Maldi microflex lt instrument

Two matrices were prepared according to the double layer method of instrument maker’s manual using sinapic acid (SA). Matrix I consisted of SA saturated in EtOH at a concentration of 10 mg/500 μl and matrix II consisted of SA saturated in 0.3% trifluoroacetic acid (TFA) in 50% acetonitrile (ACN) at a concentration of 200 mg/10 ml. Matrix I (1 μl) was spotted onto a MSP 96 target polished steel (Bruker Daltonics, Bremen, Germany) and allowed to air dry for approximately 5 min at room temperature. Each sample (1 μl) was diluted into 50 μl of matrix II and 1–1.5 μl of the sample/matrix II mixture was deposited onto the top of the matrix thin layer and then dried at room temperature. Gliadins were measured on a MALDI Microflex LT instrument equipped with a 60 Hz nitrogen laser (Bruker Daltonics, Bremen, Germany). Mass spectra were recorded in the linear positive mode and externally calibrated using a mixture of peptide/protein standards. To increase detection sensitivity, the following conditions were used: mass range 22,491–61,376 Da, sample rate 1.00 GS/s, laser shots 100, laser power 85%, laser frequency 60, and detector gain 33X. Each extracted sample was analyzed at least five times.