Post-translational Modifications of Recombinant Proteins Determined by LC/Electrospray Mass Spectrometry and High Performance Tandem Mass Spectrometry

Abstract

This chapter presents a study analyzing posttranslational modifications of recombinant proteins determined by liquid chromatography (LC)/electrospray mass spectrometry (ESMS) and high-performance tandem mass spectrometry (HPTMS). Human platelet-derived growth factor B-chain (hPDGF-B) and human low-affinity nerve growth factor receptor (hNGF-R) were expressed and purified. The purified proteins were reduced, carboxymethylated, and digested with either trypsin or trypsin/chymotrypsin. LC/ESMS experiments were performed using a Carlo Erba Phoenix 20 dual syringe pump system (Fisons) interfaced to a VG Biotech BIO-Q electrospray mass spectrometer. Peptide separation was carried out using an Aquapore 300 column. High-energy CID experiments were carried out on a Kratos Concept IIHH high-performance tandem mass spectrometer equipped with electro-optical multichannel array detection. The results of this study demonstrated that LC/ESMS can be used to readily identify peptides in a protein digest, which show anomalies to the predicted primary structure at the low picomole level and at high speed.