Chapter 5 - Supplemental material: methods 265 A Methods 1. Acquisition of Mass Spectra from Serum Samples a. Processing of Serum Samples Samples were thawed and 3 µl aliquots of each test sample and quality control/reference serum (a pooled sample obtained from serum of five healthy patients, “SerumP3”) spotted onto serum cards (Therapak). The cards were allowed to dry for 1 hour at ambient temperature after which the whole serum spot was punched out with a 6mm skin biopsy punch (Acuderm). Each punch was placed in a centrifugal filter with 0.45 µm nylon membrane (VWR). One hundred µl of HPLC grade water (JT Baker) was added to the centrifugal filter containing the punch. The punches were vortexed gently for 10 minutes then spun down at 14,000 rcf for two minutes. The flow-through was removed and transferred back on to the punch for a second round of extraction. For the second round of extraction, the punches were vortexed gently for three minutes then spun down at 14,000 rcf for two minutes. Twenty µl of the filtrate from each sample was then transferred to a 0.5 ml eppendorf tube for mass spectral analysis. All subsequent sample preparation steps were carried out in a custom designed humidity and temperature control chamber (Coy Laboratory). The temperature was set to 30 °C and the relative humidity at 10%. An equal volume of freshly prepared matrix (25 mg of sinapinic acid per 1 ml of 50% acetonitrile: 50% water plus 0.1% TFA) was added to each 20 µl serum extract and the mix vortexed for 30 sec. The first three aliquots (3 x 2 µl) of sample: matrix mix were discarded into the tube cap. Eight aliquots of 2 µl sample: matrix mix were then spotted onto a stainless steel MALDI target plate (SimulTOF). The MALDI target was allowed to dry in the chamber before placement in the MALDI mass spectrometer. b. Qualification of Mass Spectrometer The instrument qualification procedure is performed periodically to assess the reproducibility and quality of spectra compared to a defined ‘Gold Standard’ run. The machine qualification sample set used for this analysis contains 40 serum samples representative of expected diversity in human serum. The serum samples are prepared as a batch and spectra acquired as defined for the experimental samples. Spectral processing is performed using parameters defined in the machine qualification standard operating procedures, with methods similar to those
RkJQdWJsaXNoZXIy MTk4NDMw