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. 2017 Nov;16(11):2006-2016.
doi: 10.1074/mcp.RA117.000023. Epub 2017 Aug 18.

High Sensitivity Quantitative Proteomics Using Automated Multidimensional Nano-flow Chromatography and Accumulated Ion Monitoring on Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

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High Sensitivity Quantitative Proteomics Using Automated Multidimensional Nano-flow Chromatography and Accumulated Ion Monitoring on Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

Paolo Cifani et al. Mol Cell Proteomics. .
Free PMC article

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.

Conflict of interest statement

The authors have no competing financial interests

Figures

Fig. 1.
Fig. 1.
A, AIM acquisition enables detection of 2–5 yoctomoles/ms of target peptides serially diluted in neat solvent, and seven order of magnitude of linear dynamic range. The MS intensity within 10 ppm from the theoretical m/z was recorded for peptide NSPGLLVSPGNLNK (m/z 709.3981) with maximum injection time (max IT) 25 (red), 250 (black), and 2500 (gray) ms (n = 7, error bars represent standard deviation of independent experiments). Noise levels recorded at baseline level (i.e. with no target peptide infused) is displayed for each maximum injection times as continuous line. Overlapping data points were horizontally offset when needed for clarity. B, Ion accumulation times obtained for each target peptide with maximum injection time set at 5s. Ion injection time in the baseline samples (i.e. with no target present) is denoted as “B.” Comparison of spectra recorded with target flow 10e7 (C) and 100 ymol/ms (D) reveals that target accumulation is practically limited by coisolated contaminant ions (ion intensity on absolute linear scale).
Fig. 2.
Fig. 2.
Overview of the automated 2D chromatography system.
Fig. 3.
Fig. 3.
A, Automated 2D chromatography system enables efficient resolution of target peptides. Average recovery and SCX fractionation efficiency of peptides are plotted relative to 1D (n = 4, error bars represent standard deviation of fraction of target in best SCX fraction. Quantification by PRM). B, 2D chromatography reduces total ion current from coisolated contaminant ions, in absence of synthetic targets (n = 3, error bars = standard deviation). C, 2D chromatography improves ion accumulation, in absence of synthetic targets (n = 3, error bars = standard deviation). Maximum ion injection time (200 ms, dashed line) is achieved for phosphorylated peptides. D, 2D chromatography improves the ratio of MS signal specific for target ions (specific ion current), over total ion current within the scan (10 fmol target peptide, currents at apex of chromatographic peak). E, Improvement of SIC/TIC by SCX-RP chromatography is statistically significant (p = 0.0054, paired t test, n = 6).
Fig. 4.
Fig. 4.
The improvement in sensitivity from 2D depends on efficiency of fractionation. A, Phosphorylated peptide N(pS)PGLLVSPGNLNK is efficiently resolved from isobaric contaminants, resulting in a 2 amol AIM sensitivity after SCX compared with 1 fmol in 1D. B, PRM analysis achieves 100 amol LOD both in 1D and 2D. Peptide NSPGLLVSPGNLNK is poorly resolved by SCX from the bulk of doubly charged tryptic peptides, resulting in similar sensitivity in 1D (black) and 2D (red) by both AIM (C) and PRM (D). Horizontal lines represent average noise levels (n = 3) for 1D (black) and 2D (red) in AIM. Error bars represent technical variability as standard deviation (n = 3) measured at noise level, 10 amol and 100 fmol. Overlapping data points are horizontally offset when needed for clarity. Vertical arrows mark the LOD in 1D (black) and 2D (red) respectively.
Fig. 5.
Fig. 5.
Cellular amount of endogenous peptides from protein MEF2C in 1 μg sample, from ∼10,000 cells, as determined by 1D (black) and 2D (red) using A, AIM and B, PRM acquisition. Quantification of endogenous phosphorylated peptides was exclusively achievable using SCX. Each dot represents an independent measurements (full horizontal lines indicate average). LOD for each assay, established from serially diluted isotopologs, is denoted with dashed lines in correspondence.

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