4800 maldi tof tof system

UPLC-MS was performed on a Waters XEVO QTOF G2 Mass Spectrometer, with an Acquity H-class solvent manager, FTN-sample manager and TUV-detector. The system was equipped with a reversed phase C18-column (Waters, Acquity PST 130A, 1.7 μm 2.1 × 50 mm i.d.), column temperature 40°. Mobile phases consisted of 0.1% formic acid in water (solvent A) and in 90% acetonitrile (solvent B). FTN-purge solvent was 10% acetonitrile in water. Gradient condition: 10%–55% solvent B over 15 min monitored at 220 nm.
Semi-Preparative reversed-phase HPLC was performed on a Waters Delta Prep System equipped with a Waters 2487 Absorbance Detector, using a Vydac C-18 column (250 × 10 mm, 10 μm). A linear gradient of acetonitrile in water/0.1%TFA was used to elute the peptide. Flowrate was 12 mL/min. Finally, Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI TOF)-MS was performed on an Applied Biosystems 4800 MALDI TOF/TOF system in reflector mode using α-cyano-4-hydroxycinnamic acid as matrix material. Both peptides were isolated as single peaks in the LC/MS analysis with VEGF-TAMRA giving an observed mass of 664.34 [M+4H]⁴⁺ (expected 664.53) and VEGF-TAMRA-alk giving an observed mass of 678.57 [M+4H]⁴⁺ (expected 678.79), with m/z ratios resulting in a monoisotopic mass of 2652.28 (expected 2652.33) and 2709.28 (expected 2709.35), respectively.

Matrix assisted laser desorption/ionization (MALDI) analysis of unconjugated and conjugated antibodies was performed using a previously described matrix combination.64 MALDI-MS analysis was performed in positive-ion mode with a 4800 MALDI–TOF/TOF system (Applied Biosystems, Framingham, MA, USA). Laser intensity was set to 3,000–5,000. Mass range was set to 20,000–200,000. As expected at this mass range, the peak widths were broad, and multiply-charged peaks were observed. We calculated an average mass based on clearly discernible multiply-charged peaks and used that information to determine the approximate conjugation efficiency.