Maldi tof mass spectrometer

The RING finger peptide derived from the PHD domain of FANCL (with amino acid sequences of DCGICYAYQL DGTIPDQVCD NSQCGQPFHQ ICLYEWLRGL LTSRQSFNII FGECPYCSK) was obtained from New England Peptide, Inc. (Gardner, MA) and purified by HPLC. The peptide was dissolved in 20 mM Tris-HCl (pH 6.8) containing 1 mM dithiothreitol. Aliquots of 100 μM peptide were incubated with 200 μM NaAsO₂ on ice for 1 h and subsequently diluted by 100 fold. The resultant solution was mixed with an equal volume of 2,5-dihydroxybenzoic acid matrix solution followed by spotting onto a sample plate.^{4 (link)} The sample was then analyzed on a Voyager DE STR matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometer (Applied Biosystems, Foster City, CA) in the linear, positive-ion mode.

Lenses were dissected out of capsule and carefully partitioned as outer cortex (OC), inner cortex (IC), outer nucleus (ON) and inner nucleus (IN). The tissues were homogenized in 4 mM Tris-HCl, 5 mM DTT, 5mM EDTA, pH 8.0 with protease inhibitor cocktail (Sigma). Homogenates were centrifuged at 110,000 g for 30 min at 4 °C. Pellets were extracted with 7M urea in homogenizing buffer and spun down as above. Membrane pellets were washed with dH₂O and dissolved in a mixture of formic acid and isopropanol (7:3), then a solution of 50% acetonitrile / 0.1%TFA containing sinapinic acid (20mg/ml) was added. The mixture of each sample (0.5µl) was dried on sample target for analysis and run on a Voyager-DE STR (Applied Biosystems) MALDI-TOF mass spectrometer system operated in linear mode. The mass scale (m/z 5000–35000) was calibrated with a mixture of myoglobin and carbonic anhydrase and approximately 150 laser shots were used to produce each spectrum.