Protein Characterization is primarily focused on analyzing recombinant proteins for generation of peptide maps and identification of important post-translational modifications. The PCL uses HPLC, LC-MS, LC-MS/MS, and other tools to analyze samples.
The PCL has extensive experience characterizing vitamin-K dependent proteins (such as Factor IX, prothrombin, Protein C, Factor X, Factor VII) and has also characterized recombinant proteins from E. coli and yeast fermentations.
PCL also analyzes tissue homogenates, cell culture supernatants, blood plasma fractions, and other complex samples for protein identification applications.
PCL characterizes the following modifications found in proteins:
- N-glycosylation – site occupancy, microheterogeneity, and N-glycan structure by MS and MS/MS, level of sialylation.
- O-glycosylation – site occupancy, microheterogeneity.
- Phosphorylation – LC-MS/MS confirmation of low-level phosphorylation in pure recombinant proteins and from blood plasma.
- Sulfation - LC-MS/MS confirmation of low-level phosphorylation in pure recombinant proteins.
- Gamma-carboxylation – use of CID with varying collision energy levels to determine sites of gamma-carboxylation in peptides with 3 or less Gla residues.
- Disulfide bond mapping.
- Deamidation of Asn or Gln
- Oxidation of Met and Trp
- Amino acid mutation or substitution – quantitative analysis of purified proteins produced in E. coli, where codon usage and mis-incorporation can become an issue.
- Agilent Technologies 6210 LC/MS
- Agilent Technologies 1200 Series Autosampling System
- Eppendorf 5804R Centrifuge
- 3 Waters 2695 Separations Module Systems
- 3 Waters 2996 Photodiode Array Detectors
- 2 Waters 2475 Flourescence Detectors
- LabConCo CentriVap Concentrator System
- Beckman Coulter AD340