4800 plus maldi tof tof mass spectrometer

Recombinant human TSLP (SRP4896, Sigma-Aldrich) was treated with β-tryptase (G563A, Promega) or chymase (S-C8118, Sigma-Aldrich) in limited proteolysis conditions and analyzed by Matrix Assisted Laser Desorption/Ionization-Mass spectrometry (MALDI-MS) in linear mode. The reactions were carried out on 1 μg of TSLP. Tryptase was added with an enzyme:substrate ratio of 1:1000 (w/w) for 30 min, while chymase was used at an enzyme:substrate ratio of 1:100 (w/w) for 30 min. These experimental conditions were set up based on optimal hydrolysis conditions for each protease [82 (link)]. For MALDI-MS analyses, 0.5 μL of each peptide mixture was mixed with an equal volume of α-cyano-4-hydroxycinnamic acid as matrix (10 mg/mL) in 0.2% trifluoroacetic acid (TFA) in 70% acetonitrile, loaded onto the metallic sample plate, and air-dried. The peptide mixture was analyzed in linear mode by a 4800 plus MALDI TOF-TOF mass spectrometer (AB SCIEX, Toronto, ON, Canada) and using the 4000 Series Explorer (TM) software (AB SCIEX, Toronto, ON, Canada) (version 3.5) to detect the released fragments, in order to identify the cleavage sites on TSLP. Mass calibration was performed using the MH⁺ and MH₂²⁺ ions of a protein mixture containing insulin and apomyoglobin to ensure accurate mass determination and calibration [99 (link)].

The insulin lispro derivative and endoproteinase Glu-C derived peptides were analyzed in a reflector mode with the use of 4800 Plus MALDI-TOF/TOF mass spectrometer (AB SCIEX, Framingham, USA). α-cyano-4-hydroxy-cinnamic acid matrix was exploited. External calibration was performed with a 4700 proteomics analyzer calibration mixture provided by AB SCIEX. Data Explorer Software, Version 4.9, and General Protein/Mass Analysis for Windows, version 8.2, were applied for assignment of a, b and y ions to the MS/MS spectrum of acetylated peptide. DeNovo Explorer (GPS Explorer TM Software, Version 3.6) was exploited to match the MS/MS spectrum to the annotated peptide sequence.

The selected protein spots were excised and placed in Eppendorf tubes with 50 μl of destaining solution (50% v/v methanol and 5% v/v acetic acid), followed by washes with Milli-Q H₂O. Gel fragments were dehydrated by incubation in 100 μl of acetonitrile (ACN) for 10 min, and the supernatant was then removed; this step was repeated once. Dry gel pieces were rehydrated with 200 ng of trypsin (Promega V528A) in 50 mM NH₄HCO₃ and 5% ACN and incubated overnight at 37 °C. The resulting peptides were extracted with 40 μl of 50% ACN and 5% formic acid, and the solution volume was reduced in a concentrator (Eppendorf 5301). Peptides from each sample were desalted on C18 columns (ZipTipC18). A 1:1 mixture of peptide solution and matrix solution (5 mg/ml CHCA 50% v/v and TFA 0.1% v/v) was analyzed using a 4800 Plus MALDI TOF/TOF mass spectrometer (Sciex). The search was performed with the enzyme specificity of trypsin, and one missed cleavage was allowed. The detected protein threshold was 66%, and the precursor mass tolerance was 0.5–1 Da. The MS data were compared with the Rattus norvegicus database (downloaded in September 2016) using Protein Pilot™ software (version 2.0.1) and the Mascot algorithm^{43 (link)}.