocyanines14 also have some analytical restrictions for detection of small and polar analytes due to their advantaged detection for hydrophobic and negativeionized analytes (e.g., lipids). Despite the limitations of the MALDI-IMS technique, the visualization of absorbable bioactive small peptides in the intestinal membrane is of great benefit, because no direct evidence of the absorption process (intact or degradable) has been obtained so far. The aim of the present study is to achieve the visualization of small peptides (1 000 Da.19 To overcome the limitations of MS techniques, developments in chemical derivatization 20 and matrix reagents21,22 have been proposed. Contrary to these analytical advances, an updated analytical technique, MALDI-IMS, can simultaneously visualize analytes of interests in biological tissues without any use of antibodies; however, it is limited in its application, particularly toward small compounds in tissues. The difficulty in detecting small compounds may be caused by interfering peaks, e.g., from matrix clusters with alkali metal ions.8,9 With this in mind, we proposed phytic acid as a new matrix additive that possesses six phosphate groups for the efficient detection and visualization of tissue-distributed bioactive small dipeptides. The anticipated advantages of this matrix additive method are (1) an improvement in the ionization efficiency of small peptides because of the encapsulation of ionization-suppressing salts and (2) less degradation of analyte dipeptides in tissues during chemical derivatization such as sulfonation at 50 °C for 1 h11 and less redistribution of analytes on the tissues by the solvent-washing method.10 Matrix for Enhanced Detection of Dipeptides by MALDI-MS. Prior to the investigation of the matrix additive method for dipeptide visualization by MALDI-IMS, the matrix reagents for dipeptide detection by MALDI-MS were
investigated. Four matrix reagents (CHCA, DHB, SA, and THAP; 5.0 mg/mL) were individually sprayed (90 cycles) onto a peptide-spotted (2.0 nmol) or nonspotted intestinal sectionITO glass slide. As shown in Figure 1, for the nonspotted intestinal sections, the matrix reagents DHB, SA, and THAP did not show any significant ion peaks corresponding to the target dipeptides at 147.1 m/z ([M + H]+ of Gly-Sar) and 281.2 m/z ([M + H]+ of Val-Tyr), suggesting that the matrix interference from these three reagents is too great for dipeptide detection by the present MALDI-MS. Since Gly-Sar was not found in nature,16 it was apparent that CHCA was far from an ideal candidate for dipeptide detection because of its prominent interfering molecular ion peak at 147 m/z, which matches with the molecular ion peak of synthetic Gly-Sar. Detection of endogenous Val-Tyr by the present MALDI-IMS analysis was also excluded from the observed Val-Tyr in the peptide-spotted section (Figure 1), since the level of endogenous tissue Val-Tyr was negligible (400 pmol/spot in the present MALDI-IMS (data not shown). The phytic acid-aided enhancement in MALDI-MS signal was also observed for SA matrix (1.8- and 5.3-fold higher detections of Gly-Sar and Val-Tyr in SA/5.0 mM phytic acid compared to those in SA only, see Figure S-2 in the Supporting Information), indicating that phytic acid-MALDI-IMS has potential for extensive application to small analytes. Extensive application of phytic acid-MALDI-IMS for other bioactive small peptides such as Trp-His27 is thus now in progress. Visualization of Dipeptide Absorption across Intestinal Membrane by MALDI-IMS. Using the phytic-acid-aided MALDI-IMS, the distribution of Gly-Sar and Val-Tyr in the SD rat intestinal membrane was visualized after either 30- or 60min transport. As shown in Figure 4, the inner distribution or absorption of Gly-Sar, a PEPT1 transporter model, toward the basolateral side with transporting time was visualized in this study for the first time. Much more peaks in THAP/phytic acid compared to those in THAP would be caused by the phytic
importance of the encapsulation of salts by matrix additives bearing carboxyl and phosphate groups for the improved MALDI-MS detection of phosphopeptides.15 However, in the present study, only phytic acid bearing six phosphate groups enhanced the dipeptide detection, while the other acids did not show any effect at the same concentration of additives. We speculate that the phytic-acid-induced enhancement for dipeptide detection by MALDI-MS may occur because of the strongly acidic six phosphate groups, causing an efficient trap of tissue salts that affect the ionization of small peptides. The much higher formation constant (or chelation power, Kf) of phytic acid with Na+ over other acids (log Kf, phytic acid-Na+, 68.2;25 EDTA-Na+, 1.8;26 NTA-Na+, 1.229) suggested a more efficient encapsulation of tissue salts by phytic acid in the intestinal tissue sections. The efficient elimination of Na+ and K+ adduct signals when using alkylphosphonic acids as matrix additives greatly improved the phosphopeptide ion response in positive-mode MALDI-MS,15 revealing the importance of salt elimination by chelating compounds such as phytic acid in improving the MALDI-MS detection. In this study, reduced peak intensities corresponding to matrix cluster ions (i.e., [M + K]+, [M + Na + K − H]+ and [M + 2K − H]+, except [M + Na]+) were also observed in spectrum with phytic acid, compared to those without phytic acid (see Table S-1 in the Supporting Information), showing the effective suppression of alkaline metal adducts by phytic acid. In addition, more uniform and fine crystals of matrix were observed when THAP/phytic acid matrix was sprayed (Figure S-1 in the Supporting Information), implying that phytic acid could also prevent inhomogeneous detection of analytes by the hot-spot of matrix crystals in MALDI-IMS. No adduct ions of phytic acid (e.g., [M + H]+ 660.9 m/z, [M + Na]+ 683.9 m/z) were observed in the intestinal section ranging from 100 to 1 000 m/z in the present MALDI-MS detection (Figure S-1 in the Supporting 10037
dx.doi.org/10.1021/ac402252j | Anal. Chem. 2013, 85, 10033−10039
Analytical Chemistry
Technical Note
Notes
acid-induced enhancement detection of endogenous biological compounds including ADP (508.0 m/z) and AMP (348.2 m/ z). The successful visualization of Val-Tyr after a 60-min transport was also observed, while less clear observation was achieved after 30 min. Because the imaging pictures shown in Figure 4 were individually obtained from different intestinal sections in each separate transport experiment, a comparative or quantitative analysis between each picture is meaningless; however, the lesser visualization of Val-Tyr after 30 min suggested that the transport of Val-Tyr across the intestinal membrane was lower than that of Gly-Sar. The LC−MS analysis of each dipeptide in the basolateral side after 30 and 60 min also revealed a lower intestinal transport of Val-Tyr; the transported amount of Val-Tyr was estimated to be 300 times lower than that of Gly-Sar (Figure S-3 of the Supporting Information). The MALDI-MS technique allows any ionizable compounds within a given mass range to be visualized simultaneously. Thus, we could speculate that the lower transport ability of Val-Tyr was caused by its rapid enzymatic degradation, because Tyr ([M + H]+ 182.2 m/z, a possible ValTyr degradation product) was predominantly visualized after 30 min (Figure 4). This finding indicates that great attention must be paid to the degradation (probably by peptidases located at the intestinal microvillus) of peptides that can be absorbed intact via PEPT1 transport.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This study was supported in part by a Grant-in-Aid for Young Scientists (B) (Grant No. 24780130) to M.T., a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (Grant No. 22248014) to T.M., Kyushu University Interdisciplinary Programs in Education and Project in Research Development to M.T., and Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST to M.T. and T.M. The cost of publication was supported in part by the Research Grant for Young Investigators of Faculty of Agriculture, Kyushu University. The authors thank Mr. Yutaro Morita and Ms. Kaori Miyazaki for their technical assistance.
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CONCLUSIONS In this study, we have established phytic-acid-aided MALDIIMS for the visualization of small peptides (
