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-μm 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.

Tandem Analysis of Transcriptome and Proteome Changes after a Single Dose of Corticosteroid: A Systems Approach to Liver Function in Pharmacogenomics

“…Although previous studies have been conducted in identifying differences in the changes at the transcriptional and translational levels for one or selected few proteins, global assessment through high throughput methodologies have never tried to address this issue (Nouri-Nigjeh et al, 2014;Qu et al, 2006;Sukumaran et al, 2011). Studies that have characterized and compared genomic and proteomic data have almost always been single time-point studies that do not provide the relevant temporal information necessary to identify and characterize transcriptional and translational differences (de Godoy et al, 2008;Gry et al, 2009).…”

“…Protein quantification was based on the area under the curve (AUC) of the ion-current peaks. A more extensive description of the experimental setup and the analytical methodology can be found in our published report (Nouri-Nigjeh et al, 2014).…”

“…Recently, we were able to develop high-throughput methodology to perform comprehensive and accurate profiling of the tissue proteome using an ion currentbased liquid chromatography/mass spectrometry (LC/MS) strategy (Tu et al, 2012). Using this methodology, we characterized the temporal changes in the expression of thousands of proteins in rat liver after methylprednisolone (MPL) administration (Nouri-Nigjeh et al, 2014).…”

Temporal Effects of Combined Birinapant and Paclitaxel on Pancreatic Cancer Cells Investigated via Large-Scale, Ion-Current-Based Quantitative Proteomics (IonStar)

“…They circumvent the frequent lack of concordance between mRNA and protein expression (21,22) and could reveal biomarkers of PDAC tumor cell drug sensitivity, which genomic and transcriptomic profiling has been unable to provide (7). A unique ion current-based proteomics strategy was an important enabling factor in this study, in that it permits reproducible and reliable quantification of the large number of samples required for comprehensive temporal analysis of cellular drug responses (23,24), provides extremely-low levels of missing data (15) and the ability to quantify subtle changes in protein abundance (15,25), and has well-controlled falsepositive discovery rates (26,27). Using this strategy, we performed quantitative proteomic analysis of PDAC cells treated with birinapant and paclitaxel, alone and in combination.…”

Phosphorylation of Tyr188 in the WW domain of YAP1 plays an essential role in YAP1-induced cellular transformation

“…22,23 The digests were eluted on a 100 cm column with a 2-hr gradient with CID and ETD activation by Fusion Tribrid mass spectrometer (Thermo Scientific), using a previously described condition. 24 Raw files were searched with Proteome Discoverer (Thermo Scientific) against YAP1 sequence and Carbamidomethyl (57.021Da) was set as static modification on Cystine (C), and oxidation (15.995Da ) on methionine (M); phosphorylation (79.966 Da) on serine (S), threonine (T) and tyrosine (Y) were set as dynamic side chain modifications. The search results were merged with Scaffold (Proteome Software), and filtered to achieve a local peptide probability >95%.…”