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I received a degree of B.S. in Pharmaceutical Sciences (2009) and Master of Pharmaceutical Sciences (2011) at Tohoku University in Japan. I have worked at SHIONOGI & CO., LTD. from 2011. Furthermore, I received a degree of Ph.D. in veterinary medicine (2020) at Hokkaido University in Japan. My professional fields are virology, analytical chemistry, and lipid biology. I want to contribute to develop new drugs against new emerging or reemerging viral infectious diseases, including COVID-19.
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) utilizes host proteases, including a plasma membrane-associated transmembrane protease, serine 2 (TMPRSS2) to cleave and activate the virus spike protein to facilitate cellular entry. Although TMPRSS2 is a well-characterized type II transmembrane serine protease (TTSP), the role of other TTSPs on the replication of SARS-CoV-2 remains to be elucidated. Here, we have screened 12 TTSPs using human angiotensin-converting enzyme 2-expressing HEK293T (293T-ACE2) cells and Vero E6 cells and demonstrated that exogenous expression of TMPRSS11D and TMPRSS13 enhanced cellular uptake and subsequent replication of SARS-CoV-2. In addition, SARS-CoV-1 and SARS-CoV-2 share the same TTSPs in the viral entry process. Our study demonstrates the impact of host TTSPs on infection of SARS-CoV-2, which may have implications for cell and tissue tropism, for pathogenicity, and potentially for vaccine development.
Mai Kishimoto; Kentaro Uemura; Takao Sanaki; Akihiko Sato; William Hall; Hiroaki Kariwa; Yasuko Orba; Hirofumi Sawa; Michihito Sasaki. TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein. Viruses 2021, 13, 384 .
AMA StyleMai Kishimoto, Kentaro Uemura, Takao Sanaki, Akihiko Sato, William Hall, Hiroaki Kariwa, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki. TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein. Viruses. 2021; 13 (3):384.
Chicago/Turabian StyleMai Kishimoto; Kentaro Uemura; Takao Sanaki; Akihiko Sato; William Hall; Hiroaki Kariwa; Yasuko Orba; Hirofumi Sawa; Michihito Sasaki. 2021. "TMPRSS11D and TMPRSS13 Activate the SARS-CoV-2 Spike Protein." Viruses 13, no. 3: 384.
The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.
Michihito Sasaki; Kentaro Uemura; Akihiko Sato; Shinsuke Toba; Takao Sanaki; Katsumi Maenaka; William W. Hall; Yasuko Orba; Hirofumi Sawa. SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells. PLOS Pathogens 2021, 17, e1009233 .
AMA StyleMichihito Sasaki, Kentaro Uemura, Akihiko Sato, Shinsuke Toba, Takao Sanaki, Katsumi Maenaka, William W. Hall, Yasuko Orba, Hirofumi Sawa. SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells. PLOS Pathogens. 2021; 17 (1):e1009233.
Chicago/Turabian StyleMichihito Sasaki; Kentaro Uemura; Akihiko Sato; Shinsuke Toba; Takao Sanaki; Katsumi Maenaka; William W. Hall; Yasuko Orba; Hirofumi Sawa. 2021. "SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells." PLOS Pathogens 17, no. 1: e1009233.
Dengue virus (DENV) infection is one of the most important infectious diseases in tropical and subtropical regions around the world. Previously, we performed an initial phenotypic screening of 7000 compounds using DENV type 2 (DENV2)-infected BHK-21 cells to identify small molecules which could inhibit virus replication. In this study, we describe two novel compounds with anti-DENV2 activity, tentatively named Compound-X and Compound-Y. Both compounds possess a quinolone skeleton, and the EC50s of Compound-X and Compound-Y against DENV2 were 3.9 μM and 9.2 μM, respectively. Based on a DENV replicon assay, it was suggested that these compounds have anti-DENV2 activity by inhibition of a step in virus replication. Furthermore, using mutational analysis we obtained compounds-resistant to DENV2 infection and identified a mutation, V130A in the NS5 methyltransferase (MTase) domain. However, these compounds did not inhibit MTase activity. In addition, incorporation of an additional NS1 N246D mutation with the NS5 V130A mutation in DENV2 resulted in recovery of viral replication and a further reduction of the sensitivity to the quinolone compounds by an unknown mechanism. Therefore further investigations are required to clarify the antiviral mechanisms of these quinolone compounds.
Haruaki Nobori; Kentaro Uemura; Shinsuke Toba; Takao Sanaki; Takao Shishido; William W. Hall; Yasuko Orba; Hirofumi Sawa; Akihiko Sato. Identification of quinolone derivatives as effective anti-Dengue virus agents. Antiviral Research 2020, 184, 104969 .
AMA StyleHaruaki Nobori, Kentaro Uemura, Shinsuke Toba, Takao Sanaki, Takao Shishido, William W. Hall, Yasuko Orba, Hirofumi Sawa, Akihiko Sato. Identification of quinolone derivatives as effective anti-Dengue virus agents. Antiviral Research. 2020; 184 ():104969.
Chicago/Turabian StyleHaruaki Nobori; Kentaro Uemura; Shinsuke Toba; Takao Sanaki; Takao Shishido; William W. Hall; Yasuko Orba; Hirofumi Sawa; Akihiko Sato. 2020. "Identification of quinolone derivatives as effective anti-Dengue virus agents." Antiviral Research 184, no. : 104969.
Dengue fever is an acute febrile infectious disease caused by dengue virus (DENV). Despite the significant public health concerns posed by DENV, there are currently no effective anti-DENV therapeutic agents. To develop such drugs, a better understanding of the detailed mechanisms of DENV infection is needed. Both lipid metabolism and lipid synthesis are activated in DENV-infected cells, so we used lipid screening to identify potential antiviral lipid molecules. We identified 1-stearoyl-2-arachidonoyl-phosphatidylinositol (SAPI), which is the most abundant endogenous phosphatidylinositol (PI) molecular species, as an anti-DENV lipid molecule. SAPI suppressed the cytopathic effects induced by DENV2 infection as well as the replication of all DENV serotypes without inhibiting the entry of DENV2 into host cells. However, no other PI molecular species or PI metabolites, including lysophosphatidylinositols and phosphoinositides, displayed anti-DENV2 activity. Furthermore, SAPI suppressed the production of DENV2 infection-induced cytokines and chemokines, including C-C motif chemokine ligand (CCL)5, CCL20, C-X-C chemokine ligand 8, IL-6, and IFN-β. SAPI also suppressed the TNF-α production induced by LPS stimulation in macrophage cells differentiated from THP-1 cells. Our results demonstrated that SAPI is an endogenous inhibitor of DENV and modulated inflammatory responses in DENV2-infected cells, at least in part via TLR 4.—Sanaki, T., Wakabayashi, M., Yoshioka, T., Yoshida, R., Shishido, T., Hall, W. W., Sawa, H., Sato, A. Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositol in vitro. FASEB J. 33, 13866-13881 (2019). www.fasebj.org
Takao Sanaki; Masato Wakabayashi; Takeshi Yoshioka; Ryu Yoshida; Takao Shishido; William W. Hall; Hirofumi Sawa; Akihiko Sato. Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositolin vitro. The FASEB Journal 2019, 33, 13866 -13881.
AMA StyleTakao Sanaki, Masato Wakabayashi, Takeshi Yoshioka, Ryu Yoshida, Takao Shishido, William W. Hall, Hirofumi Sawa, Akihiko Sato. Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositolin vitro. The FASEB Journal. 2019; 33 (12):13866-13881.
Chicago/Turabian StyleTakao Sanaki; Masato Wakabayashi; Takeshi Yoshioka; Ryu Yoshida; Takao Shishido; William W. Hall; Hirofumi Sawa; Akihiko Sato. 2019. "Inhibition of dengue virus infection by 1-stearoyl-2-arachidonoyl-phosphatidylinositolin vitro." The FASEB Journal 33, no. 12: 13866-13881.
Most advanced knee osteoarthritis (OA) patients experience chronic pain resistant to cyclooxygenase (COX) inhibitors. However, the cells and molecules involved in this advanced OA pain remain poorly understood. In this study, we developed a rat model of advanced knee OA by modification of the monoiodoacetate-induced OA pain model and examined involvement of synovial macrophages in advanced OA pain. COX inhibitors, such as celecoxib and naproxen, and a steroid were ineffective, but an opioid and anti-nerve growth factor (NGF) antibody were effective for pain management in the advanced OA model. Similar to advanced OA patients, histological analysis indicated severe bone marrow damages, synovitis, and cartilage damage and an increase of macrophages with high expression of interleukin-1β (IL-1β), NGF, nitric oxide synthase (Nos) 1, Nos2, and COX-2 in the knee joint of the advanced OA model. Intravenous injection of clodronate liposomes depleted synovial macrophages, which decreased the level of not only proinflammatory mediator IL-1β but also NGF in the knee joint, leading to pain suppression in the advanced OA model. These data suggest the involvement of synovial macrophages in advanced knee OA pain resistant to COX inhibitors by increasing proinflammatory mediators, and that drugs targeting synovial macrophages might have potent analgesic effects.
Yusuke Sakurai; Masahide Fujita; Shiori Kawasaki; Takao Sanaki; Takeshi Yoshioka; Kenichi Higashino; Soichi Tofukuji; Sosuke Yoneda; Tatsuya Takahashi; Ken Koda; Toshiyuki Asaki; Minoru Hasegawa; Yasuhide Morioka. Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors. Pain 2018, 160, 895 -907.
AMA StyleYusuke Sakurai, Masahide Fujita, Shiori Kawasaki, Takao Sanaki, Takeshi Yoshioka, Kenichi Higashino, Soichi Tofukuji, Sosuke Yoneda, Tatsuya Takahashi, Ken Koda, Toshiyuki Asaki, Minoru Hasegawa, Yasuhide Morioka. Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors. Pain. 2018; 160 (4):895-907.
Chicago/Turabian StyleYusuke Sakurai; Masahide Fujita; Shiori Kawasaki; Takao Sanaki; Takeshi Yoshioka; Kenichi Higashino; Soichi Tofukuji; Sosuke Yoneda; Tatsuya Takahashi; Ken Koda; Toshiyuki Asaki; Minoru Hasegawa; Yasuhide Morioka. 2018. "Contribution of synovial macrophages to rat advanced osteoarthritis pain resistant to cyclooxygenase inhibitors." Pain 160, no. 4: 895-907.
Transient receptor potential vanilloid 4 (TRPV4) receptor modulates pain, and this has been noted in several animal models. However, the involvement of TRPV4 in osteoarthritic (OA) pain remains poorly understood. This study assessed the functional changes in TRPV4 and the expression of its endogenous ligand 5,6-epoxyeicosatrienoic acid (5,6-EET) in a rat monoiodoacetate (MIA)-induced OA pain model (MIA rats). Monoiodoacetate-treated rats showed reduced grip strength as compared to sham-treated rats, and this loss in function could be recovered by the intraarticular administration of a TRPV4 antagonist (HC067047 or GSK2193874). By contrast, the intraarticular administration of the TRPV4 agonist, GSK1016790A, increased the pain-related behaviors in MIA rats but not in sham rats. TRPV4 expression was not increased in knee joints of MIA rats; however, the levels of phosphorylated TRPV4 at Ser824 were increased in dorsal root ganglion neurons. In addition, 5,6-EET was increased in lavage fluids from the knee joints of MIA rats and in meniscectomy-induced OA pain model rats. 5,6-EET and its metabolite were also detected in synovial fluids from patients with OA. In conclusion, TRPV4 was sensitized in the knee joints of MIA rats through phosphorylation in dorsal root ganglion neurons, along with an increase in the levels of its endogenous ligand 5,6-EET. The analgesic effects of the TRPV4 antagonist in the OA pain model rats suggest that TRPV4 may be a potent target for OA pain relief.
Mikie Hinata; Sunao Imai; Takao Sanaki; Junji Tsuchida; Takeshi Yoshioka; Kenichi Higashino; Miyuki Yamamoto; Masayuki Imai; Masahiko Soga; Narumi Horita; Isao Fukuda; Minoru Ikeda; Shoji Yamane; Atsushi Morita; Toshiyuki Kanemasa; Gaku Sakaguchi; Minoru Hasegawa; Masabumi Minami; Yasuhide Morioka. Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis. Pain 2018, 159, 939 -947.
AMA StyleMikie Hinata, Sunao Imai, Takao Sanaki, Junji Tsuchida, Takeshi Yoshioka, Kenichi Higashino, Miyuki Yamamoto, Masayuki Imai, Masahiko Soga, Narumi Horita, Isao Fukuda, Minoru Ikeda, Shoji Yamane, Atsushi Morita, Toshiyuki Kanemasa, Gaku Sakaguchi, Minoru Hasegawa, Masabumi Minami, Yasuhide Morioka. Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis. Pain. 2018; 159 (5):939-947.
Chicago/Turabian StyleMikie Hinata; Sunao Imai; Takao Sanaki; Junji Tsuchida; Takeshi Yoshioka; Kenichi Higashino; Miyuki Yamamoto; Masayuki Imai; Masahiko Soga; Narumi Horita; Isao Fukuda; Minoru Ikeda; Shoji Yamane; Atsushi Morita; Toshiyuki Kanemasa; Gaku Sakaguchi; Minoru Hasegawa; Masabumi Minami; Yasuhide Morioka. 2018. "Sensitization of transient receptor potential vanilloid 4 and increasing its endogenous ligand 5,6-epoxyeicosatrienoic acid in rats with monoiodoacetate-induced osteoarthritis." Pain 159, no. 5: 939-947.
Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICR(TRPV3+/+) and ICR(TRPV3-/-) mice indicated that higher ear edema responses to topical application of AA were observed in ICR(TRPV3+/+) mice compared with ICR(TRPV3-/-) mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.
Takao Sanaki; Erika Kasai-Yamamoto; Takeshi Yoshioka; Shota Sakai; Kohei Yuyama; Takuji Fujiwara; Yoshito Numata; Yasuyuki Igarashi. Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3. Journal of Oleo Science 2017, 66, 591 -599.
AMA StyleTakao Sanaki, Erika Kasai-Yamamoto, Takeshi Yoshioka, Shota Sakai, Kohei Yuyama, Takuji Fujiwara, Yoshito Numata, Yasuyuki Igarashi. Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3. Journal of Oleo Science. 2017; 66 (6):591-599.
Chicago/Turabian StyleTakao Sanaki; Erika Kasai-Yamamoto; Takeshi Yoshioka; Shota Sakai; Kohei Yuyama; Takuji Fujiwara; Yoshito Numata; Yasuyuki Igarashi. 2017. "Direct Involvement of Arachidonic Acid in the Development of Ear Edema via TRPV3." Journal of Oleo Science 66, no. 6: 591-599.
To evaluate the precise role of sphingomyelin synthase 2 (SMS2) in sphingomyelin (SM) metabolism and their anti-inflammatory properties, we analyzed species of major SM and ceramide (Cer) (18:1, 18:0 sphingoid backbone, C14 - C26 N-acyl part) in SMS2 knockout and wild-type mouse plasma and liver using HPLC-MS. SMS2 deficiency significantly decreased very long chain SM (SM (d18:1/22:0) and SM (d18:1/24:0 or d18:0/24:1)) and increased very long chain Cer (Cer (d18:1/24:0 or d18:0/24:1) and Cer (d18:1/24:1)), but not long chain SM (SM (d18:1/16:0), SM (d18:1/18:0 or d18:0/18:1) and SM (d18:1/18:1)) in plasma. To examine the effects of SM on inflammation, we studied the role of very long chain SM in macrophage activation. Addition of SM (d18:1/24:0) strongly upregulated several macrophage activation markers, SM (d18:1/6:0) and Cer (d18:1/24:0) however, did not. It was suggested that very long chain SM but not long chain SM were decreased in SMS2-deficient mice liver and plasma. And the exogenously added very long chain SM (d18:1/24:0) could activate macrophages directly, suggesting a novel role of plasma very long chain SM in modulating macrophage activation and resulting inflammation.
Hideaki Sakamoto; Tetsuya Yoshida; Takao Sanaki; Shuhei Shigaki; Hirotoshi Morita; Miki Oyama; Masaru Mitsui; Yoshikazu Tanaka; Toru Nakano; Susumu Mitsutake; Yasuyuki Igarashi; Hiroshi Takemoto. Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response. Biochemical and Biophysical Research Communications 2017, 482, 202 -207.
AMA StyleHideaki Sakamoto, Tetsuya Yoshida, Takao Sanaki, Shuhei Shigaki, Hirotoshi Morita, Miki Oyama, Masaru Mitsui, Yoshikazu Tanaka, Toru Nakano, Susumu Mitsutake, Yasuyuki Igarashi, Hiroshi Takemoto. Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response. Biochemical and Biophysical Research Communications. 2017; 482 (2):202-207.
Chicago/Turabian StyleHideaki Sakamoto; Tetsuya Yoshida; Takao Sanaki; Shuhei Shigaki; Hirotoshi Morita; Miki Oyama; Masaru Mitsui; Yoshikazu Tanaka; Toru Nakano; Susumu Mitsutake; Yasuyuki Igarashi; Hiroshi Takemoto. 2017. "Possible roles of long-chain sphingomyelines and sphingomyelin synthase 2 in mouse macrophage inflammatory response." Biochemical and Biophysical Research Communications 482, no. 2: 202-207.
RationaleTargeted oxidized fatty acid analysis has been widely used to understand the roles of fatty acids in the development of diseases. However, because of the extensive structural diversity of fatty acids, it is considered that unknown lipid metabolites will remain undetected. Here, to discover and identify unknown lipid metabolites in biological samples, a global analytical system and a method of synthesizing lipid standards were investigated.MethodsOxidized fatty acids in mouse lung tissues were extracted using mixed-mode spin columns. Separation was achieved via ultra-high-performance liquid chromatography, mass spectrometric (MS) analysis was conducted in full scan mode using a Q Exactive Plus instrument equipped with an electrospray ionization probe, and structure analysis was carried out by high-resolution data-dependent tandem mass spectrometry (dd-MS2). In addition, lipid standards, which are not commercially available, were synthesized by bioconversion using Bacillus circulans.ResultsOxidized fatty acids in mouse lung tissues were analyzed by high-resolution accurate-mass analysis, and multiple unknown molecules were discovered and tentatively identified using high-resolution dd-MS2. Among these molecules, 21-hydroxydocosahexaenoic acid (21-HDoHE) and 22-HDoHE, which are not commercially available, were synthesized by bioconversion. By comparing the exact masses, retention times, and characteristic fragment ions of the synthesized standards, 21-HDoHE and 22-HDoHE were definitively identified in the mouse lung tissue.ConclusionsOur strategy of global analysis and bioconversion can be used for the discovery and identification of unknown lipid molecules. Copyright © 2016 John Wiley & Sons, Ltd.
Takao Sanaki; Yoko Inaba; Takuji Fujiwara; Takeshi Yoshioka; Keisuke Matsushima; Kazuyuki Minagawa; Kenichi Higashino; Toru Nakano; Yoshito Numata. A hybrid strategy using global analysis of oxidized fatty acids and bioconversion byBacillus circulans. Rapid Communications in Mass Spectrometry 2016, 30, 751 -762.
AMA StyleTakao Sanaki, Yoko Inaba, Takuji Fujiwara, Takeshi Yoshioka, Keisuke Matsushima, Kazuyuki Minagawa, Kenichi Higashino, Toru Nakano, Yoshito Numata. A hybrid strategy using global analysis of oxidized fatty acids and bioconversion byBacillus circulans. Rapid Communications in Mass Spectrometry. 2016; 30 (6):751-762.
Chicago/Turabian StyleTakao Sanaki; Yoko Inaba; Takuji Fujiwara; Takeshi Yoshioka; Keisuke Matsushima; Kazuyuki Minagawa; Kenichi Higashino; Toru Nakano; Yoshito Numata. 2016. "A hybrid strategy using global analysis of oxidized fatty acids and bioconversion byBacillus circulans." Rapid Communications in Mass Spectrometry 30, no. 6: 751-762.
Lipidomics by liquid chromatography/mass spectrometry has been used for a better understanding of the roles of oxidized fatty acids in the development of various diseases. However, further work is required to improve the sample preparation process and the peak tailing of cysteinyl-leukotrienes. In this study, we evaluated various mobile phases and extraction conditions. The addition of phosphoric acid to the mobile phase improved the peak tailing of cysteinyl-leukotrienes. The extraction conditions were also optimized by spin-column possessing an anion-exchange and reversed-phase properties. The extraction efficiency of the modified extraction system was examined using 62 lipids, and 13 deuterated lipids were investigated to evaluate matrix effects and recovery from mouse lung homogenate samples. Extraction efficiencies of ≥70% were obtained for almost all of the lipids. Good results with standard deviations of 0.99 for all the lipids tested. This newly developed method therefore represents a powerful tool to analyze lipids.
Takao Sanaki; Takuji Fujihara; Ryo Iwamoto; Takeshi Yoshioka; Kenichi Higashino; Toru Nakano; Yoshito Numata. Improvements in the High-Performance Liquid Chromatography and Extraction Conditions for the Analysis of Oxidized Fatty Acids Using a Mixed-Mode Spin Column. Modern Chemistry & Applications 2015, 3 .
AMA StyleTakao Sanaki, Takuji Fujihara, Ryo Iwamoto, Takeshi Yoshioka, Kenichi Higashino, Toru Nakano, Yoshito Numata. Improvements in the High-Performance Liquid Chromatography and Extraction Conditions for the Analysis of Oxidized Fatty Acids Using a Mixed-Mode Spin Column. Modern Chemistry & Applications. 2015; 3 (3):.
Chicago/Turabian StyleTakao Sanaki; Takuji Fujihara; Ryo Iwamoto; Takeshi Yoshioka; Kenichi Higashino; Toru Nakano; Yoshito Numata. 2015. "Improvements in the High-Performance Liquid Chromatography and Extraction Conditions for the Analysis of Oxidized Fatty Acids Using a Mixed-Mode Spin Column." Modern Chemistry & Applications 3, no. 3: .
Peptide mass fingerprinting (PMF) has been widely used as an efficient analytical strategy for protein identification. This is most commonly used with a combination of protein digestion using sequence-specific proteases and MALDI-TOFMS. Then database searches are performed comparing the pattern of the experimentally obtained masses with the pattern of the theoretical peptide masses of proteins stored in the database. The positive ionization mode has been mainly used for MALDI analyses with a few exceptions for phosphopeptides, oligonucleotides, etc. Therefore, nonphosphorylated peptides that have low pI values could be missed from PMF using the positive ionization mode. Here, we introduce optimal conditions for negative ionization of peptides and the practical advantages of negative ionizations in PMF. Angiotensin I (pI 6.9) and bovine serum albumin (BSA) tryptic digests were used as model peptides. Eight matrix candidates and seven additives were examined in terms of sensitivity, robustness and reproducibility. The combination of DHB and phosphoric acid was the best condition for negative ionization of peptides and was found to be compatible with the positive ionization mode. Using 150 mM DHB/1% phosphoric acid, the coverage (% by amino acid count) of BSA tryptic digest (0.6 pmol per spot) totaled 67.2% (negative + positive). The 24.1% of peptides (pI range 4.1–6.2) were detected only by negative ionization, which indicated that acidic peptides were efficiently recovered by the negative ion mode. This methodology has been successfully employed to analyze other proteins without false positive identifications.
Takao Sanaki; Mao Suzuki; Seon Hwa Lee; Takaaki Goto; Tomoyuki Oe. A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: concomitant use of negative ionization. Analytical Methods 2010, 2, 1144 -1151.
AMA StyleTakao Sanaki, Mao Suzuki, Seon Hwa Lee, Takaaki Goto, Tomoyuki Oe. A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: concomitant use of negative ionization. Analytical Methods. 2010; 2 (8):1144-1151.
Chicago/Turabian StyleTakao Sanaki; Mao Suzuki; Seon Hwa Lee; Takaaki Goto; Tomoyuki Oe. 2010. "A simple and efficient approach to improve protein identification by the peptide mass fingerprinting method: concomitant use of negative ionization." Analytical Methods 2, no. 8: 1144-1151.