Pigment epithelium-derived factor (PEDF) is a soluble glycoprotein is produced in many tissues in the body, including the retina, which has neurotrophic and anti-angiogenic effects. Two glycoforms (PEDF-A and PEDF-B) of the human recombinant protein potentially differ in their biological activity. To better understand PEDF structure-function relationships, PEDF-A and PEDF-B were produced in HEK293 cells and purified to homogeneity by cation-exchange chromatography. In this work, the structure of PEDF was investigated using mass spectrometry (MS). PEDF was digested in solution by trypsin. The digests were then analyzed using MALDI-TOF mass spectrometry and high performance liquid chromatography (HPLC) electrospray ionization tandem mass spectrometry (ESI MS/MS), including triple quadrupole, ion-trap and quadrupole time-of-flight mass spectrometry. The mass spectra were searched against protein sequence databases using the MASCOT search engine. Over 98% of the PEDF amino acid sequence was identified. The PEDF glycopeptide was found by detecting oxonioum ions and the carbohydrate group determined to be N-linked with a mass of 2352.5. The N-terminal peptide of PEDF was identified and the blocked N-terminal amino acid determined to be a pyroglutamyl group.
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