Autoflex 2 mass spectrometer

Manufactured by Bruker

Sourced in Germany, United States

The Autoflex II is a high-performance matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer. It is designed for the analysis of a wide range of biomolecules, including proteins, peptides, and oligonucleotides. The Autoflex II provides accurate mass measurement and high sensitivity, making it a versatile tool for applications in proteomics, genomics, and other life science research areas.

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Lab products found in correlation

Sequencing grade modified trypsin, by Promega (2 mentions) Nicolet is5, by Thermo Fisher Scientific (1 mentions) Avance 400 spectrometer, by Bruker (1 mentions) Data analysis 3, by Bruker (1 mentions) Mtp 384 ground steel, by Bruker (1 mentions) Plusone 2d quant kit, by GE Healthcare (1 mentions) Mascot software, by Matrix Science (1 mentions) J 725 cd spectropolarimeter, by Jasco (1 mentions) Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (1 mentions) Kod plus mutagenesis kit, by Toyobo (1 mentions)

Close spelling variants of this product

Autoflex II TOF/TOF mass spectrometer Autoflex II MALDI-TOF mass spectrometer Autoflex II MALDI-TOF/TOF mass spectrometer Autoflex mass spectrometer Autoflex II spectrometer Autoflex II Bruker spectrometers

8 protocols using autoflex 2 mass spectrometer

Spectroscopic Characterization of Compounds

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The ¹H and ¹³C NMR spectra were registered with a Bruker Avance-400 spectrometer (Bruker Daltonics, Bremen, Germany) in chloroform-d1 or methanol-d4, using the residual signals of chloroform or CHD₂OD as internal standards. MALDI-TOF mass-spectra were registered on a Bruker Daltonics Autoflex II mass-spectrometer (Bruker Daltonics, Bremen, Germany) in positive ion mode with a dithranol matrix and polyethyleneglycols as internal standards. FTIR spectra were registered on a Nicolet iS 5 (Thermo Fisher Scientific, Waltham, MA, USA) with iD3 ATR accessory (ZnSe).
The pH measurements were carried out using the «Mettler Toledo» apparatus (Greifensee, Switzerland) with a combined electrode LE438 in a glass cell. The electrode was calibrated with commercial buffers (pH = 4.01 and 7.00). UV-vis spectra were registered with a Hitachi U-2900 device (Tokyo, Japan) in a quartz cuvette (Hellma, l = 1 cm). Fluorescence spectra were registered with Horiba Jobin Yvon Fluoromax-2 apparatus (Edison, NJ, USA) in a quartz cuvette (Hellma, l= 1 cm). Luminescence quantum yields were determined relative to quinine sulfate solution in 0.05 H₂SO₄ (Φ = 0.53(2)) according to a standard procedure [78 (link)].

Kurashov I.A., Kharlamova A.D., Abel A.S., Averin A.D, & Beletskaya I.P. (2023). Polyoxa- and Polyazamacrocycles Incorporating 6,7-Diaminoquinoxaline Moiety: Synthesis and Application as Tunable Optical pH-Indicators in Aqueous Solution. Molecules, 28(2), 512.

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Affinity Purification and Mass Spectrometry

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HCT116 cells were lysed with binding buffer [50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM DTT, and 0.43 mM ABSF] at 4°C for 30 min and then centrifuged. The supernatants were used as whole-cell extracts of HCT116 cells. The extracts were incubated with agent-immobilized or empty beads at 4°C for 4 h and then washed three times with binding buffer. Bound proteins were eluted with Laemmli dye and subjected to SDS-PAGE. Proteins were stained with aqueous AgNO₃, and each strip containing a protein was cut out and digested with Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). After in-gel digestion, the peptide fragments from each strip were analyzed using an Autoflex II mass spectrometer (Bruker Daltonics, Billerica, MA, USA).

Watanabe M., Boku S., Kobayashi K., Kurumida Y., Sukeno M., Masuda M., Mizushima K., Kato C., Iizumi Y., Hirota K., Naito Y., Mutoh M., Kameda T, & Sakai T. (2022). A chemoproteoinformatics approach demonstrates that aspirin increases sensitivity to MEK inhibition by directly binding to RPS5. PNAS Nexus, 1(2), pgac059.

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MALDI-TOF Mass Spectrometry of Released Glycans

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The released glycans (1 μL after resuspending in 20 μL ultra-pure water) were spotted with 1 μL of saturated 2',4',6'-Trihydroxyacetophenone monohydrate (THAP) in 50/50 acetonitrile / water and analyzed on a Bruker Autoflex II mass spectrometer (Bruker Daltonics, GmbH, Bremen Germany) in linear positive mode. Spectra were calibrated externally with peptide standards with identical matrix. Mass spectra data were processed using Data Analysis 3.4 (Bruker Daltonics). After 1 round of 0.2 width (m/z) Gaussian smoothing, peak lists (2.5% threshold from base peak intensity) were exported into GlycoMod (http://web.expasy.org/glycomod) for glycan composition identification. Potential monosaccharide residues were restricted to Hex, HexNAc, DeoxyHex, and NeuAc. After initial searches for sodium adducts, a secondary was performed for multiply sodiated species, which was predominant for sialic acid containing glycans. Glycan structures matching within 1000 ppm were considered and potential hits were filtered to those present in the UniCarbKB database [22 (link)]. For the major species a weak signal was also observed for the potassium adducted species. At this level of mass accuracy the combination of potassium and deoxyhexose results in the same nominal mass as sodium and hexose, and such ambiguity for the major peaks is indicated in Table 2.

Guttman M., Váradi C., Lee K.K, & Guttman A. (2015). Comparative Glycoprofiling of HIV gp120 Immunogens by Capillary Electrophoresis and MALDI Mass Spectrometry. Electrophoresis, 36(0), 1305-1313.

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MALDI-TOF MS Protocol for DNA Analysis

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The
matrix for MALDI-TOF MS was 1:1 mixture
of 3-hydroxypicolinic acid (3HPA) in 1:1 acetonitrile/H₂O saturated solution and 0.5 M ammonium citrate aqueous solution.
1 μL of DNA sample was mixed with 1 μL of matrix solution.
A spot of 1 μL of the sample–matrix mixture was placed
on a MALDI target plate (MTP 384 ground steel, Bruker) and allowed
to air dry at room temperature. The spectrum was measured using a
matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer
(MALDI-TOF MS) on Bruker autoflex II mass spectrometer (negative mode)
with dT₈ ([M – H]⁻: 2370.603)
and dT₁₇ ([M – H]⁻: 5108.376)
as an external calibration standard.

Ishizuka T., Yamashita A., Asada Y, & Xu Y. (2017). Studying DNA G-Quadruplex Aptamer by 19F NMR. ACS Omega, 2(12), 8843-8848.

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Rabdosianone I-Binding Protein Purification

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Purification and identification of rabdosianone I-binding proteins were performed as previously described [24 (link)]. Briefly, HT-29 cells were lysed with binding buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 1 mM DTT, and 0.43 mM ABSF at 4 °C for 30 min, and centrifuged. The supernatants were used as whole-cell extracts of HT-29 cells. The extracts were incubated with the agent-fixed beads or empty beads at 4 °C for 4 h. The beads were washed 3 times with binding buffer. The bound proteins were eluted with Laemmli dye and subjected to SDS-PAGE. The proteins were stained by aqueous AgNO₃, and each strip, including the protein, was cut off to apply Sequencing Grade Modified Trypsin (Promega, Madison, WI, USA). The peptide fragments from each strip were analyzed using an Autoflex II mass spectrometer (Bruker Daltonics, Billerica, MA, USA) after in-gel digestion.

Watanabe M., Yamada Y., Kurumida Y., Kameda T., Sukeno M., Iizuka-Ohashi M., Sowa Y., Iizumi Y., Takakura H., Miyamoto S., Sakai T, & Mutoh M. (2021). Rabdosianone I, a Bitter Diterpene from an Oriental Herb, Suppresses Thymidylate Synthase Expression by Directly Binding to ANT2 and PHB2. Cancers, 13(5), 982.

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Proteomic Analysis of Heart Tissue

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Heart tissue was cut into 1 mm pieces, washed with PBS containing a protease inhibitor cocktail (5 mM EDTA, 100 µM PMSF, 100 µM TLCK, 100 µM TPCK, 1 µM pepstatin A, 100 µM leupeptin) at 4°C, and centrifuged three times at 10,000 rpm with supernatant discarding. Lysis buffer (7 M urea, 2 M thiourea, 2% Triton X-100, 1% DTT and the protease inhibitor cocktail) was then added in the proportion of 6 mL to 1 g of tissue, followed by vortexing for 2 min and sonication in a refrigerated bath for 2 min three times. The material was left at 4°C for 20 min and finally centrifuged at 14,000 rpm for 15 min. The supernatant (heart extract) was submitted to protein quantitation using the PlusOne 2D Quant Kit (GE Life Sciences). The protein profiles of heart extracts (100 µg) were analyzed by 2DE [100] (link). Peptide mass fingerprinting [63] (link) was used for protein identification via an Autoflex II mass spectrometer (Bruker Daltonics) and the Mascot software (Matrix Science).

Guimaro M.C., Alves R.M., Rose E., Sousa A.O., de Cássia Rosa A., Hecht M.M., Sousa M.V., Andrade R.R., Vital T., Plachy J., Nitz N., Hejnar J., Gomes C.C, & L. Teixeira A.R. (2014). Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation. PLoS Neglected Tropical Diseases, 8(12), e3384.

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CD and MALDI-TOF Analysis of Cytochrome c552

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CD spectra of WT and A5F/M11V/Y32F/Y41E/I76V HT holo and apo cyt c₅₅₂ without a His-tag were measured with a J-725 CD spectropolarimeter (Jasco, Japan) using a 0.1-cm-path-length quartz cell at 25 °C. MALDI-TOF mass spectra of HT cyt c₅₅₂ were obtained with an Autoflex II mass spectrometer (Bruker Daltonics) using sinapinic acid as a matrix in linear mode. Additional details on the experimental procedures are provided in supplementary information.

Hayashi Y., Yamanaka M., Nagao S., Komori H., Higuchi Y, & Hirota S. (2016). Domain swapping oligomerization of thermostable c-type cytochrome in E. coli cells. Scientific Reports, 6, 19334.

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Site-Directed Mutagenesis of Cytochrome c

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Amino acid substitution of Met80 was performed by PCR-based in vitro mutagenesis of the original plasmid vector using M80A-F and M80A-R primers (Table S2). Mutated DNA was purified using the KOD-Plus-Mutagenesis kit (Toyobo). DNA sequencing was carried out with the BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA) and an ABI 3100 Avant generic analyzer (Applied Biosystems, Inc.). Recombinant WT and M80A cyt c were overexpressed using E. coli Rosetta 2(DE3) pLysS cells (Novagen).
Purification of WT and M80A human cyt c were performed according to the methods described elsewhere [13, 31] . The masses of WT and M80A cyt c were checked by MALDI-TOF mass measurements with an Autoflex II mass spectrometer (Bruker Daltonics) using sinapinic acid as a matrix in a linear mode.

Hirota S., Yamashiro N., Wang Z, & Nagao S. (2017). Effect of methionine80 heme coordination on domain swapping of cytochrome c. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry, 22(5).

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