Maldi tof tof 5800 mass spectrometry

The MMP-2/-9-cleavable octapeptide Fmoc–Gly–Pro–Leu–Gly–Leu–Ala–Gly–Gly was evaluated by enzymatic digestion with the use of collagenase IV (containing MMP-2/-9). In the first step, 1 mL of HBSS was added to 1 g of collagenase IV (205 u/mg) and mixed to complete dissolution. Activation of collagenase IV was performed using 1 mM APMA in 0.15 M NaOH for 1.5 h at 37 °C [24 (link),43 ]. The octapeptide stock solution was added to activated 1 mM collagenase IV and incubated at 37 °C. The aliquots were removed and analyzed by HPLC, between 2 and 12 h. The HPLC Dionex Ultimate 3000 (Waltham, MA, USA) was equipped with a bioZen Intact XB-C8 column (3.6 μm, 2.1 × 150 mm; Phenomenex, Aschaffenburg, Germany). The flow rate was set at 0.5 mL/min with a mobile phase consisting of solvent A (0.1% TFA) and solvent B (100% ACN). The gradient was as follows: 0–5 min, 0% of solvent B; 5–8 min, 0–30% of solvent B. The hydrolysis product was detected by MALDI TOF/TOF 5800 mass spectrometry (AB Sciex, Darmstadt, Germany).

The appropriate band identified to bind with LBD1 was excised from SDS-PAGE gel, placed into a clean microcentrifuge tube, and then digested with trypsin as described [47 (link)]. The digested band was run on a MALDI-TOF/TOF 5800 mass spectrometry (AB SCIEX, USA) to obtain the corresponding peptide mass fingerprinting (PMF) using GPS Explorer 3.5 (Applied Biosystems, USA) and Mascot (Matrix Science, UK). PMF data were further indexed using Andromeda search engine to assign the corresponding peptide sequences in an integrated silkworm proteome database [48 (link), 49 ].

Morpholine (0.5 mL) was added to a stirred solution of Fmoc–Leu–Ala–Gly–Gly–DOX (50 mg, 47 µmol) in DMF (0.5 mL), and the stirring was carried on for a further 2 h. Afterward, the mixture was neutralized with trifluoroacetic acid (0.6 mL). The raw product was isolated on a semi-preparative HPLC System (Shimadzu, LC 20AD, Shimadzu, Canby, OR, USA) using a Gemini NX Column (5.0 μm, 10 × 150 mm) to give 24 mg in a 61% yield. The flow rate was set at 4.0 mL/min with the mobile phase consisting of solvent A (0.1% formic acid) and solvent B (100% ACN). The gradient was as follows: 0–15 min, 10–90% of solvent B; 15–20 min, 90% of solvent B. The chemical structure of the product was confirmed using MALDI TOF/TOF 5800 mass spectrometry (AB Sciex, Darmstadt, Germany). As a matrix, 2,5-dihydroxybenzoic acid was used. The measurement was done in reflector positive ion mode. Samples were prepared using the dried droplet preparation method by mixing 0.8 mL of an analyte solution with 0.8 mL of the matrix solution (directly on a plate). MS spectra were acquired from 789 to 961 m/z for a total of 1000 laser shots by a 1 kHz OptiBeam laser (AB Sciex, Darmstadt, Germany).