HPLC, UHPLC

Getting the best of both worlds: Ensuring the perfect partnership of chromatographic separation and MS detection for protein quantification

Jun 18 2010

Author: M.Quaglia, G.O’Connor on behalf of LGC Standards

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The absolute quantification of proteins by stable isotopically labelled peptides as calibrators and selected reaction monitoring experiments has become a method commonly applied for the quantification of standard proteins [1,2], proteins in complex matrices [3] or post-translational modifications [4]. Assuring traceability, or the anchoring of the “absolute amount of substance” to the Système international d’unités (SI), and robustness in protein quantification is still a complex task however.

This article describes the digestion steps and the chromatographic and mass spectrometry conditions which need to be optimised when traceable quantification of proteins with minimised uncertainty has to be performed. Examples will be given where optimising chromatographic and mass spectrometry conditions can help improve the overall confidence for the absolute quantification of proteins using stable isotopically labelled peptides as standards. The performance of a number of columns and critical mass spectrometry parameters such as number of points across the peak and scan time, and their influence on ratio measurements, and therefore measurement uncertainty will be discussed.

INTRODUCTION
Liquid chromatography mass spectrometry (LC-MS) based proteomics has evolved as an important tool to support molecular and cellular biology. While improvements in LCMS technical platforms and bio-informatic
tools have significantly contributed to the development of robust methods for biomarker discovery and cell expression profiling, an increasing interest has emerged in protein quantification for clinical, environmental and
nutritional applications...

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