4800 plus maldi tof tof analyzer

Each of the selected differential protein spots was excised manually by using pipette tips and subsequently de-stained with 100 mM NH₄HCO₃ in 30% acetonitrile solution (ACN, Solarbio, Beijing, China). After being aspirated and freeze-dried, the samples were digested with 5 μL of 2.5–10 ng/μL sequencing-grade modified trypsin (Promega, Madison, WI, USA) overnight at 37 °C. The Peptides were extracted three times with 60% ACN and 0.1% trifluoroacetic acid (TFA, Solarbio, Beijing, China), freeze-dried and then resuspended in 5 mg/mL cyano-4-hydroxycinnamic acid (Bio-Rad) in 50% ACN and 0.1% TFA for matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI TOF-MS) analysis. Peptide mass was determined using MALDI TOF-MS on a 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems, Massachusetts, Framingham, MA, USA). Peptide mass maps were acquired in a positive ion reflector mode (2 kV accelerating voltage) with 355-nm laser shots for per spectrum. The minimum signal-to-noise ratio was set at 50. MS spectra for all samples were measured with an overall mass/charge (m/z) range of 800 to 4000, and eight of the most intense ions were selected as precursors for the tandem mass spectrometry (MS/MS) acquisition.

After image analysis, gels containing the additional load of unlabeled proteins from the epididymal adipose tissue were stained with Colloidal Coomassie Brilliant Blue G (GE Healthcare) and matched to the fluorescent 2D-DIGE images. Selected spots were picked and in-gel digestion of protein samples was performed using the protocol described previously by our group
[15 (link)]. The peptide mixtures were analyzed using matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS; 4800 Plus MALDI TOF/TOF™ Analyzer, Applied Biosystems, Foster City, CA) operating in positive-ion reflector mode. Protein database search was performed with the Paragon Method using Protein Pilot software (Applied Biosystems) to identify excised proteins.

To determine the reactive site at which SW-AT-1 was cleaved by chymotrypsin, the purified rSW-AT-1 (280 µg/ml) was mixed with chymotrypsin (100 µg/ml) for 10 min at room temperature, and the reaction mixture was desalted using a C₁₈ zip-tip column (Millipore) and eluted with 70% acetonitrile, 0.1% formic acid. The sample was mixed with an equal volume of saturated sinapinic acid matrix on a MALDI plate, air-dried, and subjected to mass determination on a mass spectrometer (4800 Plus MALDI TOF/TOF Analyzer, Applied Biosystems, USA). The spectra were calibrated using bovine serum albumin as an external standard. The molecular mass of a peak that was absent in the control spectra of rSW-AT-1 and chymotrypsin alone was compared with calculated values of carboxyl-terminal peptides to deduce the cleavage site in SW-AT-1.

Bands of interest from the Blue Safe stained SDS-PAGE gel were cut out with a sterile scalpel and immersed in 5% acetic acid solution. Proteins were then gel reduced, alkylated, and digested with trypsin according to Sechi and Chait [18 (link)]. Analysis of peptides from protein digestion was performed using the 4800 Plus MALDI TOF/TOF Analyzer mass spectrometer (Applied Biosystems, MDS Sciex, Toronto, ON, Canada), at the Proteomic Unit of Complutense University of Madrid (Spain). Peptide mass fingerprint and some peptide fragmentation spectra were combined and searched for in the MASCOT v2.3 search engine (http://www.matrixscience.com) through the Global protein Server (Applied Biosystems) against the NCBI database (17,919,084 sequences; 6,150,218,869 residues) without taxonomy restriction or search parameters: carbamidomethylcysteine was used as the fixed modification and oxidized methionine was used as the variable modification; peptide mass tolerance 80 ppm; 1 missed trypsin cleavage site allowed, and MS/MS fragments tolerance, 0.3 Da.
The applied probability filter was that set by the search engine software itself, in this case MASCOT, which uses its own probability algorithm. Thus, in all protein identification, the probability scores were greater than the score fixed by MASCOT as significant with a p-value lower than 0.05.

The structure of the purified ACE inhibitor peptide was identified using a 4800 plus MALDI TOF/TOF™ Analyzer (Applied Biosystems, Beverly, MA, USA). The sample was mixed with a matrix solution (α-cyano-4-hydroxycinnamic acid solution) and prepared with 50% acetonitrile containing 0.1% TFA [57 (link)]. Tandem mass spectrometry (MS) experiments were conducted by collision-induced dissociation, and the peptide sequencing was obtained via tandem MS analysis.