MIP/NIP were analysed using MALDI-TOF-MS (ultrafleXtreme instrument, Bruker Daltonik GmbH, Bremen Germany) equipped with a laser (operating at wavelength of 355 nm with an accelerating voltage of 25 kV, a maximum energy of 43.2 μJ, and a repetition rate of 2000 Hz) in linear positive ion mode for data acquisition. Three different organic matrix solutions were tested, namely α-cyano-4-hydroxycinnamic acid, sinapinic acid and 2,5dihydroxybenzoic acid (DHB) (Bruker Daltonik, Bremen, Germany), with DHB diluted in 0,1% trifluoroacetic acid (Sigma-Aldrich) being considered the optimal solution, since less background was produced in the final spectrum [18] . Matrix (0.5 µL) was applied on the prepared MIP and/or NIP polymerized layer (as previously described in section 2.2) and dried under atmospheric pressure and ambient temperature (25 °C). The laser frequency was set to 1000 Hz and laser energy was optimized prior to each measurement. Calibration was done externally using a protein standard mixture I and II (Bruker Daltonics, Bremen, Germany) in the range of m/z 1-90 kDa. A total of 500 spectra were summed for each spot using the Random Walk raster pattern, with no evaluation criteria and were analysed with the Flex Analysis software (Version 3.4).
2 5 dihydroxybenzoic acid dhb
Manufactured by Bruker
Sourced in Germany
2,5-dihydroxybenzoic acid (DHB) is a chemical compound that can be used as a matrix in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. It is a solid, crystalline substance that absorbs ultraviolet light and can facilitate the ionization of analytes for mass spectrometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Methanol, by Fujifilm (3 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
α cyano 4 hydroxycinnamic acid hcca, by Bruker (2 mentions)
Ultrapure water, by Fujifilm (2 mentions)
Sinapinic acid, by Bruker (1 mentions)
Protein standard mixture 1 and 2, by Bruker (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
Bovine serum albumin fraction 5, by Solarbio (1 mentions)
Trichloroacetic acid, by Merck Group (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Pepsin, by Solarbio (1 mentions)
N glycosidase f pngase f, by Roche (1 mentions)
Phosphoric acid, by Thermo Fisher Scientific (1 mentions)
Concentrated nitric acid, by Thermo Fisher Scientific (1 mentions)
Lc ms grade water, by Thermo Fisher Scientific (1 mentions)
Acetonitrile acn, by Thermo Fisher Scientific (1 mentions)
Dopamine hydrochloride, by Merck Group (1 mentions)
Cholesterol, by Merck Group (1 mentions)
15 protocols using 2 5 dihydroxybenzoic acid dhb
1
MALDI-TOF-MS Analysis of Molecularly Imprinted Polymers
2
Okra Antioxidant and Glycoprotein Analysis
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Okra was purchased from a local market in Jinzhou, Liaoning Province, China. 2,2-Diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic acid (TCA), and dithiothreitol were purchased from Sigma-Aldrich (St. Louis, Mo, USA). Pepsin and bovine serum albumin fraction V were purchased from Solarbio Life Sciences (Beijing, China). N-glycosidase F (PNGase F) and N-glycoamidase A (PNGase A) were purchased from Roche CustomBiotech (Penzberg, Germany). All the lectins were purchased from Calbiochem (Merck Biosciences GmbH, Germany). 2,5-Dihydroxy-benzoic acid (DHB) was purchased from Bruker Daltonik GmbH (Germany). All other chemicals and reagents used were of analytical grade.
3
Lipid Characterization by Mass Spectrometry
4
Mass Spectrometry Calibration Standards
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Methanol (MeOH), ethanol (EtOH), chloroform (CHCl3), ultrapure water, and potassium acetate (CH3COOK) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Calibration standard peptides (human bradykinin and angiotensin II) were purchased from Bruker Daltonics (Billerica, MA, USA). 2,5-Dihydroxybenzoic acid (DHB) was purchased from Bruker Daltonics (Billerica, MA, USA) or Sigma-Aldrich (St. Louis, MO, USA). 9-Aminoacridine hemihydrate (9-AA) was purchased from Acros Organics (NJ, USA).
5
Glycoprotein Analysis by Mass Spectrometry
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Ribonuclease B (RNase B, from bovine pancreas), trypsin (from bovine pancreas), Glu-C (from staphylococcus aureus V8), protease inhibitor cocktail, formic acid (FA), urea, trifluoroacetic acid (TFA), succinic anhydride (SA), acetic anhydride (AA), butyraldehyde, dithiothreitol (DTT) and iodoacetamide (IAA) were bought from Sigma-Aldrich (St. Louis, MO). Acetonitrile (ACN, HPLC grade) was purchased from Merck (Darmstadt, Germany). 2, 5-Dihydroxybenzoic acid (DHB) was obtained from Bruker (Daltonios, Germany). PNGase F was bought from New England Biolabs (Ipswich, MA). Water was purified by a Milli-Q system (Millipore, Milford, MA). All other chemicals and solvents were analytical-grade.
Fused-silica capillary (150 μm i.d. × 375 μm o.d.) was obtained from Sino Sumtech (Handan,China). Click maltose modified matrixes (5 μm, 100 Å) were kindly donated by Prof. Xinmiao Liang (Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China). Daiso C18 particles (5 μm, 100 Å) were ordered from Daiso (Osaka, Japan).
Fused-silica capillary (150 μm i.d. × 375 μm o.d.) was obtained from Sino Sumtech (Handan,China). Click maltose modified matrixes (5 μm, 100 Å) were kindly donated by Prof. Xinmiao Liang (Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, China). Daiso C18 particles (5 μm, 100 Å) were ordered from Daiso (Osaka, Japan).
6
Characterization of Protein Standards
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The standard proteins bovine serum albumin (BSA, Z98%, molecular weight 66 400 u) and immunoglobulin G from human serum (IgG, Z95%, molecular weight 150 000 u) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetone, acetonitrile, acetonitrile with 0.1% trifluoroacetic acid (TFA) and water with 0.1% TFA (all LC-MS grade) were obtained from Merck (Darmstadt, Germany). Both MALDI matrices a-cyano-4hydroxycinnamic acid (a-CHCA) and 2,5-dihydroxybenzoic acid (DHB) were received from Bruker Daltonics (Bremen, Germany). Formic acid (Z99%, LC-MS grade) was acquired from VWR Chemicals (Darmstadt, Germany). Water was purified using an on-site purification system Milli-Q Integral 5 (Merck Millipore, Darmstadt, Germany).
7
Peptide Synthesis and Modification
8
IgG Purification from Human Serum
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Immunoglobulin G from human serum (IgG, 56834-25MG), albumin from bovine serum (BSA, A3059), trypsin from bovine pancreas (T1426), acetonitrile (ACN), trifluoroacetic acid (TFA), DL-dithiothreitol (DTT), iodoacetamide (IAA), sodium chlorite (NaClO 2 ), Eppendorf LoBind® microcentrifuge tubes (Lobind tube), and all salts used in the present work were purchased from Sigma-Aldrich (Stockholm, Sweden). H 2 O used in the present work was purified to have a resistivity of 18.2 MΩ cm at 25 °C with a Millipore Synergy® 185 system (Bedford, MA, USA). 2,5-Dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (HCCA) were obtained from Bruker Daltonics (Bremen, Germany). Anonymized and pooled human plasma from volunteers was stored in -80 °C before use. DEAE Affi-Gel® Blue Gel (Affi-Gel) was from Bio-RAD (Hercules, USA).
9
Optimizing Molecular Characterization Techniques
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Nine-aminoacridine (9AA) was purchased from Merck Schuchardt (Hohenbrunn, Germany). 2, 5-dihydroxybenzoic acid (DHB) was purchased from Bruker Daltonics (Germany). Methanol, ethanol, and ultrapure water (Wako Pure Chemical Industries, Osaka, Japan) were used for the preparation of all solvents. All chemicals used in this study were of the highest purity available. For the Kawamoto method (Kawamoto and Shimizu, 2000 (link)), adhesive film (Cryofilm type IIC) was purchased from Leica Microsystems (Wetzlar, Germany). Standard chemicals of L-carnosine, inosine monophosphate, taurine, hydrocortisone, and corticosterone were purchased from Wako Pure Chemical Industries. Dopamine (Sigma Aldrich, St. Louis, MO, USA) and L-DOPA (Toronto Research Chemicals, Toronto, Canada) were also purchased and used in the study.
10
MALDI-TOF MS Characterization of Purified MTs
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MTs were purified by using FPLC according to our previous study [44 (link)]. After isolation, MTs were analyzed using MALDI-TOF MS (Ultraflex III instrument, Bruker Daltonik, Leipzig, Germany) equipped with a laser, operating at wavelength of 355 nm with an accelerating voltage of 25 kV, a maximum energy of 43.2 µJ, and a repetition rate of 2000 Hz. The matrixes used for analyses were α-cyano-4-hydroxycinnamic acid (HCCA) and 2,5-dihydroxybenzoic acid (DHB) (Bruker Daltonik, Leipzig, Germany) prepared in acetonitrile solution (30% w/w) with the addition of trifluoroacetic acid (0.1% w/w). Matrix and substance solutions for analysis were mixed in ratio of 1:1 (v/v). Obtained homogeneous solution (1 µL) was dried under atmospheric pressure and ambient temperature (25 °C). MS spectra were typically acquired by averaging 20 sub-spectra from a total of 500 shots of the laser (Smartbeam 2. Version: 1_0_38.5, Bruker Daltonik, Leipzig, Germany).
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