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Maxis 4g etd

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The MaXis 4G ETD is a high-resolution quadrupole time-of-flight (Q-TOF) mass spectrometer designed for advanced analytical applications. It features enhanced Electron Transfer Dissociation (ETD) capabilities for improved structural characterization of biomolecules.

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Lab products found in correlation

Esi calibration mixture, by Agilent Technologies (6 mentions) Dataanalysis 4, by Bruker (4 mentions) Ultimate 3000 system, by Bruker (2 mentions) Modified trypsin, by Promega (2 mentions) Dionex ultimate 3000 system, by Thermo Fisher Scientific (2 mentions) Instantblue coomassie stain, by Abcam (1 mentions) Proteinscape 3, by Bruker (1 mentions) Sequencing grade modified trypsin, by Promega (1 mentions) Cary 60 spectrophotometer, by Agilent Technologies (1 mentions) Mars 14, by Agilent Technologies (1 mentions) 2 d quant kit, by GE Healthcare (1 mentions) Nano advance, by Bruker (1 mentions) Cary 60, by Agilent Technologies (1 mentions) Ultimate 3000, by Bruker (1 mentions) Pngase f, by Roche (1 mentions) Congo red, by Merck Group (1 mentions) Acclaim pepmap μ precolumn, by Thermo Fisher Scientific (1 mentions)

12 protocols using maxis 4g etd

1

Profiling Antibody Glycosylation via Mass Spectrometry

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1 µg antibodies were digested with PNGaseF following manufacturer’s instructions (NEB, cat. #P0704). Samples were reduced with 5% β-mercaptoethanol before performing SDS-PAGE. Proteins were identified using InstantBlue™ Coomassie stain (Expedeon, cat. #ab119211). PNGaseF assays were performed in triplicate.
For mass spectrometry, pN6 antibody was trypsin-digested and analyzed by liquid chromatography–electrospray ionization–mass spectrometry as described in Teh et al. [33 (link)]. Briefly, samples were resuspended in 80 mM ammonium formiate buffer and run on a BioBasic C18 column with a 5% to 40% 80%-acetonitrile for 45 min, followed by a 15 min gradient from 40 to 90% 80%-acetonitrile, that facilitates elution of large peptides, at a flow rate of 6 µL/min. Peptide identification was performed with maXis 4G ETD (Bruker, Germany) in positive ion mode. Manual glycopeptide searches were made using DataAnalysis 4.0 (Bruker).
Moore C.M., Grandits M., Grünwald-Gruber C., Altmann F., Kotouckova M., Teh A.Y, & Ma J.K. (2021). Characterisation of a highly potent and near pan-neutralising anti-HIV monoclonal antibody expressed in tobacco plants. Retrovirology, 18, 17.
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2

Protein Identification by Shotgun Proteomics

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Relevant protein bands were cut out and digested in gel. S-alkylation with iodoacetamide and digestion with sequencing grade modified trypsin (Promega) were performed. The peptide mixture was analysed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion, DDA mode (= switching to MSMS mode for eluting peaks). MS-scans were recorded (range: 150–2200 Da) and the 6 highest peaks were selected for fragmentation. Instrument calibration was performed using ESIcalibration mixture (Agilent). For separation of the peptides a Thermo BioBasic C18 separation column (5 μm particle size, 150°0.360 mm) was used. A gradient from 95% solvent A and 5% solvent B (Solvent A: 65 mM ammonium formiate buffer, B: 100% ACCN) to 32% B in 45 min was applied, followed by a 15 min gradient from 32% B to 75% B, at a flow rate of 6 μL∙min−1. The analysis files were converted using Data Analysis 4.0 (Bruker) to XML files, which are suitable to perform MS/MS ion searches with MASCOT (embedded in ProteinScape 3.0, Bruker) for protein identification. Only proteins identified with at least 2 peptides with a protein score higher than 80 were accepted. For searches the SwissProt database was used.
Gmeiner C., Saadati A., Maresch D., Krasteva S., Frank M., Altmann F., Herwig C, & Spadiut O. (2015). Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase. Microbial Cell Factories, 14, 1.
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3

Coproheme Decarboxylation Activity Assay

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Experimental conditions for the determination of the coproheme decarboxylation activity via titration with hydrogen peroxide monitored by UV-vis absorption and mass spectrometric analysis have been described in detail previously (5 (link)). Experiments were performed in 100 mM phosphate buffer (pH 7) using a Cary 60 spectrophotometer for UV-vis absorption. 10 μL samples were drawn from the 1200 μL reaction mix, deactivated by the addition of 10 mM cyanide and analyzed using a Dionex Ultimate 3000 system directly linked to a quadrupole time-of-flight mass spectrometer (maXis 4G ETD; Bruker, Billerica, MA) equipped with the standard electrospray ionization source in the positive ion mode. MS scans were recorded within a range from m/z 400 to 3800, and the instrument was tuned to detect both the rather small free heme derivatives and intact proteins in a single run. A Thermo ProSwift RP-4H analytical separation column (250 × 0.200 mm) (Thermo Fisher Scientific, Vienna, Austria) was used to separate the analytes. A gradient from 99% solvent A and 1% solvent B (solvent A, 0.05% trifluoroacetic acid; solvent B, 80.00% acetyl cyanide and 20% solvent A) to 65% B in 11 min was applied, followed by a 2 min gradient from 65% B to 95% B, at a flow rate of 8 μL min−1 and at 65°C. A blank run (5.0 μL of H2O) was performed after each sample to minimize carryover effects.
Sebastiani F., Michlits H., Lier B., Becucci M., Furtmüller P.G., Oostenbrink C., Obinger C., Hofbauer S, & Smulevich G. (2021). Reaction intermediate rotation during the decarboxylation of coproheme to heme b in C. diphtheriae. Biophysical Journal, 120(17), 3600-3614.
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4

Coproheme to Heme b Conversion

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H2O2-induced
conversion of supplied coproheme
to MMD and heme b was investigated by titration of
1000 μL of the enzyme solution in 50 mM phosphate buffer, pH
7, with around 15 μM apo-enzyme and 10 μM coproheme in
a Cary 60 spectrophotometer (Agilent Technologies) with a resolution
of 1.5 nm. Subequimolar amounts of H2O2 were
added, and spectra were taken after each titration step. Samples from
this solution (10 μL) were drawn and analyzed using a Dionex
Ultimate 3000 system directly linked to a QTOF mass spectrometer (maXis
4G ETD, Bruker) equipped with the standard ESI source in the positive
ion mode. MS scans were recorded within a range from m/z 400 to 3800, and the instrument was tuned to
detect both the rather small free heme derivatives and intact proteins
in a single run. For separation of the analytes a Thermo ProSwift
RP-4H analytical separation column (250 × 0.200 mm) was used.
A gradient from 99% solvent A and 1% solvent B (solvent A, 0.05%
trifluoroacetic acid (TFA); solvent B, 80.00% acetyl cyanide (ACN)
and 20% solvent A) to 65% B in 11 min was applied, followed by a 2
min gradient from 65% B to 95% B, at a flow rate of 8 μL min–1 and at 65 °C. A blank run (5.0 μL of H2O) was performed after each sample to minimize carryover effects.
Relative amounts of formed MMD and heme b as well
as oxidized coproheme and heme b upon addition of
hydrogen peroxide were determined.
Michlits H., Lier B., Pfanzagl V., Djinović-Carugo K., Furtmüller P.G., Oostenbrink C., Obinger C, & Hofbauer S. (2020). Actinobacterial Coproheme Decarboxylases Use Histidine as a Distal Base to Promote Compound I Formation. ACS Catalysis, 10(10), 5405-5418.
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5

Proteomic Profiling of Cerebrospinal Fluid

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Total protein concentration in CSF was determined by using the 2D Quant kit (GE Healthcare Life Sciences, UK), according to the manufacturer’s protocol, and 400 µg total protein was used as input for profiling. All samples were loaded on an affinity removal column for the depletion of the 14 most abundant proteins (MARS-14, Agilent Technologies, Santa Clara, CA, USA). After tryptic digestion, CSF samples were fractionated in 20 fractions using high pH reversed-phase C18 LC and each fraction was subsequently analyzed by nanoflow liquid chromatography (Bruker Daltonics; nano-Advance) connected online to an ultra-high resolution quadrupole time-of-flight tandem mass spectrometer (Qq-TOF; Bruker Daltonics; maXis 4G ETD) as described previously50 (link).
Raw MS data were analyzed by MaxQuant software version 1.551 (link) with pre-defined Qq-ToF parameter settings against the RefSeq (release 55) human protein sequence database. We set cysteine carbamidomethylation as a fixed modification, whereas N-terminal acetylation, methionine oxidation, and deamidation of glutamine and/or asparagine were set as variable modifications. For further statistical analysis, only peptides with intensity above the detection limit in at least 75% of the samples in one of the groups (PD, MSA, or non-neurological controls) were used.
Marques T.M., van Rumund A., Kersten I., Bruinsma I.B., Wessels H.J., Gloerich J., Kaffa C., Esselink R.A., Bloem B.R., Kuiperij H.B, & Verbeek M.M. (2021). Identification of cerebrospinal fluid biomarkers for parkinsonism using a proteomics approach. NPJ Parkinson's Disease, 7, 107.
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6

PNGase F Protein Separation and MS Analysis

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Twenty microliter PNGase F digested sample (β = 0.30 mg·mL−1) was analyzed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD; Bruker, Billerica, MA, USA) equipped with the standard ESI source in the positive ion mode. MS scans were recorded within a range from 400 to 3800 m/z. Instrument calibration was performed using ESI calibration mixture (Agilent). For separation of the proteins, a Thermo ProSwift™ RP‐4H Analytical separation column (250 × 0.200 mm) was used. A gradient from 80% solvent A and 20% solvent B (A: 0.05% trifluoroacetic acid, B: 80% acetonitrile, and 20% A) to 65% B in 20 min was applied, followed by a 15‐min gradient from 65% B to 95% B, at a flow rate of 8 μL·min−1 and at 60 °C. Deconvolution of summed spectra was done using the MaxEnt algorithm in Data Analysis 4.0.
Sádio F., Stadlmayr G., Stadlbauer K., Gräf M., Scharrer A., Rüker F, & Wozniak‐Knopp G. (2019). Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds. Febs Letters, 594(3), 477-490.
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7

Characterization of CdChdC Variants by UV-Vis and MS

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The CdChdC wild-type and Y135A mutant activity was calculated using the UV–Vis electronic absorption spectra recorded by means of a Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) with a scan rate of 600 nm min−1 and a resolution of 1.5 nm. To analyze their respective activities, 18 μM of recombinant enzyme was added to 9 μM of coproheme in 1000 μL of 100 mM phosphate buffer solution, pH 7, to form CdChdC–coproheme complexes. The CdChdC–coproheme complexes were eventually titrated to heme b complexes by adding small aliquots of a 1 mM H2O2 stock solution. The complete heme b formation was followed by the recording of the electronic absorption spectra of the wild-type and variant upon the addition of three equivalents (eqs.) of H2O2.
Samples from this solution (10 μL) were drawn and analyzed using a Dionex Ultimate 3000 system directly linked to a QTOF mass spectrometer (maXis 4G ETD, Bruker), which was equipped with the standard ESI source in the positive ion mode using an optimized protocol to simultaneously detect small masses of porphyrins and the entire protein [7 (link)].
Patil G., Michlits H., Furtmüller P.G, & Hofbauer S. (2023). Reactivity of Coproheme Decarboxylase with Monovinyl, Monopropionate Deuteroheme. Biomolecules, 13(6), 946.
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8

Glycoprotein Analysis via LC-ESI-MS

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20 μL of glycosylated or PNGaseF digested sample (β = 0.30 mg/mL) was analyzed using a Dionex Ultimate 3000 LC-ESI-MS system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion, MS mode (range: 750–5000 Da) mode. Instrument calibration was performed using ESI calibration mixture (Agilent). For separation of the proteins a Thermo ProSwift™ RP-4H Analytical separation column (250 * 0.200 mm) was used. A gradient from 20 to 80% acetonitrile in 0.05% trifluoroacetic acid at a flow rate of 8 μL/min was applied in 30 min gradient time. Deconvolution of summed spectra was done using the MaxEnt algorithm in Data Analysis 4.0 (Bruker).
Benedetti F., Stracke F., Stadlmayr G., Stadlbauer K., Rüker F, & Wozniak-Knopp G. (2021). Bispecific antibodies with Fab-arms featuring exchanged antigen-binding constant domains. Biochemistry and Biophysics Reports, 26, 100959.
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9

IgG N-Glycan Analysis by UPLC-MS

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The protein A purified IgGs were digested with PNGase F (Roche) to release all N-glycans and were analysed using a Dionex Ultimate 3000 system directly linked to a QTOF instrument (maXis 4G ETD, Bruker) equipped with the standard ESI source in the positive ion mode. MS-scans were recorded within a range from 400 to 3800 m/z. Instrument calibration was performed using ESIcalibration mixture (Agilent). For separation of the proteins a Thermo ProSwift™ RP-4H Analytical separation column (250 × 0.200 mm2) was used. A gradient from 80% solvent A and 20% solvent B (Solvent A: 0.05% TFA, B: 80% ACCN and 20% solvent A) to 62.5% B in 15 min was applied, followed by a 5 min gradient from 62.5% B to 95% B, at a flow rate of 8 μL/min and 65°C. The analysis files were deconvoluted (Maximum Entropy Method, low mass: 40 000, high mass: 200 000, instrument resolv. power: 10 000) using DataAnalysis 4.0 (Bruker) and manually annotated. The maximum peak intensity of the deconvoluted spectra was taken to calculate the prevalence of each chain pairing variant relative to all detected heterodimeric IgGs. Traces of detected heavy chain homodimers were not included in the calculation. The prevalence of the variant with both light chains mispaired was estimated as described elsewhere (Yin et al., 2016 (link)).
Bönisch M., Sellmann C., Maresch D., Halbig C., Becker S., Toleikis L., Hock B, & Rüker F. (2017). Novel CH1:CL interfaces that enhance correct light chain pairing in heterodimeric bispecific antibodies. Protein Engineering, Design and Selection, 30(9), 685-696.
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10

Fungal Cellulolytic Enzyme Screening

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In this study, seven fungal strains were screened for cellulolytic enzyme activities by using a plate assay in carboxymethyl cellulose (CMC; Sigma Aldrich CHEMIE, Steinheim, Germany) agar using Congo red (Sigma Aldrich, St. Louis, MO, USA) as the dye. Afterwards, screening experiments for lignocellulolytic and starch degrading enzyme activity were undertaken by using sequential solid state and submerged cultivation containing pretreated corn cobs as the inducer substrate. Scale up of cultivations by selected strains was performed, and enzyme activities were monitored for 14 days. The obtained supernatants were used for partial purification and LC-ESI-MS (maXis 4G ETD; Bruker, Billerica, MA, USA) was used to identify the enzymes present in the cocktails.
Marđetko N., Trontel A., Novak M., Pavlečić M., Ljubas B.D., Grubišić M., Tominac V.P., Ludwig R, & Šantek B. (2021). Screening of Lignocellulolytic Enzyme Activities in Fungal Species and Sequential Solid-State and Submerged Cultivation for the Production of Enzyme Cocktails. Polymers, 13(21), 3736.
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