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Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats.

ABSTRACT: A proteome-level time-series study of drug effects (i.e., pharmacodynamics) is critical for understanding mechanisms of action and systems pharmacology, but is challenging, because of the requirement of a proteomics method for reliable quantification of many biological samples. Here, we describe a highly reproducible strategy, enabling a global, large-scale investigation of the expression dynamics of corticosteroid-regulated proteins in livers from adrenalectomized rats over 11 time points after drug dosing (0.5-66 h, N = 5/point). The analytical advances include (i) exhaustive tissue extraction with a Polytron/sonication procedure in a detergent cocktail buffer, and a cleanup/digestion procedure providing very consistent protein yields (relative standard deviation (RSD%) of 2.7%-6.4%) and peptide recoveries (4.1-9.0%) across the 60 animals; (ii) an ultrahigh-pressure nano-LC setup with substantially improved temperature stabilization, pump-noise suppression, and programmed interface cleaning, enabling excellent reproducibility for continuous analyses of numerous samples; (iii) separation on a 100-cm-long column (2-?m particles) with high reproducibility for days to enable both in-depth profiling and accurate peptide ion-current match; and (iv) well-controlled ion-current-based quantification. To obtain high-quality quantitative data necessary to describe the 11 time-points protein expression temporal profiles, strict criteria were used to define "quantifiable proteins". A total of 323 drug-responsive proteins were revealed with confidence, and the time profiles of these proteins provided new insights into the diverse temporal changes of biological cascades associated with hepatic metabolism, response to hormone stimuli, gluconeogenesis, inflammatory responses, and protein translation processes. Most profile changes persisted well after the drug was eliminated. The developed strategy can also be broadly applied in preclinical and clinical research, where the analysis of numerous biological replicates is crucial.

SUBMITTER: Nouri-Nigjeh E

PROVIDER: S-EPMC4139173 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats.

Nouri-Nigjeh Eslam E Sukumaran Siddharth S Tu Chengjian C Li Jun J Shen Xiaomeng X Duan Xiaotao X DuBois Debra C DC Almon Richard R RR Jusko William J WJ Qu Jun J

Analytical chemistry 20140806 16

A proteome-level time-series study of drug effects (i.e., pharmacodynamics) is critical for understanding mechanisms of action and systems pharmacology, but is challenging, because of the requirement of a proteomics method for reliable quantification of many biological samples. Here, we describe a highly reproducible strategy, enabling a global, large-scale investigation of the expression dynamics of corticosteroid-regulated proteins in livers from adrenalectomized rats over 11 time points after ...[more]

PMID: 25072516

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Project description:In-depth and reproducible protein measurement in many biological samples is often critical for pharmaceutical/biomedical proteomics but remains challenging. MS1-based quantification using quadrupole/ultrahigh-field Orbitrap (Q/UHF-Orbitrap) holds great promise, but the critically important experimental approaches enabling reliable large-cohort analysis have long been overlooked. Here we described an IonStar experimental strategy achieving excellent quantitative quality of MS1 quantification. Key features include: (i) an optimized, surfactant-aided sample preparation approach provides highly efficient (>75% recovery) and reproducible (<15% CV) peptide recovery across large cell/tissue cohorts; (ii) a long column with modest gradient length (2.5 h) yields the optimal balance of depth/throughput on a Q/UHF-Orbitrap; (iii) a large-ID trap not only enables highly reproducible gradient delivery as for the first time observed via real-time conductivity monitoring, but also increases quantitative loading capacity by >8-fold and quantified >25% more proteins; (iv) an optimized HCD-OT markedly outperforms HCD-IT when analyzing large cohorts with high loading amounts; (v) selective removal of hydrophobic/hydrophilic matrix components using a novel selective trapping/delivery approach enables reproducible, robust LC-MS analysis of >100 biological samples in a single set, eliminating batch effect; (vi) MS1 acquired at higher resolution (fwhm = 120 k) provides enhanced S/N and quantitative accuracy/precision for low-abundance species. We examined this pipeline by analyzing a 5 group, 20 samples biological benchmark sample set, and quantified 6273 unique proteins (?2 peptides/protein) under stringent cutoffs without fractionation, 6234 (>99.4%) without missing data in any of the 20 samples. The strategy achieved high quantitative accuracy (3-6% media error), low intragroup variation (6-9% media intragroup CV) and low false-positive biomarker discovery rates (3-8%) across the five groups, with quantified protein abundances spanning >6.5 orders of magnitude. Finally, this strategy is straightforward, robust, and broadly applicable in pharmaceutical/biomedical investigations.

Dataset Information

Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats.

Publications

Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats.

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