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Abstract
Quantitative proteomics using high-resolution and accuracy mass spectrometry promises to transform our understanding of biological systems and disease. Recent development of parallel reaction monitoring (PRM) using hybrid instruments substantially improved the specificity of targeted mass spectrometry. Combined with high-efficiency ion trapping, this approach also provided significant improvements in sensitivity. Here, we investigated the effects of ion isolation and accumulation on the sensitivity and quantitative accuracy of targeted proteomics using the recently developed hybrid quadrupole-Orbitrap-linear ion trap mass spectrometer. We leveraged ultrahigh efficiency nano-electrospray ionization under optimized conditions to achieve yoctomolar sensitivity with more than seven orders of linear quantitative accuracy. To enable sensitive and specific targeted mass spectrometry, we implemented an automated, two-dimensional (2D) ion exchange-reversed phase nanoscale chromatography system. We found that automated 2D chromatography improved the sensitivity and accuracy of both PRM and an intact precursor scanning mass spectrometry method, termed accumulated ion monitoring (AIM), by more than 100-fold. Combined with automated 2D nano-scale chromatography, AIM achieved subattomolar limits of detection of endogenous proteins in complex biological proteomes. This allowed quantitation of absolute abundance of the human transcription factor MEF2C at ∼100 molecules/cell, and determination of its phosphorylation stoichiometry from as little as 1 μg of extracts isolated from 10,000 human cells. The combination of automated multidimensional nano-scale chromatography and targeted mass spectrometry should enable ultrasensitive high-accuracy quantitative proteomics of complex biological systems and diseases.
Highlights
The emerging ability to measure cellular and physiological states accurately and quantitatively promises to transform our understanding of biology and disease [1]
Sensitivity of mass spectrometric detection is in principle determined by two factors: the minimum number of ions necessary to produce a measureable electronic signal, and the baseline instrumental noise
In the case of high-resolution mass analyzers coupled to external ion storage devices, the number of ions delivered to the mass analyzer can be increased by prolonged ion accumulation prior to detection
Summary
The emerging ability to measure cellular and physiological states accurately and quantitatively promises to transform our understanding of biology and disease [1]. Accurate measurements of human disease states are enabling improved diagnostic markers and refined mechanisms of disease pathophysiology [4], [5]. In large part, these advances were made possible by the development of increasingly accurate and sensitive methods for quantitative analysis of proteins and their post-translational modifications in complex biological proteomes. Selected reaction monitoring (SRM) utilizes quadrupole mass analyzers to filter specific precursor and fragment ions produced by collision-induced dissociation (CID) [6]-[9] This method benefits from high-efficiency continuous ion beams, and relatively high sensitivity of direct dynode detection, but is subject to interference effects due to the relatively low unit-mass resolution of quadrupole mass analyzers. SRM methods require specialized approaches to control for variable specificity, hindering their widespread use [7], [10]-[12]
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