Sonifier slpe cell disruptor

Identification of relevant mass spectral peaks is necessary to gain functional insight into immunological processes. In order to identify the endogenous peptides observed with MSI, up to 10 lung tissue slices from the same piece of tissue were, as soon as possible after sectioning, collected in peptide extraction solvent consisting of methanol:water:formic acid (90:9:1 v:v:v) and shaked on ice for 30 min. The sample was kept on ice during the whole extraction procedure. First, the peptide sample was sonicated with a bar sonicator twice for 15 s each (Branson Sonifier SLPe cell disruptor). After 15 min centrifugation at 14,000 rcf by 4 °C, the supernatant was collected and methanol could be evaporated by using a vacuum centrifuge concentrator (Savant SPD1010 SpeedVac Concentrator, Thermo Scientific). The lipids were removed by re-extraction with n-hexane. From the remaining aqueous fraction, the peptides were concentrated using an ultra-0.5 mL 10 K centrifugal filter device (Merck) and desalting was performed by solid phase extraction with a Pierce C18 Spin Column (Thermo Scientific) according to the manufacturer’s procedure. The eluted sample was again dried using the vacuum centrifuge concentrator and the sample pellets were stored at −20 °C prior to LC-MS/MS analysis.