Maldi tof mass spectrometer

The amino acid sequences of the BSA-binding peptide and MMP-3-responsive peptide (NFF-3) were integrated into a new single peptide and selected as the linker, named the PN peptide. The PN peptide was conjugated to 4-arm-PEG_10,000-MAL (PEG-Mal) by a nucleophilic substitution reaction to obtain the targeting compound PN-PEG. Briefly, the PN peptide and PEG-Mal (molar ratio 5:1) were dissolved in freshly distilled dimethyl sulfoxide (DMSO) and phosphate-buffered saline (PBS), respectively. After 18 h of incubation at room temperature, the reaction mixture was placed in a dialysis bag with a cut-off molecular weight (MW) of 8000–14,000 Da and dialyzed against deionized water for 48 h to remove the unreacted ligand²⁴ (link). Then, the solution was lyophilized and stored at −20 °C. The conjugation of the PN peptide with PEG-Mal was confirmed using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (Bruker Daltonics, Germany). Acetonitrile:water (7:3) with 0.1% trifluoroacetate was used as the solvent, and 10 mg/mL of α-cyano-4-hydroxycinnamic acid was used as the matrix²⁵ .

High-purity grade reagents and solvents were used for the synthesis of ethyl 2-(2-(arylidene)hydrazinyl)thiazole-4-carboxylates (1a–k) and alkyl 2-((2-(arylidene)hydrazinyl)-4-oxo-4,5-dihydrothiazol-5-yl)acetates (2a–p). All synthesized esters of 1,3-thiazoles and thiazolidin-4-ones were initially characterized by their physical parameters, like the change in color, melting points, and the R_f (retardation factor) values. Spectroscopic techniques, like FT-IR, NMR and high-resolution mass spectrometry, were used for the confirmation of the synthesized compounds. To evaluate the purity of all synthesized compounds, the R_f values were calculated by thin-layer chromatography using silica gel 60 HF254 pre-coated aluminum sheets (Merck). Melting points were recorded on DMP-300 A&E Lab UK, apparatus. Characteristic functional groups were determined by the FT-IR spectrophotometer using ATR. Bruker 300 MHz, Varian VNMRS 400 MHz, and 500 MHz spectrometers were used to check protons and carbon signals. HRMS was recorded using a MALDI-TOF mass spectrometer, Bruker. The purity of the selected compound was calculated by qualitative HNMR method using qHNMR normalization (100%) method, established by Pauli et al. The spectra were recorded using this optimized protocol.^{32 (link)}

MALDI-TOF-MS in reflection positive-ion mode was employed for the profiling of plasma N-glycans [26 ,27 (link)]. Specifically, 1 μL of glycan samples was combined with an equal volume of matrix containing 5 mg/mL super-DHB with 1 mM NaOH in 50% ACN on the AnchorChip target plate (Bruker Daltonics, Bremen, Germany), followed by air-drying the mixture. The N-glycomes were subsequently detected using a rapifleXtreme MALDI-TOF mass spectrometer equipped with a Smartbeam-3D laser, under the control of flexControl 4.0 (Bruker Daltonics). Calibration of the instrument was performed using a peptide calibration standard (Bruker Daltonics), and the mass range was set from m/z 1000 to m/z 5000. Laser shots were accumulated 5000 times for each spectrum. An automatic acquisition mode and random walk pattern at a laser frequency of 5000 Hz were selected for the acquisition of sample spectra.