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Efficient mass spectral analysis of active transporters overexpressed in Escherichia coli Mamiyo Kawakami, Narinobu Juge, Yuri Kato, Hiroshi Omote, Yoshinori Moriyama, and Takaaki Miyaji J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00777 • Publication Date (Web): 19 Jan 2018 Downloaded from http://pubs.acs.org on January 21, 2018

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Journal of Proteome Research

Journal of Proteome Research: Article

Efficient Mass Spectral Analysis of Active Transporters Overexpressed in Escherichia coli Mamiyo Kawakami1, Narinobu Juge1-3, Yuri Kato2, Hiroshi Omote4, Yoshinori Moriyama4, and Takaaki Miyaji1,2*

1

Department of Molecular Membrane Biology, Okayama University Graduate School of

Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan 2

Advanced Science Research Center, Okayama University, Okayama 700-8530, Japan

3

Precursory Research for Embryonic Science and Technology, Japan Science and

Technology Agency, Kawaguchi 332-0012, Japan 4

Department of Membrane Biochemistry, Okayama University Graduate School of

Medicine, Dentistry and Pharmaceutical Sciences, Okayama 700-8530, Japan *

To whom correspondence should be addressed.

E-mail: [email protected]

Tel: +81-86-251-7260

1 ACS Paragon Plus Environment

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Abstract Structural analysis of purified active membrane proteins can be performed by mass spectrometry (MS). However, no large-scale expression systems for active eukaryotic membrane proteins are available. Moreover, because membrane proteins cannot easily be digested by trypsin and ionized, they are difficult to analyze by MS. Here, we developed a method for mass spectral analysis of eukaryotic membrane proteins combined to an overexpression system in Escherichia coli. Vesicular glutamate transporter 2 (VGLUT2/SLC17A6) with a soluble α-helical protein and histidine tag on the N- and C-terminus, respectively, was overexpressed in E. coli, solubilized with detergent, and purified by Ni-NTA affinity chromatography. Proteoliposomes containing VGLUT2 retained glutamate transport activity. For MS analysis, the detergent was removed from purified VGLUT2 by trichloroacetic acid precipitation, and VGLUT2 was then subjected to reductive alkylation and tryptic digestion. The resulting peptides were detected with 88% coverage by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS with or without liquid chromatography. Vesicular excitatory amino acid transporter and vesicular acetylcholine transporter were also detected with similar coverage by the same method. Thus, this methodology could be used to analyze purified eukaryotic active transporters. Structural analysis with chemical modifiers by MS could have applications in functional binding analysis for drug discovery.

Keywords: vesicular glutamate transporter, membrane protein, vesicular neurotransmitter transporter, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, sequence coverage, trypsin digestion


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Introduction Membrane proteins play important roles in biological processes. In particular, transporters are essential for the delivery of mediators, metabolites, drugs, and ions. Because transporter dysfunction is closely related to many diseases, transporters have been evaluated as potential targets of drug discovery.1,2 Although functional and structural analyses of such proteins require the establishment of overexpression systems, this methodology has not been developed owing to the difficulties of handling transporter proteins. Notably, eukaryotic membrane proteins can be overexpressed as inclusion bodies in Escherichia coli and are difficult to solubilize using detergents while retaining transport activity.3,4 Leviatan et al. developed a large-scale eukaryotic membrane protein expression system in E. coli, which could be used for functional analysis of transporters.5 Briefly, cDNA encoding transporter proteins with soluble α-helix tags at the N- and C-termini was transformed into E. coli, and membrane fractions were solubilized with mild detergent and purified by Ni-NTA affinity column chromatography. The soluble α-helix proteins of E. coli were expected to facilitate the folding of the eukaryotic membrane protein, and proteoliposomes containing purified eukaryotic transporter retained active and passive transport activities. This technique was effective for identification of transport substrates, specific inhibitors, and essential amino acid residues in various organisms.6-10 Moreover, the transport functions of various transporters could be analyzed by changing the buffer composition.11, 12 Although mass spectrometry (MS) with chemical modifiers is effective for the structural analysis of transporters, no MS-based methods have been developed for the detection of peptides derived from transporters to analyze the structures of eukaryotic membrane proteins. Specifically, it is difficult to digest membrane proteins for MS and further ionize the peptides in detergent-containing solution.13,14 Although previous reports have demonstrated the detection of prokaryotic membrane



proteins by MS,15 Cox et al. developed an efficient digestion method and mass spectral analytical approach for eukaryotic vesicular glutamate transporter (VGLUT) 1 involving denaturation and solubilization with sodium dodecyl sulfate (SDS), detergent removal, reductive alkylation, and tryptic digestion in solution.16 Although this method yielded high-coverage peptides, the purified VGLUT1 was not active due to denaturation by SDS. As this method cannot be applied to functional studies such as those on ligand binding, another approach for MS is necessary. Vesicular neurotransmitter transporters are responsible for vesicular storage and release of neurotransmitters and are therefore essential for neurotransmission.6 Accordingly, because many disease states are caused by dysfunction of such transporters, the development of a simple and efficient method for analysis of membrane proteins by MS may have applications in functional binding analysis for drug discovery. In this study, we developed an efficient matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS method for detection and functional/structural analysis of active membrane proteins, such as vesicular neurotransmitter transporters.

Materials and Methods cDNA The cDNAs encoding rat VGLUT2 (accession no. NM053427.1), mouse vesicular excitatory amino acid transporter (VEAT; accession no. NM172773), and human vesicular acetylcholine transporter (VAChT; accession no. BC007765.2) were cloned by polymerase chain reaction.11,17

Antibodies Mouse polyclonal antibodies against histidine-tagged recombinant proteins conjugated with peroxidase (diluted 1:500; cat no. 11 965 085 001; Roche, Basel, Switzerland) were used for


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western blotting.5

Expression and Purification of Vesicular Neurotransmitter Transporters in E. coli Vesicular neurotransmitter transporters were expressed and purified as previously described.5 Briefly, E. coli C43 (DE3) cells were transformed with the expression vectors and grown in TB medium containing 30 µg/mL kanamycin sulfate at 37°C. E. coli cells were grown until A600 reached 0.6–0.8, and isopropyl-β-D-thiogalactopyranoside was then added to a final concentration of 1 mM followed by incubation for an additional 16 h at 18°C. The cells were then harvested by centrifugation and suspended in buffer consisting of 70 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM KCl, 15% glycerol, and 2 mM phenylmethylsulfonyl fluoride. The cell suspension was then disrupted by sonication with a tip sonicator (Sonics & Materials Inc., Newtown, CT, USA) and centrifuged at 5856 × g at 4°C for 10 min to remove large inclusion bodies and cell debris. The resulting supernatant was carefully collected and centrifuged again at 150000 × g for 1 h at 4°C. The pellet was suspended in the same buffer, and protein concentration was adjusted to 10 mg/mL. Membranes were then treated with 2 w/v% Fos-choline 14 (Affymetrix, Santa Clara, CA, USA) and centrifuged at 150000 × g at 4°C for 1 h. The supernatant containing recombinant protein was obtained; diluted twofold with the same buffer as above; and then applied to a column containing 1 mL nickel-NTA Superflow resin (Qiagen, Valencia, CA, USA) equilibrated with buffer consisting of 70 mM Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM KCl, and 15% glycerol. After incubation for 3 h at 4°C, the column was washed with 15 mL washing buffer consisting of 70 mM Tris-HCl (pH 8.0), 20 mM imidazole, 100 mM NaCl, 10 mM KCl, 20% glycerol, and 0.1w/v% n-decyl-β-D-thiomaltopyranoside (DTM; Affymetrix). The protein was eluted with 3 mL buffer consisting of 20 mM Tris-HCl (pH 8.0), 250 mM imidazole, 100 mM NaCl, 10 mM KCl, 20% glycerol, and 0.1w/v% DTM



and then stored at –80°C, whereby the protein was stable without loss of activity for at least a few months.

Reconstitution Aliquots of 20 µg purified protein were mixed with 500 µg liposomes and frozen at –80°C for at least 15 min. The mixtures were then thawed quickly and diluted 60-fold with reconstitution buffer containing 20 mM MOPS-Tris (pH 7.0), 150 mM sodium acetate, and 5 mM magnesium acetate. Reconstituted proteoliposomes were pelleted by centrifugation at 200000 × g for 1 h at 4°C and then suspended in 0.2 mL reconstitution buffer. Asolectin liposomes were prepared as described previously.5 Soybean lecithin (10 mg/mL; Sigma Type IIS, St. Louis, MO, USA) was suspended in buffer containing 20 mM MOPS-NaOH (pH 7.0) and 1 mM dithiothreitol. The mixture was sonicated until clear with a tip sonifier and stored at –80°C until use.

Transport Assay The reaction mixture (130 µL) consisting of 0.5 µg protein incorporated into proteoliposomes, 20 mM MOPS-Tris (pH 7.0), 140 mM potassium acetate, 5 mM magnesium acetate, 10 mM KCl, 2 µM valinomycin, and 100 µM [2,3-3H] L-glutamate (0.5 MBq/µmol; PerkinElmer, Waltham, MA, USA) was incubated at 27°C. At the indicated time points, the proteoliposomes were separated from the external medium using centrifuge columns containing Sephadex G-50 (fine) (GE Healthcare, Fairfield, CT, USA) to terminate transport. The radioactivity in the eluate was measured with a liquid scintillation counter (PerkinElmer).5,11

Intact Protein Analysis Intact protein analysis was performed as previously described, with modifications.16 The protein used for intact protein analysis was neither reduced nor alkylated. Purified VGLUT2 protein (20


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µg) was precipitated in 10% trichloroacetic acid (TCA) at –30°C for 30 min. The protein pellet was collected by centrifugation at 15000 × g for 5 min and washed twice with a 1:1 (v/v) solution of ethanol:ether at room temperature. The washed pellet was briefly air-dried and immediately resuspended in 5 µL of 50% formic acid, 25% acetonitrile, 15% isopropanol, and 10% water (FAPH buffer). The solution was analyzed immediately by MALDI-TOF MS as described below and was not stored.

Sample Preparation by Digestive Enzymes Samples were prepared as previously described, with modifications.16 Protein (10 µg) was precipitated in 10% TCA at –30°C for 30 min. The protein pellet was collected by centrifugation at 15000 × g for 5 min and washed twice with a 1:1 (v/v) solution of ethanol:ether at room temperature. The washed pellet was briefly air-dried, and the protein pellet was then reduced with 5 mM tris (2-carboxyethyl) phosphine (TCEP) at room temperature for 1 h and alkylated with 100 mM iodoacetamide in the dark for 1 h. The protein was then precipitated in 10% TCA at –30°C for 30 min. The protein pellet was collected by centrifugation at 15000 × g for 5 min and washed three times with a 1:1 (v/v) solution of ethanol:ether at room temperature. For trypsin digestion, the washed pellet was briefly air-dried and resuspended in 20 µL of solution containing 50 mM ammonium bicarbonate (AMBIC) buffer, pH 8.0. Trypsin (Promega, Madison, WI, USA) was added at a 1:20 ratio and digestion was allowed to proceed at 37°C for 24 h. Digested peptides were dried in a SpeedVac and resuspended in FAPH buffer. For chymotrypsin digestion, the washed pellet was briefly air-dried and resuspended in 20 µL of solution containing 100 mM Tris-HCl and 10 mM CaCl2, pH 8.0. Chymotrypsin



(Promega) was added at a 1:20 ratio and digestion was allowed to proceed at 25°C for 24 h. Digested peptides were dried in a SpeedVac and resuspended in FAPH buffer. For trypsin and Asp-N digestion, the washed pellet was briefly air-dried and resuspended in 20 µL of solution containing 50 mM AMBIC buffer, pH 8.0. Trypsin was added at a 1:20 ratio and digestion was allowed to proceed at 37°C for 24 h. Digested peptides were dried in a SpeedVac and resuspended in 20 µL of solution containing 50 mM AMBIC buffer, pH 8.0. Asp-N (Promega) was added at a 1:20 ratio and digestion was allowed to proceed at 37°C for 24 h. Digested peptides were dried in a SpeedVac and resuspended in FAPH buffer.

MALDI-TOF MS MALDI-TOF MS analysis was performed with an UltrafleXtreme MALDI-TOF/TOF Mass Spectrometer system (Bruker Daltonics, Karlsruhe, Germany). For intact protein analysis, an aliquot of sample was spotted onto an MTP 384 target plate polished steel TF plate using the double-layer preparation method. Matrix solution (0.5 µL, a saturated solution of sinapinic acid in ethanol) was spotted onto the target plate. An aliquot of 1 µL of the sample elute was mixed with 4 µL of freshly prepared matrix solution (a saturated solution of sinapinic acid in acetonitrile [ACN]:0.1% trifluoroacetic acid [TFA] 1:2), and 1 µL of this mixture was deposited onto the matrix spot and allowed to dry. External calibration was performed using protein standards II (Bruker Daltonics) and the bovine serum albumin (BSA; Sigma) dimer ([2M+H]+ = 132861.0 Da). Spectral masses obtained in linear positive mode were acquired with a range of m/z 10000–200000. For peptide analysis, samples were spotted onto an MTP AnchorChip 600/384 TF plate using the thin-layer affinity α-cyano-4-hydroxycinnamic acid (HCCA) AnchorChip preparation method. After the preparation of thin-layer affinity HCCA on the target plate,


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samples were diluted 1:20 in 0.1% TFA, and 2 µL was spotted onto the target plate. Similarly, external calibration was performed using peptide calibration standard II (Bruker Daltonics) diluted 1:7 in 0.1% TFA, and 1 µL was spotted onto the target plate. Peptide masses obtained in reflector positive mode were acquired with a range of m/z 300–6000. Mass accuracy was set at 20 ppm. Two to four thousand laser shots were accumulated for each spectrum. The MS and MS/MS spectra were assigned using BioTools software v.3.2 (Bruker Daltonics), and the results were searched against the NCBI protein databases using Mascot (www.matrixscience.com). The following specified parameters were applied for database search: taxonomy (rat VGLUT2, mouse VEAT, and human VAChT); proteolytic enzyme (trypsin); peptide mass tolerance (± 100 ppm); global modification (carbamidometyl [Cys]); and maximum missed cleavage (1).

Liquid Chromatography (LC)-MS/MS LC-MS/MS was performed as previously described, with modifications.18 The peptides were separated with an easy-nanoLC-liquid chromatographer (Bruker Daltonics), which was coupled to a Proteineer fc II collector (Bruker Daltonics). Solvent A consisted of water containing 0.1% TFA, and solvent B consisted of ACN containing 0.1% TFA. Separation was performed on a C-18 column (EASY-Column; 10 cm, i.d., 75 µm, 3 µm; C18-A2; Thermo Fisher Scientific, Waltham, MA, USA) with a linear gradient of 2% to 98% solvent B in 48 min and a flow rate of 300 nL/min. The separated peptides were mixed with the HCCA matrix directly in the Proteineer fc II fraction collector and spotted onto an MTP Anchorchip 384 BC target plate (Bruker Daltonics) with an interval of two spots per min. The HCCA matrix used for spotting was prepared as follows: 748 µL TA95 (ACN:0.1%TFA 95:5), 36 µL saturated HCCA matrix-TA90 (ACN:0.1%TFA 90:10), 8 µL of 10% TFA, and 8 µL of 100 mM NH4H2PO4. One per four spots was spotted with peptide calibration standard II (Bruker Daltonics) diluted 1:200



in the HCCA matrix. MS data were acquired with an UltrafleXtreme MALDI-TOF/TOF Mass Spectrometer (Bruker Daltonics) in reflector positive mode. The acquisition was assisted by applying WARP-LC software v.1.2 (Bruker Daltonics) for automatic TOF-MS spectra acquisition followed by optimized precursor selection for subsequent MS/MS experiments. MS/MS data acquired through WARP-LC were searched on MASCOT using Protein Scope (Bruker Daltonics) with the search restricted to rat for VGLUT2, mouse for VEAT, and human for VAChT on the NCBI database. The search was limited to trypsin peptides with carbamidometyl cysteine and an MS/MS tolerance of ± 0.7 Da. The peptide tolerance was set to ± 100 ppm. The peptide charge was defined as 1, and one missed cleavage was allowed.

Results and Discussion Overexpression and Purification of VGLUT2 in E. coli As the first step in this study, the cDNA encoding rat VGLUT2 was cloned into E. coli expression vectors with α-helix soluble proteins and 6× His tags and was overexpressed in E. coli. The membrane fractions were solubilized, and VGLUT2 was purified via Ni-NTA affinity column chromatography. The final fraction contained the major protein band of the expected apparent molecular mass of 94000, as determined by Coomassie Brilliant Blue (CBB) staining and immunoblotting using anti-6× His antibodies (Figure 1A). The purified VGLUT2 was incorporated into proteoliposomes, and we observed VGLUT2-mediated glutamate transport in a membrane potential-dependent manner; this transport was completely inhibited by 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and acetoacetate, two known inhibitors of SLC17 transporters such as VGLUT (Figure 1B).11

MALDI-TOF MS Analysis of Intact VGLUT2 Protein


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For MALDI-TOF MS analysis of the intact protein, the detergent was removed by TCA precipitation. After washing, the pellet was resuspended in FAPH buffer, mixed 1:4 with sinapinic acid matrix, and spotted on the target plate. The MALDI spectrum of the intact protein contained a singly charged monomer by protonation (Figure 1C), with a mass of 93541.338 ± 10.8, and an error between predicted (93501.8) and experimental mass of -0.04%.

Trypsin Digestion of VGLUT2 and the Peptide Detection by MALDI-TOF MS Trypsin digestion of the purified VGLUT2 protein was performed to analyze the peptides by MALDI-TOF MS. VGLUT2 protein was reduced with TCEP and alkylated with iodoacetamide. After TCA precipitation and washing with ethanol:ether (1:1 [v/v]), the pellet containing 10 µg denatured VGLUT2 protein was resuspended in AMBIC buffer, and trypsin was added at an enzyme:protein ratio of 1:20. The sample was evaluated by MALDI-TOF MS and analyzed using the Mascot search engine (Figure 2). The MALDI-TOF data obtained from 33 digested VGLUT2 peptides were matched significantly with Rattus novegicus VGLUT2 (gi|16758166). The score of Mascot search matching was 237, and sequence coverage was 70% (Table 1 and Figure 2B). Nine peptide fragments generated from VGLUT2 by trypsin cleavage were subjected to MS/MS analysis for sequencing. Using MS/MS ion search (MIS), five peptides were fully identified as VGLUT2-derived peptides, and four peptides were partially identified as VGLUT2-derived peptides (Table 2). Furthermore, the VGLUT2-derived peptides were analyzed by LC-MS/MS to improve the detection efficiency. The sequence coverage was 87% using LC-MS, and 21 peptide fragments were detected as VGLUT2-derived peptides using LC-MS/MS (Figure 2B, Table S1 and 2).

MALDI MS Analysis of Other Transporters



We tested the above method on other vesicular neurotransmitter transporters, such as mouse VEAT and human VAChT. These transporters were overexpressed in E. coli, and the membrane fractions were solubilized and purified via Ni-NTA affinity column chromatography. The final fraction contained major protein bands at the apparent molecular mass of 75000, as determined by CBB staining and immunoblotting (Figures 3A and 4A).5,17 We previously showed that proteoliposomes containing purified transporters retained membrane potential-dependent aspartate and H+/acetylcholine transport activity.5,17 Similar to VGLUT2, the MALDI spectrum of the intact protein displayed a singly charged monomer (Figures 3B and 4B). VEAT had a mass of 86713.050 ± 6.4, and the error between predicted (86706.0) and experimental mass was -0.01%. VAChT had a mass of 89327.099 ± 12.3, and the error between predicted (89298.2) and experimental mass was -0.03%. Although the major protein bands of VEAT and VAChT are present at slightly lower than expected apparent molecular weight, the present results strongly suggest that these major bands contain proteins whose molecular weight was 87000 and 89000, respectively. Trypsin digestion of the purified VEAT and VAChT proteins was performed to analyze the peptides by MALDI MS, as performed for the VGLUT2 experiment (Figures 3C and 4C). The MALDI-TOF data obtained from digested VEAT and VAChT were matched significantly with Mus musculus VEAT (gi|27370146) and Homo sapiens VAChT (gi|507744). Mascot search scores were 108 and 138, and sequence coverage was 54% and 56%, respectively (Tables 3 and 4, Figures 3D and 4D). A total of 19 and 21 peptide fragments generated from VEAT and VAChT by trypsin cleavage were subjected to MS/MS analysis for sequencing, respectively. Using MIS, one and zero peptides were fully identified as VEAT- and VAChT-derived peptides, and five and seven peptides were partially identified as VEAT- and VAChT-derived peptides, respectively (Tables 5 and 6). Furthermore, the VEAT- or


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VAChT-derived peptides were analyzed by LC-MS/MS. Twelve and eight peptide fragments were detected as VEAT- and VAChT-derived peptides, respectively (Tables 5 and 6). Sequence coverage was 52% and 57% using MALDI-TOF MS with LC, respectively (Tables S2 and S3). To improve sequence coverage, we further examined the combinatorial effects of digestive enzymes such as chymotrypsin, or trypsin and Asp-N. In total, 73 and 89 peptide fragments were matched with VEAT- and VAChT-derived peptides digested by chymotrypsin, and trypsin + Asp-N, respectively (Tables S4 and S5). Using MIS, three and zero peptides were fully identified as VEAT- and VAChT-derived peptides, and four and eleven peptides were partially identified as VEAT- and VAChT-derived peptides, respectively (Tables S6 and S7). Accordingly, sequence coverage was improved to 90 and 91%, respectively (Figures 3D and 4D). A combination of digestive enzymes could improve sequence coverage due to the difference of cleavage sites dependent on the amino acid sequence of transporters. Thus, we developed an efficient method for mass spectral analysis of eukaryotic transporters with fully retained activity in combination with an overexpression system for membrane proteins in E. coli (Figure 5).

Further Applications of the Methodology Given that our methodology allows for the purification of fully active transporters, its combination with chemical modifiers is expected to enable the identification of the binding peptide of the chemical modifier. VGLUT is responsible for the accumulation of glutamate in synaptic vesicles and is essential for glutamatergic chemical transmission.19,20 Membrane potential-dependent glutamate transport is activated by the chloride ion in an allosteric manner and is reversibly inhibited by ketone bodies, such as acetoacetate.11 This metabolic anion



switch-off inhibits the excess excitatory neurotransmission and reduces the occurrence of epileptic seizures.11 Although VGLUT is a good drug target for epilepsy, the structure of VGLUT remains unknown. Interestingly, the chemical modifier DIDS inhibits VGLUT in a Cl--dependent manner,11,21 and identification of DIDS binding sites can therefore help elucidate the structure of the Cl- binding site in VGLUT. To facilitate drug discovery, further mass spectral studies with chemical modifiers are required to identify the Cl--binding site in VGLUT. Similarly, this methodology will help to characterize the function and structure of VEAT and VAChT.

Conclusions We developed an efficient method for mass spectral analysis of eukaryotic membrane proteins that avoids denaturation and is combined with an overexpression system in E. coli. Based on coverage of the amino acid sequence, this method was comparable to analysis of eukaryotic membrane proteins with denaturation.16 Our methodology can provide a new approach for analyzing membrane proteins in combination with chemical modifiers, facilitating applications in functional binding analysis for drug discovery.

Supporting Information: The Supporting Information is available free of charge on the ACS Publications website. Table S1:

Summary of LC-MS data for trypsin-digested peptides from VGLUT2.

Table S2:

Summary of LC-MS data for trypsin-digested peptides from VEAT.

Table S3:

Summary of LC-MS data for trypsin-digested peptides from VAChT.

Table S4:

Summary of MS data for chymotrypsin and trypsin+Asp-N digested peptides from VEAT.


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Table S5:

Summary of MS data for chymotrypsin and trypsin+Asp-N digested peptides from VAChT.

Table S6:

Summary of MS/MS data for digested peptides from VEAT in Table S4.

Table S7:

Summary of MS/MS data for digested peptides from VAChT in Table S5.

Acknowledgments We wish to thank Prof. Nathan Nelson (Tel Aviv University) for providing the vectors and the Advanced Science Research Center, Okayama University, Japan and Bruker Daltonics, Kanagawa, Japan for their help in the mass spectral study. This work was supported in part by AMED under Grant Number JP17gm5910019, Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research (C) (no. 26460067), and JSPS Challenging Research (Exploratory) (no. 17K19489) to T.M.

Author Contributions M.K. and T.M. designed the experiments, analyzed the data, wrote the paper, and performed the experiments. N.J., Y.K., H.O., and Y.M. designed the experiments, analyzed the data, and wrote the paper.

Competing Financial Interest Statement The authors declare that they have no competing financial interests.



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9.

Miyaji T.; Kuromori T.; Takeuchi Y.; Yamaji N.; Yokosho K.; Shimazawa A.; Sugimoto E.; Omote H.; Ma J.F.; Shinozaki K.; Moriyama Y. AtPHT4;4 is a chloroplast-localized ascorbate transporter in Arabidopsis. Nature Commun. 2015, 6, DOI: 10.1038/ncomms6928.

10. Juge N.; Moriyama S.; Miyaji T.; Kawakami M.; Iwai H.; Fukui T.; Nelson N.; Omote H.; Moriyama Y. Plasmodium falciparum chloroquine resistance transporter is an H+-coupled polyspecific nutrient and drug exporter. Proc. Natl Acad. Sci. USA 2015, 112, 3356-3361. 11. Juge, N. et al. Metabolic control of vesicular glutamate transport and release. Neuron 2010, 68, 99-112. 12. Miyaji T. et al. Identification of a vesicular aspartate transporter. Proc. Natl Acad. Sci. USA 2008, 105, 11720-11724. 13. Loo, R.R.; Dales, N.; Andrews, P.C. Surfactant effects on protein structure examined by electrospray ionization mass spectrometry. Protein Sci. 1994, 3, 1975-1983. 14. Beavis, R.C.; Chait, B.T. Rapid, sensitive analysis of protein mixtures by mass spectrometry. Proc. Natl Acad. Sci. USA 1990, 87, 6873-6877. 15. Weinglass, A.B. et al. Elucidation of substrate binding interactions in a membrane transport protein by mass spectrometry. EMBO J. 2003, 22, 1467-1477. 16. Cox, HD.; Chao, CK.; Patel, SA.; Tompspn, CM. Efficient digestion and mass spectral analysis of vesicular glutamate transporter 1: A recombinant membarne protein expressed in yeast. J. Proteome Res. 2008, 7, 570-578. 17. Hiasa M.; Miyaji T.; Haruna Y.; Takeuchi T.; Harada Y.; Moriyama S.; Yamamoto A.; Omote H.; Moriyama Y. Identification of a mammalian vesicular polyamine transporter. Sci. Rep. 2014, 4, DOI:10.1038/srep06836. 18. Gekenidis, M.T.; Studer, P.; Wüthrich, S.; Brunisholz, R., Drissner, D. Beyond the



Matorix-Assisted Laser Desorption Ionization (MALDI) biotyping workflow: in search of microorganism-specific tryptic peptides enabling discrimination of subspecies. Appl. Environ. Microbiol. 2014, 80, 4234–4241. 19. Fremeau, R.T. Jr; Voglmaier, S.; Seal, R.P., Edwards, R.H. VGLUTs define subsets of excitatory neurons and suggest novel roles for glutamate. Trends Neurosci. 2004, 27, 98-103. 20. Omote, H.; Miyaji, T.; Juge, N.; Moriyama, Y. Vesicular neutotransmitter transporter: Bioenergetics and regulation of glutamate transport. Biochemistry 2011, 50, 5558-5565. 21. Hartinger, J.; Jahn, R. An anion binding site that regulates the glutamate transporter of synaptic vesicles. J. Biol. Chem. 1993, 268, 23122-23127.


Page 18 of 35

Page 19 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure Legends Figure 1. Purification and MALDI mass spectrum of the recombinant VGLUT2 protein. (A) Purification of recombinant vesicular glutamate transporter 2 (VGLUT2). The purified fraction (5 and 0.5 µg) was analyzed by SDS-PAGE on 10% gels and visualized by Coomassie Brilliant Blue staining (CBB) (left) and immunoblotting using anti-6× His antibodies (right). The molecular mass of purified β-VGLUT2-α protein (94000 Da) was of the expected size due to YbeL (β, 121 amino acids) and YaiN (α, 95 amino acids) conjugation at the N- and C-termini. The positions of marker proteins are indicated on the left. The position of the recombinant protein is indicated by an arrow. (B) Glutamate uptake for 2 min in the absence or presence of 2 µM valinomycin (Val), 2 µM Val and 2 µM DIDS, or 2 µM Val and 1 mM acetoacetate. N = 3, data represent the mean ± SEM. **P < 0.01 (two-tailed paired Student’s t-test). (C) Spectrum of purified intact VGLUT2 (20 µg) resulting from the accumulation of 10000 laser shots in linear mode. The position of the recombinant protein is indicated by an arrow. N = 13, data represent the mean ± SEM.

Figure 2. MALDI mass spectrum of VGLUT2 peptides digested in solution by trypsin. (A) The purified VGLUT2 (10 µg) was subjected to reductive alkylation and digested with trypsin. The mass spectrum identified peptides below 3700 Da as derived from VGLUT2. The m/z (lower) and assigned peptide sequences (upper) are labeled. (B) Putative membrane topology and sequence coverage of VGLUT2. Transmembrane regions are shown in colored boxes. + and - indicate detection by LC-MS and MS, respectively.

Figure 3. Purification and MALDI mass spectrum of intact and digested VEAT protein. (A) Purification of recombinant mouse vesicular excitatory amino acid transporter (VEAT). The



purified fraction (5 and 0.5 µg) was analyzed by SDS-PAGE on 10% gels and visualized by Coomassie Brilliant Blue (CBB) staining (left) and immunoblotting using anti-6× His antibodies (right). The molecular mass of purified β-VEAT-β protein (87000 Da) was of the expected size due to YbeL conjugation at the N- and C-termini. The positions of marker proteins are indicated on the left. The position of the recombinant protein is indicated by an arrow. (B) The spectrum of purified intact VEAT (20 µg) results from accumulation of 10000 laser shots in linear mode. The position of the recombinant protein is indicated by an arrow. N = 13, data represent the mean ± SEM. (C) The purified VEAT (10 µg) was subjected to reductive alkylation and digested with trypsin. The mass spectrum identified peptides below 4500 Da as derived from VEAT. The m/z (lower) and assigned peptide sequences (upper) are labeled. (D) Putative membrane topology and sequence coverage of VEAT. Transmembrane regions are shown in color boxes. + and - indicate detection by LC-MS and MS, respectively, following trypsin digestion. * indicates detection by other digestive enzymes with MS.

Figure 4. Purification and MALDI mass spectrum of intact and digested VAChT protein. (A) Purification of recombinant human vesicular acetylcholine transporter (VAChT). The purified fraction (5 and 0.5 µg) was analyzed by SDS-PAGE on 10% gels and visualized by Coomassie Brilliant Blue (CBB) staining (left) and immunoblotting using anti-6× His antibodies (right). The molecular mass of purified β-VAChT-β protein (89000 Da) was of the expected size due to YbeL conjugation at the N- and C-termini. The positions of marker proteins are indicated on the left. The position of the recombinant protein is indicated by an arrow. (B) The spectrum of purified intact VAChT (20 µg) results from accumulation of 10000 laser shots in linear mode. The position of the recombinant protein is indicated by an arrow. N = 13, data represent the mean ± SEM. (C) The purified VAChT (10 µg) was subjected to reductive alkylation and


Page 20 of 35

Page 21 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


digested with trypsin. The mass spectrum identified peptides below 4300 Da as derived from VAChT. The m/z (lower) and assigned peptide sequences (upper) are labeled. (D) Putative membrane topology and sequence coverage of VAChT. Transmembrane regions are shown in color boxes. + and – indicate detection by LC-MS and MS, respectively, following trypsin digestion. * indicates detection with other digestive enzymes using MS.

Figure 5. Schematic representation of mass spectral analysis of eukaryotic active transporters. Upper and lower schemes indicate the methods for the digested peptides and intact protein, respectively. IAA = iodoacetamide; TCA = trichloroacetic acid; HCCA = α-cyano-4-hydroxycinnamic acid; SA = sinapinic acid.



Page 22 of 35

Table 1. Summary of MS data for trypsin-digested peptides from VGLUT2 in Fig. 2. N.D. = not detected; CAM = carbamidomethyl cysteine.

Residues 1–5

Observed

Calculated

∆Mass

mass (Da)

mass (Da)

(Da)

N.D.

593.297

S/N

Intensity

0.042

14.4

1390.61

Missed cleavage

Modification

6–7

N.D.

303.178

6–13

882.595

882.553

8–13

N.D.

598.393

14–17

446.284

446.261

0.023

12.0

539.17

18–22

536.302

536.283

0.019

6.5

342.62

23–29

836.485

836.463

0.022

150.8

13033.09

30–33

488.333

488.308

0.024

20.2

923.89

34–38

660.364

660.343

0.021

11.4

753.67

35–38

532.268

532.248

0.020

15.9

782.34

39–48

N.D.

1134.553

39–55

1927.013

1926.991

0.022

11.7

2184.29

1

49–55

N.D.

811.457

56–69

1694.808

1694.782

0.026

23.7

4540.71

1

57–69

1566.710

1566.687

0.023

253.4

45770.56

3CAM

71–88

2018.069

2018.061

0.008

8.3

1555.78

1CAM

89–106

2013.031

2013.001

0.030

93.2

15275.34

1CAM

110–112

N.D.

359.266

117–148

3659.847

3659.779

0.069

5.1

201.06

149–153

544.336

544.321

0.015

20.2

1008.71

149–174

2684.541

2684.529

0.013

6.2

777.77

154–174

2159.243

2159.226

0.017

36.2

5452.41

175–184

1249.679

1249.652

0.028

459.1

69081.85

175–204

3458.806

3458.787

0.019

7.3

350.38

185–204

2228.172

2228.154

0.018

19.2

2848.36

205–211

868.497

868.468

0.029

486.6

44347.70

214–270

N.D.

6343.081

271–279

1097.551

1097.523

0.028

68.3

8770.98

271–280

1253.660

1253.624

0.036

22.2

3443.78

281–298

1953.968

1953.948

0.020

10.3

1946.32

301–304

559.317

559.299

0.017

138.8

6922.27

301–305

687.350

687.394

-0.044

5.5

402.00

306–322

2036.030

2036.014

0.016

28.0

4660.02

323–347

N.D.

3014.523

1

1

3CAM

1

1CAM 1

2CAM 1CAM

1CAM


1

1 1CAM

Page 23 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


348–374

2847.624

2847.599

0.025

33.7

2732.52

377–385

1018.609

1018.590

0.019

16.0

1903.12

387–409

2526.239

2526.231

0.008

10.8

1310.02

410–440

3188.765

3188.726

0.039

11.9

805.03

441–469

N.D.

2940.507

472–502

3563.903

3563.837

474–502

N.D.

3320.704

503–515

N.D.

1545.671

516–542

3162.426

3162.385

543–560

N.D.

1967.852

561–576

1921.905

1921.882

562–576

N.D.

1793.787

579–582

N.D.

499.168

1CAM

1CAM 0.066

5.4

243.11

0.041

17.0

1126.58

0.023

45.6

7997.22


1

1CAM

1


Page 24 of 35

Table 2. Summary of MS/MS and LC-MS/MS data for trypsin-digested peptides from VGLUT2. N.D. = not detected.

Residues Sequence

Calculated

Observed mass (Da)

mass (Da)

MS/MS

LC-MS/MS

836.463

836.497

836.497

23–29

SLGQIYR

23–33

SLGQIYRVLEK

1305.753

N.D.

1305.759

39–55

ETIELTEDGKPLEVPEK

1926.991

N.D.

1927.058

56–69

KAPLCDCTCFGLPR

1694.782

N.D.

1694.823

57–69

APLCDCTCFGLPR

1566.687

1566.726

1566.708

89–106

CNLGVAIVDMVNNSTIHR

2013.001

2013.046

2013.081

544.321

544.336

N.D.

2684.529

N.D.

2684.600

149–153 LAANR 149–174 LAANRVFGAAILLTSTLNMLIPSAAR 154–174 VFGAAILLTSTLNMLIPSAAR

2159.226

N.D.

2159.289

175–184 VHYGCVIFVR

1249.652

1249.694

1249.664

185–204 ILQGLVEGVTYPACHGIWSK

2228.154

2228.174

2228.205

868.468

868.508

868.495

271–279 HPTITDEER

1097.523

1097.563

1097.551

281–298 YIEESIGESANLLGAMEK

1953.948

N.D.

1953.972

306–322 FFTSMPVYAIIVANFCR

2036.014

N.D.

2036.050

348–374 VGMLSAVPHLVMTIIVPIGGQIADFLR

2847.599

N.D.

2847.634

377–385 QILSTTTVR

1018.590

1018.609

N.D.

410–440 GVAISFLVLAVGFSGFAISGFNVNHLDIAPR

3188.726

N.D.

3188.759

474–502 EEWQYVFLIAALVHYGGVIFYALFASGEK

3320.704

N.D.

3320.689

503–515 QPWADPEETSEEK

1545.671

N.D.

1545.717

516–542 CGFIHEDELDEETGDITQNYINYGTTK

3162.385

N.D.

3162.389

561–576 KEEFVQESAQDAYSYK

1921.882

N.D.

1921.960

562–576 EEFVQESAQDAYSYK

1793.787

N.D.

1793.813

205–211 WAPPLER


Page 25 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 3. Summary of MS data for trypsin-digested peptides from VEAT in Fig. 3. N.D. = not detected; CAM = carbamidomethyl cysteine.

Residues 1–2

Observed

Calculated

∆Mass

mass (Da)

mass (Da)

(Da)

N.D.

306.160

S/N

Intensity

Missed cleavage

Modification

3–6

N.D.

498.340

7–27

2085.060

2084.937

0.123

204.9

137121.68

28–39

1349.652

1349.594

0.058

12.8

7704.99

2CAM

40–57

2151.214

2151.147

0.067

26.7

17510.25

1CAM

40–78

4409.440

4409.268

0.172

2.5

48.65

58–78

2277.236

2277.140

0.096

74.6

44883.29

79–81

N.D.

335.193

82–91

1141.600

1141.531

0.069

24.1

11095.73

92–99

929.496

929.471

0.025

6.9

2004.06

101–102

N.D.

310.177

103–132

3395.845

3395.674

0.171

4.3

625.23

133–136

N.D.

360.225

133–168

3623.360

3623.192

0.168

2.4

214.10

137–168

3282.171

3281.986

0.185

11.8

2063.39

169–195

2940.537

2940.418

0.119

4.8

1419.03

198–256

N.D.

6859.551

1

1CAM

1CAM

1

1CAM

257–263

N.D.

877.442

257–271

1796.962

1796.943

264–271

N.D.

938.520

272–278

N.D.

804.422

279–287

998.640

998.604

288–317

N.D.

3574.885

318–321

530.342

530.330

322–353

N.D.

3813.769

356–364

1109.613

1109.574

0.039

217.6

96233.72

356–365

1265.617

1265.675

-0.058

7.0

3836.23

366–445

N.D.

8093.261

446–454

1016.566

1016.538

0.029

8.9

3143.37

455–479

2864.465

2864.485

-0.020

3.7

1156.45

480–494

1715.859

1715.800

0.058

139.4

108386.04

0.019

7.4

5777.44

0.037

10.3

3454.08

0.012

5.1

638.17

1

3CAM

1CAM


1CAM


Page 26 of 35

Table 4. Summary of MS data for trypsin-digested peptides from VAChT in Fig. 4. N.D. = not detected; CAM = carbamidomethyl cysteine.

Residues 1–11

Observed

Calculated

∆Mass

mass (Da)

mass (Da)

(Da)

N.D.

1146.521

S/N

Intensity

12–16

461.279

461.272

0.006

9.7

834.83

12–29

1782.990

1782.971

0.019

4.4

2993.37

17–29

1340.754

1340.717

0.036

214.5

59930.47

31–32

N.D.

303.178

31–33

459.290

459.279

0.011

5.4

486.05

34–65

N.D.

3696.053

66–73

730.361

730.348

74–112

N.D.

3964.972

74–114

4218.413

115–123

Missed cleavage

Modification

1

1 1CAM

0.012

105.8

12064.67

4218.126

0.287

13.7

1309.23

1067.525

1067.490

0.036

8.5

1740.52

115–131

1883.042

1882.980

0.062

3.4

2583.23

124–131

N.D.

834.509

132–148

1866.153

1866.085

0.067

535.5

223998.11

149–182

3699.953

3699.885

0.068

8.2

3150.90

183–203

N.D.

2053.028

183–210

2953.560

2953.425

0.135

34.1

11208.34

204–210

919.437

919.416

0.021

30.2

4661.84

213–241

N.D.

2912.597

243–264

N.D.

2283.409

243–271

2983.845

2983.775

0.070

5.0

3227.93

265–271

N.D.

719.384

274–284

1174.701

1174.670

0.031

111.5

24384.77

285–317

N.D.

3633.914

318–347

N.D.

3399.685

348–351

430.290

430.278

352–378

N.D.

2972.528

379–412

3648.141

3647.995

413–470

N.D.

6111.319

471–477

772.479

772.468

480–482

N.D.

391.194

483–498

1799.986

483–500 501–502

1

1

1

1

1CAM

0.012

20.9

1705.12 2CAM

0.146

8.7

3187.45

0.011

34.0

4246.54

1799.918

0.068

233.9

96725.51

2069.172

2069.103

0.069

4.9

3235.21

N.D.

304.162


2CAM

1

Page 27 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


501–512

1326.693

1326.640

503–512

N.D.

1041.497

513–531

2388.019

2387.940

0.053

36.6

10374.63

0.079

124.3

48168.11


1

1CAM


Page 28 of 35

Table 5. Summary of MS/MS and LC-MS/MS data for trypsin-digested peptides from VEAT. N.D. = not detected. Calculated

Residues Sequence

Observed mass (Da)

mass (Da)

MS/MS

LC-MS/MS

7–27

GPAGNDDEESSDSTPLLPGAR

2084.937

2085.073

N.D.

28–39

QTEAAPVCCSAR

1349.594

1349.662

1349.620

40–57

YNLAILAFCGFFVLYALR

2151.147

N.D.

2151.162

58–78

VNLSVALVDMVDSNTTLTDNR

2277.140

2277.262

2277.139

82–91

ECAEHSAPIK

1141.531

N.D.

1141.542

92–99

VHHNHTGK

929.471

929.488

929.478

257–263 TISHYEK

877.442

N.D.

877.452

264–271 EYIVSSLK

938.520

N.D.

938.530

272–278 NQLSSQK

804.422

N.D.

804.434

279–287 VVPWGSILK

998.604

N.D.

998.618

356–364 WNFSTISVR

1109.574

1109.627

1109.584

446–454 SLTPDNTIR

1016.538

N.D.

1016.518

480–494 GEVQSWALSDHHGHR

1715.800

1715.877

1715.815


Page 29 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 6. Summary of MS/MS and LC-MS/MS data for trypsin-digested peptides from VAChT. N.D. = not detected. Calculated

Residues Sequence

Observed mass (Da)

mass (Da)

MS/MS

LC-MS/MS

1340.717

1340.737

N.D.

17-29

LSEAVGAALQEPR

66-73

GGGEGPTR

730.348

730.378

N.D.

115-123

YPTESEDVK

1067.490

1067.525

1067.499

124-131

IGVLFASK

834.509

N.D.

834.507

132-148

AILQLLVNPLSGPFIDR

1866.085

1866.248

1866.111

204-210

YPEEPER

919.416

919.505

919.427

243-271

VPFLVLAAVSLFDALLLLAVAKPFSAAAR

2983.775

N.D.

2983.811

274-284

ANLPVGTPIHR

1174.670

1174.686

N.D.

471-477

NVGLLTR

772.468

N.D.

772.468

483-498

DVLLDEPPQGLYDAVR

1799.918

1800.095

N.D.

501-512

ERPVSGQDGEPR

1326.640

N.D.

1326.661

513-531

SPPGPFDECEDDYNYYYTR

2387.940

N.D.

2387.937



100 75 50

∗∗

40 30 20

∗∗ 10

∗∗

0

1

m M

Ac

2

μM

-V al

35

50

et oa ce ta te

WB

D ID S

CBB

+V al

kDa 150

B Glutamate uptake (nmol/mg)

A

C

80 60

Intensity

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 35

40 20 0 60000

70000

80000

90000

m/z

100000 110000 120000

Fig.1

ACS Paragon Plus Environment

Page 31 of 35

A ×104 175-184 1249 18-22 536

205-211 868

30-33 149-153 301-305 488 544 687 35-38 14-17 301-304 34-38 532 446 559 660

Intensity

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


57-69 1566

23-29 6-13 836 882

377-385 1018

561-576 1921 39-55 1927

271-280 1253

271-279 1097

281-298 1953

56-69 1694

×104

89-106 2013

71-88 2018 306-322 2036 154-174 2159

149-174 2684

185-204 2228

387-409 2526

516-542 3162

348-374 2847

410-440 3188

175-204 3458

472-502 117-148 3563 3659

m/z

B

MESVKQRILA PGKEGIKNFA GKSLGQIYRV LEKKQDNRET IELTEDGKPL EVPEKKAPLC ±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±-----±± ±±±±±±±±±± ±±±±±±±±±±

60

DCTCFGLPRR YIIAIMSGLG FCISFGIRCN LGVAIVDMVN NSTIHRGGKV IKEKAKFNWD ±±±±±±±±±+ ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±++++ ++ ±±±±

120

PETVGMIHGS FFWGYIITQI PGGYIASRLA ANRVFGAAIL LTSTLNMLIP SAARVHYGCV

180

TMD1

TMD2

TMD3

±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± TMD4

TMD5

IFVRILQGLV EGVTYPACHG IWSKWAPPLE RSRLATTSFC GSYAGAVIAM PLAGILVQYT ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±++

240

GWSSVFYVYG SFGMVWYMFW LLVSYESPAK HPTITDEERR YIEESIGESA NLLGAMEKFK ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±++

300

TPWRKFFTSM PVYAIIVANF CRSWTFYLLL ISQPAYFEEV FGFEISKVGM LSAVPHLVMT ±±±±±±±±±± ±±±±±±±±±± ±±++++++++ ++++++++++ +++++++±±± ±±±±±±±±±±

360

IIVPIGGQIA DFLRSKQILS TTTVRKIMNC GGFGMEATLL LVVGYSHTRG VAISFLVLAV ±±±±±±±±±± ±±±±++±±±± ±±±±±+±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±±

420

GFSGFAISGF NVNHLDIAPR YASILMGISN GVGTLSGMVC PIIVGAMTKN KSREEWQYVF ±±±±±±±±±± ±±±±±±±±±± ++++++++++ ++++++++++ ++++++++++ +±±±±±±±±±

480

LIAALVHYGG VIFYALFASG EKQPWADPEE TSEEKCGFIH EDELDEETGD ITQNYINYGT ±±±±±±±±±± ±±±±±±±±±± ±±++++++++ +++++±±±±± ±±±±±±±±±± ±±±±±±±±±±

540

TKSYGATSQE NGGWPNGWEK KEEFVQESAQ DAYSYKDRDD YS

582

TMD6

TMD7

TMD8

TMD9

TMD10

TMD11

TMD12

±±++++++++ ++++++++++ ±±±±±±±±±± ±±±±±±


Fig.2


A

kDa 150

CBB

B

WB

80

100

60

Intensity

75 50

C

40 20

35

Intensity

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

0

60000

70000

80000

90000

m/z

100000 110000 120000

×105

356-364 1109 279-287 446-454 82-91 998 1016 1141 92-99 929

318-321 530

480-494 1715

356-365 1265 28-39

257-271 1796

1349

×105

7-27 2085

40-57 2151

D

455-479 2864

58-78 2277

169-195 2940

137-168 103-132 3282 3395

133-168 3623

40-78 4409

m/z TMD1

MRPLLRGPAG NDDEESSDST PLLPGARQTE AAPVCCSARY NLAILAFCGF FVLYALRVNL ****±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±±

60

SVALVDMVDS NTTLTDNRTS KECAEHSAPI KVHHNHTGKK YKWDAETQGW ILGSFFYGYI ±±±±±±±±±± ±±±±±±±±++ +±±±±±±±±± ±±±±±±±±±+ ++±±±±±±±± ±±±±±±±±±±

120

VTQIPGGYIA SRVGGKLLLG LGILGTSVFT LFTPLAADLG VVTLVVLRAL EGLGEGVTFP ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±-- ----------

180

AMHAMWSSWA PPLERSKLLT ISYAGAQLGT VISLPLSGII CYYMNWTYVF YLFGIVGIVW ---------- ----- ***** ********** ********** ***

240

FILWMWIVSD TPETHKTISH YEKEYIVSSL KNQLSSQKVV PWGSILKSLP LWAIVVAHFS **** ****** ±±±± ±±±±±±±±±± ±+++++++±± ±±±±±±± * **********

300

YNWSFYTLLT LLPTYMKEIL RFNVQENGFL SALPYFGCWL CMILCGQAAD YLRVKWNFST *** ** ±±± ±********* ********** ****** ***++±±±±±

360

ISVRRIFSLV GMVGPAVFLV AAGFIGCDYS LAVAFLTIST TLGGFASSGF SINHLDIAPS ±±±±* ********** ********** ********** ********** **********

420

YAGILLGITN TFATIPGMTG PIIAKSLTPD NTIREWQTVF CIAAAINVFG AIFFTLFAKG ********** ********** ***** ±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±±

480

EVQSWALSDH HGHRN

495

TMD2

TMD3

TMD4

TMD5

TMD6 TMD7

TMD8

TMD9

TMD10

TMD12

TMD11

±±±±±±±±±± ±±±±


Fig.3

Page 33 of 35

kDa 150

CBB

B

WB

80

100

60

Intensity

75 50

40 20

35

0

60000

70000

80000

C

90000

m/z

100000

110000

120000

×105 132-148 1866 483-498 1799 12-29 1782

348-351 430 31-33 459 12-16 461

Intensity

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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A


×104

471-477 772 66-73 730

204-210 919

115-123 1067

17-29 501-512 1340 274-284 1326 1174

115-131 1883

513-531 2388 379-412 3648

149-182 3699

183-210 243-271 2953 2983

483-500 2069

74-114 4218

D

m/z TMD1

MESAEPAGQA RAAATKLSEA VGAALQEPRR QRRLVLVIVC VALLLDNMLY MVIVPIVPDY ±±±±±±±±± ±±±±±±±±± ---******* ********** **********

60

IAHMRGGGEG PTRTPEVWEP TLPLPTPANA SAYTANTSAS PTAAWPAGSA LRPRYPTESE

120

±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± TMD2

TMD3

DVKIGVLFAS KAILQLLVNP LSGPFIDRMS YDVPLLIGLG VMFASTVLFA FAEDYATLFA ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±-- ---------- ---------- ----------

180

ARSLQGLGSA FADTSGIAMI ADKYPEEPER SRALGVALAF ISFGSLVAPP FGGILYEFAG --±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ++++++++++ ++++++++++ ++++++++++

240

KRVPFLVLAA VSLFDALLLL AVAKPFSAAA RARANLPVGT PIHRLMLDPY IAVVAGALTT + *±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±++±±±±±±± ±±±±*** **********

300

CNIPLAFLEP TIATWMKHTM AASEWEMGMA WLPAFVPHVL GVYLTVRLAA RYPHLQWLYG ***** *** ********** ********** ********** *******±±± ± *******

360

ALGLAVIGAS SCIVPACRSF APLVVSLCGL CFGIALVDTA LLPTLAFLVD VRHVSVYGSV ±± ±±±±±+±±±± ±±±±±±±±±± ±±±±±±±±±± ±±********

420

YAIADISYSV AYALGPIVAG HIVHSLGFEQ LSLGMGLANL LYAPVLLLLR NVGLLTRSRS ********** ** ****** ********** ********** ***** * ±±±±±±±+++

480

ERDVLLDEPP QGLYDAVRLR ERPVSGQDGE PRSPPGPFDE CEDDYNYYYT RS ++±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±±±±±±±±±± ±

532

TMD4

TMD5

TMD6

TMD7

TMD8

TMD9

TMD10

TMD11

TMD12


Fig.4


Overexpression

Solubilization

Purification

Reduction & Alkylation

TCA Precipitation

Page 34 of 35

Trypsin Digestion

Supernatant

N C

YbeL(β)

S

soluble α-helical protein

Overexpression

IAA

S

YaiN(α)

Solubilization

Purification

S

IAA

Pellet

TCA Precipitation

S

IAA IAA

Mass Spectrometry Peptides + HCCA

S

IAA

S

IAA

Intact protein + SA

Mass Spectrometry

Fig.5


Page 35 of 35 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 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Efficient Mass Spectral Analysis of Active ... - ACS Publications

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