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MALDI glycotyping of O-antigens from a single colony of gram-negative bacteria – Scientific Reports

Materials

All E. coli strains, American Type Culture Collection (ATCC) 25922, ATCC43888, ATCC11775, and ATCC700728, and E. tarda strain (ATCC15947) were purchased from Microbiologics (St. Cloud, MN, USA). C. freundii strain (NBRC16624) was purchased from Biological Resource Center, National Institute of Technology and Evaluation (NBRC, Tokyo, Japan). The standard method agar ”DAIGO No. 802” was obtained from Nihon Pharmaceutical Co., Ltd. (Osaka, Japan). Acetic acid, guanidine-HCl, ammonium bicarbonate, triethylamine, dioxan, DHB, sodium bicarbonate, acetonitrile (HPLC grade), HCl, and chloroform were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). The 2,2,2-trifluoroacetic acid (TFA) was purchased from Watanabe Chemical Industry Co. Ltd. (Hiroshima, Japan). The DAN and CHCA were obtained from Sigma-Aldrich Corp. (St. Louis, MO, USA). The 3-methyl-1-p-tolyltriazene and benzyloxyamine hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Blot GlycoH beads were acquired from Sumitomo Bakelite Co., Ltd. (Tokyo, Japan). Acetic anhydride was purchased from Nacalai Tesque Inc. (Kyoto, Japan). All the water used in this study was freshly prepared using Milli-Q® water (Direct-Q 3 UV; Merck Millipore, Tokyo, Japan).

Matrices

For O-antigen analysis, 2 µL of 500 mM DHB in acetonitrile/water (9:1, v/v), 4 µL of 50 mM DAN in acetonitrile/water (1:1, v/v), and 1 µL of 100 mM NaHCO3 in water were mixed and diluted to 100 µL in an acetonitrile/water (1:1 v/v) solution. The DAN/DHB/Na matrix was used within 12 h. CHCA (10 mM) matrix solution in acetonitrile/water/TFA (50:50:0.1, v/v/v) was used for ribosomal polypeptide typing. A DHB (10 mg/mL) matrix solution in chloroform/methanol (9:1, v/v) was used for lipid A typing. For O-antigen analysis using glycoblotting method, 2 µL of 500 mM DHB in acetonitrile/water (9:1, v/v) and 1 µL of 100 mM NaHCO3 in water were mixed and diluted to 100 µL in an acetonitrile/water (1:1 v/v) solution.

O-antigen typing of a single strain

All bacterial strains including E. coli, E. tarda, and C. freundii were grown on agar plates. Several bacterial colonies on the agar plate were transferred into a 1.5-mL tube. The cells were suspended in 1 mL of water, centrifuged at 10,000×g for 2 min, and the supernatant was discarded (Microcentrifuge 201324.1 Rev A; Thermo Fisher Scientific, Hampton, NH, USA). This water-washing procedure was performed twice; the bacteria were resuspended in 30 µL of water, and the cell suspension was transferred into PCR tubes. The OD of the suspension was monitored using a Photo Absorbance Meter (PAS-110-YU; Yamato Scientific Co., Ltd., Tokyo, Japan) and diluted to an OD of 1.7–1.8. From each suspension, 1.5 µL was transferred to another PCR tube and mixed with 0.5 µL of 400 mM HCl (total HCl concentration, 100 mM). The mixtures were incubated for 10 min at 90 °C and centrifuged at 20,000×g for 5 min (D3024 High-Speed Microcentrifuge; DLAB Scientific Co., Ltd., Beijing, China). The supernatant (0.35 µL) was loaded onto a MALDI target plate and dried at room temperature (20–25 ºC). The matrix solution (0.35 μL, DAN/DHB/Na) was deposited on the sample pre-spotted position and dried at room temperature.

Ribosomal polypeptide typing (formic acid-acetonitrile extraction method)

Several colonies of bacterial cells grown on an agar culture plate were transferred into a 1.5-mL tube and suspended in 250 µL of water. Ethanol (1 mL) was added, and the mixture was resuspended and centrifuged at 10,000×g for 2 min. The supernatants were removed, and the pellets were dried. The pellets were mixed with 50 µL of 70% formic acid and then with acetonitrile (50 µL). The mixture was centrifuged at 15,000×g for 2 min. 0.35 µL of the supernatant was overlaid on 0.35 µL of the CHCA matrix pre-spotted target plate and dried at room temperature.

Lipid A typing

The method up to the measurement of OD was the same as that for the O-antigen measurement. Each suspension (15 µL) was transferred into another PCR tube and suspended with 5 µL of 400 mM HCl (total HCl concentration of 100 mM). The mixture was incubated for 10 min at 90 °C and centrifuged at 20,000×g for 5 min. The supernatant was discarded, mixed with 100 µL of water, and centrifuged at 20,000×g for 5 min. The supernatant was discarded, and the remaining pellet was dried. The dried pellet was resuspended in 2 µL of water, and 0.2 µL of the suspension was loaded onto a MALDI target plate. The DHB matrix solution (0.60 μL) was deposited on the sample pre-spotted position and dried at room temperature.

O-antigen typing from a single colony

A single colony of bacterial cells grown on an agar culture plate was picked, transferred into a 1.5 mL tube, suspended in 30 µL water, and centrifuged at 15,000×g for 2 min. The supernatant (28.5 µL) was removed, and 1.5 µL of the suspension of the remaining pellets was transferred into a PCR tube and mixed with 0.5 µL of 400 mM HCl (total HCl concentration of 100 mM). These mixtures were incubated for 10 min at 90 °C and centrifuged at 20,000×g for 5 min. 0.35 µL of the supernatant was loaded on a MALDI target plate and dried at room temperature. The matrix solution (0.35 μL, DAN/DHB/Na) was deposited on the sample pre-spotted target plate and dried at room temperature.

O-antigen typing using the glycoblotting method

Treatment similar to the optimized O-antigen process was performed on the ATCC43888 strain. The strain was washed similarly to prepare a bacterial suspension with an OD of 1.9–2.0. Subsequently, 60 µL of the strain suspension was added to 20 µL of 400 mM HCl and incubated at 90 °C for 10 min. After incubation, the sample was centrifuged, and 60 µL of the supernatant was transferred to a 1.5 mL tube and dried in a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA). Blot GlycoH beads (250 µL, 10 mg/mL) were dispensed into a well of a 96-well multiScreen Solvert filter plate (Millipore, Billerica, MA, USA). The filter plate was then attached to a vacuum to remove water. The dried O-antigen sample released from ATCC43888 was dissolved in 60 µL of water, and all the solution was transferred to a well with 180 µL of 2% acetic acid in acetonitrile. The 96-well filter plate was incubated at 80 °C for 45 min. Then, the treated sample was washed with 200 µL of 2 M guanidine-HCl in 16.6 mM ammonium bicarbonate, water, and 1% triethylamine in methanol. Each washing process was performed twice, and a vacuum was performed after each step. The unreacted hydrazide functional groups on the beads were capped with acetyl groups by incubating with 100 µL of 2% acetic anhydride in methanol for 30 min at room temperature. The solution was then removed using a vacuum and washed twice with 200 µL of 10 mM HCl, methanol, and dioxane. On-bead methyl esterification was performed by adding 100 µL of 100 mM 3-methyl-1-p-tolyltriazene in dioxane into the sample well and incubating at 60 °C for 90 min. The plate was sequentially washed twice with 200 µL of dioxane, water, methanol, and water. The captured O-antigen-derived glycans on the BlotGlycoH beads were released and labeled with benzyloxyamine (BOA). The labeling and releasing process was performed by adding 20 µL of 50 mM BOA-HCl and 180 µL of 2% acetic acid in acetonitrile at 80 °C for 45 min. BOA-labeled glycans were eluted twice with 200 µL of water. The eluted solutions were mixed and dried using a SpeedVac. The dried sample was dissolved in 10 µL of water. The sample solution (0.35 µL) and DHB/Na matrix solution (0.35 µL) were loaded on a MALDI target plate and dried under air blow.

Mass spectrometry

All spectra were acquired using an Ultraflex III instrument (Bruker, Bremen, Germany) equipped with a 200 Hz Smartbeam Nd:YAG laser (355 nm) and operated with flexControl (version 3.4, https://www.bruker.com/). O-antigen spectra were acquired in the positive reflectron mode with 1000 laser shots and random walks. Lipid A spectra were acquired in the negative reflectron mode with 1000 laser shots and a random walk. Ribosomal polypeptide spectra were acquired in positive linear mode with 1000 laser shots and a random walk. The generated ions accelerated to a kinetic energy of 25.0 kV. The low-mass-ion deflector cut-off was set at 700 Da. In the LIFT-TOF/TOF mode, the fragment ions were accelerated to 8 kV in the MALDI ion source and selected at a time gate. The selected ions were further accelerated to 19 kV in the LIFT cell. The metastable PSD ions were analyzed. The raw MALDI-TOF and MALDI-TOF/TOF MS data were annotated using the flexAnalysis (version 3.4, https://www.bruker.com/).