From each purified sample, 10 µL was premixed with 10 µL matrix consisting of 5 mg/mL super-DHB in 99% ACN with 1 mM NaOH. Of this mixture, 2 µL was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany) and was left to dry. MALDI-TOF-MS spectra were recorded on an UltrafleXtreme mass spectrometer with a Smartbeam-II laser (Bruker Daltonics) in reflectron positive mode, controlled by flexControl 3.4 (Build 135). Measurements were performed within a range from m/z 1,000 to 5,000, accumulating 10,000 laser shots at a frequency of 1,000 Hz and with 100 shots per raster spot using a random walking pattern.
Mtp anchorchip
Manufactured by Bruker
Sourced in Germany
The MTP AnchorChip is a specialized sample preparation plate designed for use in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The core function of the MTP AnchorChip is to provide a standardized and consistent sample deposition surface to improve the reproducibility of MALDI-TOF analyses.
Automatically generated - may contain errors
Lab products found in correlation
Ultraflextreme, by Bruker (2 mentions)
Smartbeam 2 laser, by Bruker (1 mentions)
Axima resonance maldi qit tof ms, by Shimadzu (1 mentions)
Trypsin, by Promega (1 mentions)
α cyano 4 hydroxycinnamic acid, by Bruker (1 mentions)
Ultraflex tof tof, by Bruker (1 mentions)
Peptide calibration standard, by Bruker (1 mentions)
Autoflex 2, by Bruker (1 mentions)
Tm sprayer, by HTX Technologies (1 mentions)
Cm3050s cryostat, by Leica Biosystems (1 mentions)
Trypsin solution, by Promega (1 mentions)
Maldi tof ms, by Bruker (1 mentions)
10 protocols using mtp anchorchip
1
MALDI-TOF-MS Analysis of Purified Samples
2
MALDI-QIT-TOF MS Analysis of N-Glycans
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One microliter of collected N-glycans was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany) and allowed to dry by air. Then, one microliter 2,5-DHB (10 mg/ml) in 0.1% (v/v) TFA in 50% ACN/H2O (v/v) was added onto the sample layer, followed by recrystallized to form homogeneity of the spot surface with ethanol.
Each sample was spotted in triplicate. The samples were interrogated automatically in a “batch mode” by AXIMA Resonance MALDI-QIT-TOF MS (Shimadzu Corp. JP) equipped with a 337 nm nitrogen laser in reflector positive ionization mode. The m/z range was monitored to span from 500 to 5,000. The GlycoWorkbench software was used for the annotation of MS spectra.
Each sample was spotted in triplicate. The samples were interrogated automatically in a “batch mode” by AXIMA Resonance MALDI-QIT-TOF MS (Shimadzu Corp. JP) equipped with a 337 nm nitrogen laser in reflector positive ionization mode. The m/z range was monitored to span from 500 to 5,000. The GlycoWorkbench software was used for the annotation of MS spectra.
3
Automated Glycan Derivatization and Analysis
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The automated sample preparation consists of derivatization, hydrophilic interaction liquid chromatography (HILIC) purification, and MALDI-target plate spotting which was performed using an automated liquid handling platform.40 (link) Ethyl esterification, for stabilization and linkage-specific derivation of sialic acids, was performed with freshly prepared chemicals and solutions. Therefore, 2 μL of the released glycan samples was added to 40 μL of ethyl esterification reagent, which consisted of 0.25 M EDC and 0.25 M HOBt dissolved in 100% ethanol, followed by incubation for 1 h at 37°C.88 (link) Subsequently, 40 μL of acetonitrile was added. For the purification of the N-glycans, in-house assembled cotton HILIC microtips were used (approx. 3 mm or 180 μg cotton thread per tip). The tips were pre-washed with MQ water and 85% acetonitrile. Then, the glycans were bound to the cotton by pipetting the samples up and down 20 times. The tips were washed with 85% acetonitrile with 1% TFA followed by 85% acetonitrile and eluted in 20 μL MQ water. Subsequently, 7 μL of the purified sample plus 7 μL of sDHB matrix (2.5 mg/mL in 50% ACN with 0.1 mM NaOH)89 (link) was premixed in a 384-well plate. Then, 2 μL of the mixture was spotted onto a MALDI target plate (800/384 MTP AnchorChip, Bruker Daltonics, Bremen, Germany), and after air-drying the spots were measured by MALDI-FTICR-MS.
4
Proteomic Fingerprinting of Fluoride Effects
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Proteins of spots the intensity of which detectably differed between the fluoride-treated and control group, respectively, were identified by peptide mass fingerprinting (PMF). The spots were excised with circular plugs 2-3 mm in diameter, and transferred to 1.5 mL EP tubes. Coomassie blue-stained gel pieces were first destained with 50 μL of a 1:1 (v/v) mixture of 50 mM NH 4 HCO 3 and acetonitrile for 10 min, followed by three washes with 50 μL of MilliQ water. The gel pieces were then dehydrated with 100% acetonitrile for 5 min and dried in a SpeedVac (Thermo Savant, Holbrook, NY, USA) for 30 min. The dried gel particles were rehydrated at 4 °C for 30 min with trypsin (sequencing grade; Promega, Madison, WI, USA) in 50 mM NH 4 HCO 3 (20 g/mL), and then incubated at 37 °C overnight. The peptide mixture (1 μL) was mixed with 1 μL 10 mg/mL α-cyano-4-hydroxycinnamic acid (Sigma) and spotted onto the MTP Anchor Chip (Bruker Daltonics, Bremen, Germany) and analysed by matrixassisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) (ultraflex tof/tof, Bruker, Bremen, Germany).
5
MALDI-TOF/TOF-MS Analysis of N-Glycans
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N-glycans were dissolved in 10 μL MW and spotted on an MTP AnchorChip (Bruker Daltonics; Bremen, Germany) sample target. The samples were air-dried, and 1 μL DHB solvent (20 mg/ mL in MW) was added to recrystallize the glycans. The N-glycans were analyzed by MALDI-TOF/TOF-MS (UltrafleXtreme; Bruker Daltonics) in positive ion mode, and m/z data were analyzed and annotated using the GlycoWorkbench software program as described previously [7 (link)]. The relative variation of the different types of N-glycans were calculated by adding up all of the relative intensity of a given type of N-glycan.
6
MALDI-TOF/TOF-MS/MS Analysis of N-Glycans
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N-glycans were characterized by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS/MS) (Ultrafle Xtreme; Bruker Daltonics, Bremen, Germany). N-glycans were resuspended in 10 μL of methanol/H2O (1:1 v/v) and 1 μL of the mixture was spotted onto the microtiter plate (MTP) AnchorChip (Bruker Daltonics, Bremen, Germany) sample target and air-dried. 1 μL of 20 mg/mL 2,5-dihydroxy-benzoic acid (DHB) in methanol/H2O was spotted to recrystallize the glycans. Mass calibration was performed using peptide calibration standards (250 calibration points; Bruker). Distinct N-glycan peaks were obtained from the mass spectra using a signal-to-noise ratio >5 as criterion. Measurements were taken in positive-ion mode, and m/z data were analyzed and annotated using the GlycoWorkbench software program (http://code.google.com/p/glycoworkbench/ )[28 (link)].
7
MALDI-TOF MS Analysis of Urine Samples
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Urine samples were prepared as previously described 27 . Serial dilutions of each sample, which covered two orders of magnitude (0.35–0.0035 μL equivalents of urine) were prepared with 0.1% TFA. Protein concentration was determined by Bradford analysis to be in the range of 13–35 ng/μL. Twelve dilutions were then spotted on a 384 well MTP Anchorchip (Bruker Daltronics, Bremen, Germany) target plate in quadruplicate 28 . One microliter of sample was left to dry on the target plate, followed by 1 μL of a 5 mg/mL of α‐cyano‐4‐hydroxycinnamic acid (αCHCA) matrix (Laser Biolabs, Sophia‐Antipolis, France). The matrix concentration was previously determined to be the optimum using standard urine dilution series. No internal standards were added for quantification purposes as this has been shown to be ineffective 28 .
8
MALDI-TOF Mass Spectrometry Protocol
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Samples were spotted on a MTP AnchorChip (Bruker Daltonics, Bremen, Germany) by first crystallizing 1 L of saturated Super-DHB matrix solution (2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid), followed by addition of 1 L of sample solution. After drying, mass spectra were recorded in positive mode with a Autoflex II mass spectrometer (Bruker Daltonics, Bremen, Germany).
9
Cryosection Mouse Tissue for Lipid Analysis
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For tissue preparation, one-week old C57BL/6 control mouse pup was snap frozen at -80 oC, shaved over dry ice, and cryosectioned (20µm thickness), using a CM3050 S cryostat (Leica Biosystems, Wetzlar, Germany). The tissue was thaw-mounted onto conductive indium tin oxide coated glass slides (Delta Technologies, Loveland, CO, USA). The sample was placed on a heated metal block (75 oC) and coated with a 15 mg/mL solution of DAN dissolved in THF using a TM Sprayer (HTX Technologies, LLC, Chapel Hill, NC, USA) (0.05 mL/hr, 5 passes, 0 sec drying time, 40 oC spray nozzle). For lipid standard analysis, the standards (in chloroform) were aliquoted in vials (5mg/mL), dried down with nitrogen gas, and dissolved in 80% methanol for a final concentration of 1.0 mg/mL. The aliquots were then mixed with DHA matrix, dissolved in 90% acetonitrile to a final lipid concentration of 0.2 mg/mL. The mixtures were spotted on a polished steel target -MTP AnchorChip (Bruker Daltonik, Bremen, Germany).
10
Protein Identification by MALDI-TOF MS
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Protein spots were excised manually and transferred into 1.5 ml RNase-free centrifuge tubes. The spots were washed three times with double distilled water, de-stained by sonication in 50 mM ammonium bicarbonate, 50% (v/v) acetonitrile, dehydrated with acetonitrile and dried in a vacuum pump. The dried proteins spots were treated by 10 mM DTT for 1 h at 56°C, alkylated with 55 mM iodoacetamide for 45 min at room temperature, washed with 25 mM ammonium bicarbonate and 50% (v/v) acetonitrile, respectively. It was further dehydrated with acetonitrile and incubated with 10~15 µl trypsin solution (Promega, 10 μg/ml) at 37°C overnight. The resulting peptides were collected and mixed with an equal volume of 10 mg/ml matrix solution (a-cyano-4-hydroxycinnamic acid), saturated with 0.1% (v/v) trifluoroacetic acid in 50% (v/v) acetonitrile, spotted on an MTP Anchor Chip (Bruker, Germany), and analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS, Bruker, Germany) to acquire spectra with a mass range from 500 to 6,000 Da. External calibration was performed with standard peptides. The matrix and autolysis of trypsin were served as internal standards. The mass spectra were interpreted using the Mascot peptide mass fingerprint engine available on the website (http://www.matrixscience.com ) for protein identification.
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