Quantification of protein posttranslational modifications using stable isotope and mass spectrometry I: principles and applications.
With the increased attention to quality by design (QbD) for biopharmaceutical products, there is a demand for accurate and precise quantification methods to monitor critical quality attributes (CQAs). To address this need we have developed a mass spectrometry (MS) based method to quantify a wide range of posttranslational modifications (PTMs) in recombinant proteins using stable isotope-labeled internal standard (SILIS). The SILIS was produced through metabolic labeling where ¹⁵N was uniformly introduced at every nitrogen atom in the studied proteins. To enhance the accuracy of the method, the levels of PTMs in SILIS were quantified using orthogonal analytical techniques. Digestion of an unknown sample mixed with SILIS generates a labeled and a nonlabeled version of each peptide. The nonlabeled and labeled counterparts coelute during RP-HPLC separation but exhibit a sufficient mass difference to be distinguished by MS detection. With the application of SILIS, numerous PTMs can be quantified in a single analysis based on the measured MS signal ratios of ¹⁵N-labeled versus the nonlabeled pairs. Several examples using microbial and mammalian-expressed recombinant proteins demonstrated the principle and utility of this method. The results indicate that SILIS is a valuable methodology in addressing CQAs for the QbD paradigm.
Department of Analytical and Formulation Sciences, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA.
Xinzhao Grace
XG
Jiang
Izydor
I
Apostol
Quanzhou
Q
Luo
Jeffrey
J
Lewis
Ronald
R
Keener
Shun
S
Luo
Matthew
M
Jerums
Xin
X
Zhang
Jette
J
Wypych
Gang
G
Huang
D002853:Chromatography, Liquid; D007553:Isotope Labeling / Q000379:methods*; D007554:Isotopes / Q000737:chemistry; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
D007554:Isotopes