A general, robust method for the quality control of intact proteins using LC–ESI-MS
2007 (English)In: Journal of chromatography. B, ISSN 1570-0232, E-ISSN 1873-376X, Vol. 852, no 1-2, 188-194 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
2007. Vol. 852, no 1-2, 188-194 p.
Alkylation; Column regeneration; Electrospray ionization; Glycoprotein; Liquid chromatography; Mass spectrometry; Protein analysis; Quality control; Reduction; Xyloglucan endo-transglycosylase (XET)
IdentifiersURN: urn:nbn:se:kth:diva-8350DOI: 10.1016/j.jchromb.2007.01.011ISI: 000247286700026ScopusID: 2-s2.0-34249689690OAI: oai:DiVA.org:kth-8350DiVA: diva2:13648
QC 201008182008-05-072008-05-072010-08-18Bibliographically approved