Reactions were centrifuged at 20,817× g, one microliter of supernatant was mixed with an equal volume of matrix solution (20 mg mL−1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile plus 0.1% TFA) and spotted on a SCOUT-MTP 384 target plate (Bruker, Billerica, MA, USA). The spotted samples were air dried and then analyzed by positive-mode MALDI-TOF MS using an Ultraflex III matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker).
Scout mtp 384 target plate
Manufactured by Bruker
Sourced in United States
The SCOUT-MTP 384 target plate is a laboratory equipment designed for sample preparation and analysis. It provides a standardized platform for holding and positioning samples in a 384-well format, facilitating efficient sample processing and data acquisition.
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7 protocols using scout mtp 384 target plate
1
MALDI-TOF MS Sample Preparation
2
Enzymatic Oxidation of Cellohexaose
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Cellohexaose was
used as substrate. One hundred microliter reactions were set up with
750 μM cellohexaose, 1 mM ascorbic acid, 100 μM H2O2 (no H2O2 added in the
O2 turnover reactions), and 1 μM LsAA9 or purple LsAA9 in 5 mM MES at pH 7.0 and were
incubated at 40 °C for 2 h. The reaction was then quenched by
addition of 3 reaction volumes of ethanol (98% v/v). Reactions with
H2O2 were performed inside an N2 atmosphere
glovebox. A 1 μL amount of sample was then mixed with 2 μL
of 10 mg/mL 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic
acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were
then dried in air under a lamp before being analyzed by mass spectrometry
on a Ultraflex III matrix-assisted laser desorption ionization-time-of-flight/time-of-flight
(MALDI-TOF/TOF) instrument (Bruker), as described previously.9 (link) The purple species LsAA9 sample
used in the assay was incubated with EDTA overnight to remove Cu2+ from any LsAA9 that had not been converted
to the purple species. The resulting [Cu(EDTA)]2– complex was then removed from the solution by ultracentrifugation
through 10 kDa cutoff size-exclusion filters.
used as substrate. One hundred microliter reactions were set up with
750 μM cellohexaose, 1 mM ascorbic acid, 100 μM H2O2 (no H2O2 added in the
O2 turnover reactions), and 1 μM LsAA9 or purple LsAA9 in 5 mM MES at pH 7.0 and were
incubated at 40 °C for 2 h. The reaction was then quenched by
addition of 3 reaction volumes of ethanol (98% v/v). Reactions with
H2O2 were performed inside an N2 atmosphere
glovebox. A 1 μL amount of sample was then mixed with 2 μL
of 10 mg/mL 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic
acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were
then dried in air under a lamp before being analyzed by mass spectrometry
on a Ultraflex III matrix-assisted laser desorption ionization-time-of-flight/time-of-flight
(MALDI-TOF/TOF) instrument (Bruker), as described previously.9 (link) The purple species LsAA9 sample
used in the assay was incubated with EDTA overnight to remove Cu2+ from any LsAA9 that had not been converted
to the purple species. The resulting [Cu(EDTA)]2– complex was then removed from the solution by ultracentrifugation
through 10 kDa cutoff size-exclusion filters.
3
MALDI-TOF Mass Spectrometry Analysis of Glycans
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One microliter of reaction supernatant was mixed with an equal volume of 20 mg mL−1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile, 0.1% TFA on a SCOUT-MTP 384 target plate (Bruker). The spotted samples were then dried in a vacuum desiccator before being analyzed by mass spectrometry on an Ultraflex III matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker)54 (link).
Sample permethylation was carried out according to Ciucanu and Kerek55 (link). Spotted samples were analyzed by MS using 2,5-DHB matrix with 0.1% TFA on an AB-Sciex 4700 (for MALDI-TOF) and Ultraflex III MALDI/TOF-TOF instrument (Bruker). Data were collected using a 2-kHz smartbeam-II laser and acquired on reflector mode (mass range 300–3000 Da) for MS analysis and on LIFT-CID for MS/MS analysis using argon as collision gas. FlexControl and FlexAnalysis softwares were used for data acquisition and analysis. On average, about 10,000 shots were used to obtain high-enough resolution. MS/MS fragmentation patterns were named according to Domon and Costello56 (link).
Sample permethylation was carried out according to Ciucanu and Kerek55 (link). Spotted samples were analyzed by MS using 2,5-DHB matrix with 0.1% TFA on an AB-Sciex 4700 (for MALDI-TOF) and Ultraflex III MALDI/TOF-TOF instrument (Bruker). Data were collected using a 2-kHz smartbeam-II laser and acquired on reflector mode (mass range 300–3000 Da) for MS analysis and on LIFT-CID for MS/MS analysis using argon as collision gas. FlexControl and FlexAnalysis softwares were used for data acquisition and analysis. On average, about 10,000 shots were used to obtain high-enough resolution. MS/MS fragmentation patterns were named according to Domon and Costello56 (link).
4
Enzymatic Assays for TtAA10 Activity
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TtAA10 activity assays were set up using PASC [prepared according to Wood (1988 ▸ )] or Avicel as the main substrate. First, 1 ml samples were prepared in 50 mM sodium acetate pH 6.0 buffer containing 1 mg ml−1 Avicel or PASC and 1 µM TtAA10. Then, 1 mM ascorbate was used as the electron donor in positive controls. Where TtX183A, TtX183B or TtX183C were used as the electron source, the haem was chemically reduced first by the addition of 1 mM ascorbate, which was subsequently removed by passing the protein down a PD-10 desalting column before the protein was added to assays at 100 µM. Reactions were incubated rotating end-over-end on a tube rotator (Stuart Scientific) overnight at room temperature. Prior to mass-spectrometric analysis, the samples were centrifuged at 10 000g for 1 min to pellet any solid material.
For MALDI-MS measurements, 1 µl of sample was mixed with an equivalent volume of 10 mg ml−1 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were then dried in air under a lamp before being analysed by mass spectrometry on an Ultraflex III MALDI-TOF/TOF instrument (Bruker), as described by Hemsworth et al. (2014 ▸ ).
For MALDI-MS measurements, 1 µl of sample was mixed with an equivalent volume of 10 mg ml−1 2,5-dihydroxybenzoic acid in 50% acetonitrile, 0.1% trifluoroacetic acid on a Bruker SCOUT-MTP 384 target plate. The spotted samples were then dried in air under a lamp before being analysed by mass spectrometry on an Ultraflex III MALDI-TOF/TOF instrument (Bruker), as described by Hemsworth et al. (2014 ▸ ).
5
Enzymatic Oxidation of Substrates by LpsAA10A
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Reactions with the purified LpsAA10A were carried out by mixing 4 mg mL − 1 substrate with purified copper-loaded enzyme (2 μM) and 4 mM electron donor (gallic acid), in 50 mM ammonium acetate buffer pH 6 in 2-mL plastic reaction tubes (reaction volume: 100 μL). The tubes were incubated for 24 h at 28 °C shaking at 1000 rpm, centrifuged at 14,000 rpm and the supernatant was collected for analysis through mass spectrometry. Briefly, 1 μl of supernatant was mixed with an equal volume of matrix solution (20 mg mL− 1 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile plus 0.1% TFA), spotted on a SCOUT-MTP 384 target plate (Bruker) and analysed by positive-mode MALDI-TOF MS using an Ultraflex III matrix-assisted laser desorption ionisation-time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker).
6
Cellulose Fiber Substrate Assays
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Celery cellulose fibres prepared as described above were used in activity assays without further purification. Reactions were set up in 1 mL total volume with 2 mg of solid substrate in 10 mM ammonium acetate, pH 6.0, 1 mM ascorbic acid and 1 μM Cu(II)-LsAA9A or Cu(II)-TaAA9A and were incubated at 30 °C rotating overnight. Remaining substrate was removed by centrifugation at 14 000g for 5 min and the supernatant used for the analysis. 1 μL of sample was mixed with 2 μL of 10 mg mL -1 2,5-dihydroxybenzoic acid in 50% v/v acetonitrile, 0.1% v/v trifluoroacetic acid in water, on a SCOUT-MTP 384 target plate (Bruker). The spotted samples were then dried under a lamp in air, before being analysed by mass spectrometry on an Ultraflex III matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) instrument (Bruker), as described by Vaaje-Kolstad et al. 3 MALDI-TOF data are available on request through the Research Data York (DOI: 10.15124/e6bd5772-738f-4108-88c3-9d5f9e65e00e).
7
MALDI-TOF-TOF Analysis of Reaction Supernatant
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One microliter of reaction supernatant was mixed with an equal volume of 20 mg/mL 2,5-dihydroxybenzoic acid (DHB) in 50% acetonitrile, 0.1% TFA on a SCOUT-MTP 384 target plate (Bruker, Billerica, USA). The spotted samples were then dried in a vacuum desiccator before being analyzed by mass spectrometry on an Ultraflex III matrix-assisted laser desorption ionization time of flight/time of flight (MALDI/TOF-TOF) instrument (Bruker, Billerica, USA) (Abdul Rahman et al. 2014) .
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