title: A general, robust method for the quality control of intact proteins using LC-ESI-MS.
abstract: A simple and robust method for the routine quality control of intact proteins based on liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) is presented. A wide range of prokaryotic and eukaryotic proteins expressed recombinantly in Escherichia coli or Pichia pastoris has been analyzed with medium- to high-throughput with on-line desalting from multi-well sample plates. Particular advantages of the method include fast chromatography and short cycle times, the use of inexpensive trapping/desalting columns, low sample carryover, and the ability to analyze proteins with masses ranging from 5 to 100 kDa with greater than 50 ppm accuracy. Moreover, the method can be readily coupled with optimized chemical reduction and alkylation steps to facilitate the analysis of denatured or incorrectly folded proteins (e.g., recombinant proteins sequestered in E. coli inclusion bodies) bearing cysteine residues, which otherwise form intractable multimers and non-specific adducts by disulfide bond formation.
authors.affiliation: School of Biotechnology, Royal Institute of Technology, Albanova University Centre, 10691 Stockholm, Sweden.
authors.forename: Gustav
authors.initials: G
authors.lastname: Sundqvist
authors.forename: Maria
authors.initials: M
authors.lastname: Stenvall
authors.forename: Helena
authors.initials: H
authors.lastname: Berglund
authors.forename: Jenny
authors.initials: J
authors.lastname: Ottosson
authors.forename: Harry
authors.initials: H
authors.lastname: Brumer
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D011506:Proteins / Q000302:isolation & purification*; D011786:Quality Control; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D011506:Proteins