Abstract
Absolute quantification of protein expression and post-translational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. To address these issues, we have developed MARQUIS — Multiplex Absolute Regressed Quantification with Internal Standards — a novel mass spectrometry-based approach using a combination of isobaric tags and heavy-labeled standard peptides to construct internal standard curves for peptides derived from key nodes in signal transduction networks. We applied MARQUIS to quantify phosphorylation dynamics within the EGFR network at multiple time points following stimulation with several ligands, enabling a quantitative comparison of EGFR phosphorylation sites and demonstrating that receptor phosphorylation is qualitatively similar but quantitatively distinct for each EGFR ligand tested. MARQUIS was also applied to quantify the effect of EGFR kinase inhibition on glioblastoma patient derived xenografts. MARQUIS is a versatile method, broadly applicable and extendable to multiple mass spectrometric platforms.
Highlights
Absolute quantification of protein expression and posttranslational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response
Multiplexed relative quantification has been useful for elucidating temporal dynamics of phosphorylation signalling following growth factor stimulation, with changes in protein posttranslational modifications (PTMs) levels in stimulated versus nonstimulated conditions highlighting pathways involved in signal processing and cellular response[4]
Absolute quantification of phosphorylation incorporation has historically been performed by using radiolabelled ATP and tryptic digestion followed by two-dimensional thin-layer chromatography; it is difficult to identify the specific phosphorylation sites corresponding to each peptide, the method is limited to single protein analysis and the phosphorylation reaction must occur in vitro or in cells whose membranes have been disrupted
Summary
Absolute quantification of protein expression and posttranslational modifications by mass spectrometry has been challenging due to a variety of factors, including the potentially large dynamic range of phosphorylation response. One of the most common MS-based methods is isotope dilution, or AQUA, in which a synthetic isotope-encoded peptide is added to the sample pre-analysis and quantification is based on the relative peak heights or area under the chromatographic elution curve for the endogenous and synthetic peptide standard[7] This technique is fairly straightforward, it relies on single-point calibration and may provide erroneous estimates if the endogenous peptides span a large dynamic range across multiple conditions, or if the concentrations of these peptides fall outside of the linear response range of the instrument. One would like to combine the multiplexed capabilities of chemical labelling with standard curves internal to the sample, allowing the accurate, absolute quantification of given peptides across multiple biological conditions within a single analysis. MARQUIS enables the acquisition of accurate absolute quantification data, is applicable across multiple instrument platforms and is applicable to protein expression profiling and PTM quantification
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