Ultraflex 2 tof tof instrument

UV-exposed photo-MHC-I was purified by SEC to remove any possible fragment. In order to detect fragments of the photocleavable peptide, SEC fractions were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) using α-cyano-4-hydroxycinnamic acid (HCCA) as matrix. The samples were applied on the MALDI target using the dried-droplet technique. Spectra were recorded on an Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) in the positive reflector mode in the m/z range of 600–4000. Samples of photocleavable peptide before and after UV exposure served as a reference.

Crude cupula material from salmon and chicken was dissolved in denaturing 2 x SDS sample buffer and boiled for 5 min. Remained debris was removed by centrifugation (14,000×g, 10 min). Soluble extract was separated by SDS-PAGE under reducing conditions and the gel was stained with Coomassie Brilliant Blue. The dominant 45 kDa band was excised from the gel with a scalpel and cut into small 1 mm gel cubes.
Peptides were obtained by trypsin in-gel digestion as described previously [11] (link) and peptide masses were analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) using an Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz solid-state Smart beam laser. The mass spectrometer was operated in the positive reflector mode. Mass spectra were acquired over an m/z range of 600–4,000.
α-cyano-4-hydroxycinnamic acid (CHCA) was used as the matrix and protein digest samples were spotted using the dried-droplet technique. MS/MS spectra of selected peptides were acquired in the LIFT mode [12] (link).
Database searches were performed using Mascot (Matrix Science Ltd., http://www.matrixscience.com). Mass tolerance was typically set at ±75 ppm and we allowed for one missed cleavage. Annotation of the MS/MS spectra was done manually.

Intact protein mass of gD1, gD4, and MHC-I was determined by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) using an Ultraflex-II TOF/TOF instrument (Bruker Daltonics, Bremen, Germany) equipped with a 200 Hz solidstate Smart beam™ laser. Samples were spotted using the dried-droplet technique on sinapinic acid (SA) or 2,5-dihydroxybenzoic acid (DHB) matrix (saturated solution in 33% acetonitrile/0,1% trifluoroacetic acid). The mass spectrometer was operated in the positive linear mode, and spectra were acquired over an m/z range of 3,000-60,000. Data was analyzed using FlexAnalysis 2.4. software provided with the instrument.
Protein identity was determined by tandem mass spectrometry (MS/MS) of in-gel digested Coomassie stained protein with 12_,5 μg_/ml Glu-C and trypsin, and 10 μg_/ml Asp-N in 25 nm ammonium bicarbonate.
N-terminal c and C-terminal (z + 2) sequence ion series were generated by in-source decay (ISD) with 1,5-diaminonaphthalene (1,5-DAN) as matrix (20 mg/ml 1,5-DAN in 50% acetonitrile/0,1% trifluoroacetic acid). Spectra were recorded in the positive reflector mode (RP PepMix) in the mass range 800–4,000.