Ultraflextreme spectrometer

Microbubbles (MBs) were obtained (8.4 × 10⁸ MBs/vial) from a MicroMarker^TM, Target-Ready Contrast Agent Kit (VisualSonics Inc., Toronto, ON). Streptavidin coated magnetic beads (New England BioLabs, Whitby, ON) and MACSiMAG^TM separator magnet (Miltenyi Biotec, Auburn, CA) were used during the purification of MBs. Conjugated antibodies were analyzed by mass spectrometry on a MALDI Bruker Ultraflextreme Spectrometer. MB size and concentration were determined using Z2 Coulter counter (Beckman Coulter, Fullerton CA). The syringe pump used in the flow chamber assay was a PhD 2000 (Harvard Apparatus, Holliston, MA). Western blot images were generated using a STORM 840 imaging system (GMI Ltd., Ramsey, MN)

Apo-SOD1 (10 µM) was incubated with 1 mM cholesterol, Seco A or Seco B in 50 mM phosphate buffer pH 8.4 containing 150 mM NaCl and 100 µM DTPA for 24 h at 37 °C. The samples were then reduced with 5 mM sodium borohydride for 1 h at room temperature to stabilize possible adducts. After that, the protein was denatured at 95 °C for 5 min and the aggregates were reduced with 1 M dithiothreitol (DTT) for 1 h at room temperature. Methanol (750 µL) was added to the samples, which were left on ice for 20 min to precipitate SOD1. After centrifugation at 10,000 rpm for 5 min, the precipitate was suspended in 100 µL of 0.1% trifluoroacetic acid for MALDI-TOF analysis. Samples were mixed in a 1:4 (v/v) ratio with a saturated solution of α-cyano-4-hydroxycinnamic acid (HCCA) in 50% acetonitrile/0.1% aqueous trifluoroacetic acid (1:1, v/v). Approximately 1 µL of the resulting mixture was spotted onto a MALDI target and analyzed by MALDI-TOF MS. The analyses were performed in the linear, positive ion mode in an UltrafleXtreme spectrometer (Bruker Daltonics, Germany) using an acceleration voltage of 25 kV. The resulting spectra were analyzed by flexAnalysis software (Bruker Daltonics, Germany).

Matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF MS) was performed using a Bruker UltrafleXtreme spectrometer, equipped with a 355 nm Nd:YAG laser, with α-cyano-4-hydroxycinnamic acid as a matrix. Label-assisted laser desorption/ionization mass spectrometry (LA-LDI MS) was performed using the same apparatus as for MALDI-TOF MS. Samples dissolved in 50% aq. MeOH or MeCN/1% TFA were spotted to an MTP384 ground steel target plate and air-dried according to the manufacturer’s instructions.

We perform mass spectrometry using MALDI-TOF Bruker Ultraflextreme spectrometer to determine the mass of purified protein. Protein is mixed in 1:1 ratio with sinapinic acid which is dissolved in a mixture of acetonitrile and trifluoroacetic acid (1:1).