4.4. MALDI-TOF MS Analysis

In-solution trypsin digestion of RET (n = 12) and ONH (n = 12) tissue samples was performed (50 µg /sample; trypsin/protein amount = 1:10). A total protein amount of 5 µg per resulting peptide sample was acidified (0.1% TFA) and incubated with 29 µL conditioned magnetic RPC18 Dynabeads (Invitrogen, Carlsbad, CA, USA), followed by stepwise elution in four 6 µL fractions (0.1% TFA containing 20, 30, 40, and 50% ACN). The peptides were lyophilized and resolubilized in 0.1% TFA. Five replicates of each peptide fraction (2 µL/replicate) were spotted on a 386 MTP polished steel MALDI target plate (Bruker Daltonics, Bremen, Germany) and air-dried, followed by matrix application (19 mg of α-4-cyano-hydroxy cinnamic acid, 60% ACN; 2% TFA; 2µL/spot). Analysis was performed using an Ultraflex II MALDI-TOF–TOF MS analyzer (Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser in the instrument’s reflector mode. MS data were recorded in a detection range of 900–2200 m/z considering a signal-to-noise ratio of 6. Thereby, 500 laser shots in 50 units were accumulated for each MS spectrum with fuzzy controlled laser power (40–80%). MS data were externally calibrated using the peptide II calibration standard (Bruker Daltonics, Bremen, Germany) and internally calibrated considering autodigestive trypsin peaks. MS data inspection was realized in Flex Analysis version 2.4 (Bruker Daltonics, Bremen, Germany). Peptide reporter peaks of 27 protein candidates revealed from the BULCMS analysis were selected for MALDI-specific fragmentation. For this purpose, an in‑house established pig retina MALDI reporter peak reference list within a 20 ppm tolerance window was created and subjected to further MALDI-TOF–TOF MS/MS post-source decay fragmentation (PSD) experiments using BioTools software (version 3.0; Bruker Daltonics, Bremen, Germany). Semi-quantitative determination was realized on the raw intensities of monoisotopic SNAP-detected peptides of specific protein candidates among the technical replicates. Statistical unpaired t-tests of protein levels were performed using Statistica version 10 (Statsoft, Tulsa, OK, USA).