Quantification of protein posttranslational modifications using stable isotope and mass spectrometry. II. Performance.
In this report, we examine the performance of a mass spectrometry (MS)-based method for quantification of protein posttranslational modifications (PTMs) using stable isotope labeled internal standards. Uniform labeling of proteins and highly similar behavior of the labeled vs nonlabeled analyte pairs during chromatographic separation and electrospray ionization (ESI) provide the means to directly quantify a wide range of PTMs. In the companion report (Jiang et al., Anal. Biochem., 421 (2012) 506-516.), we provided principles and example applications of the method. Here we show satisfactory accuracy and precision for quantifying protein modifications by using the SILIS method when the analyses were performed on different types of mass spectrometers, such as ion-trap, time-of-flight (TOF), and quadrupole instruments. Additionally, the stable isotope labeled internal standard (SILIS) method demonstrated an extended linear range of quantification expressed in accurate quantification up to at least a 4 log concentration range on three different types of mass spectrometers. We also demonstrate that lengthy chromatographic separation is no longer required to obtain quality results, offering an opportunity to significantly shorten the method run time. The results indicate the potential of this methodology for rapid and large-scale assessment of multiple quality attributes of a therapeutic protein in a single analysis.
Department of Analytical and Formulation Sciences, Amgen Inc., Thousand Oaks, CA 91320, USA. qluo@amgen.com
Quanzhou
Q
Luo
Jette
J
Wypych
Xinzhao Grace
XG
Jiang
Xin
X
Zhang
Shun
S
Luo
Matthew
M
Jerums
Jeffrey
J
Lewis
Ronald
R
Keener
Gang
G
Huang
Izydor
I
Apostol
D002851:Chromatography, High Pressure Liquid; D007553:Isotope Labeling; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D015203:Reproducibility of Results; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D011506:Proteins