Molecular Dynamics Simulation-assisted Ionic Liquid Screening for Deep Coverage Proteome Analysis

Abstract

In-depth coverage of proteomic analysis could enhance our understanding to the mechanism of the protein functions. Unfortunately, many highly hydrophobic proteins and low-abundance proteins, which play critical roles in signaling networks, are easily lost during sample preparation, mainly attributed to the fact that very few extractants can simultaneously satisfy the requirements on strong solubilizing ability to membrane proteins and good enzyme compatibility. Thus, it is urgent to screen out ideal extractant from the huge compound libraries in a fast and effective way. Herein, by investigating the interior mechanism of extractants on the membrane proteins solubilization and trypsin compatibility, a molecular dynamics simulation system was established as complement to the experimental procedure to narrow down the scope of candidates for proteomics analysis. The simulation data shows that the van der Waals interaction between cation group of ionic liquid and membrane protein is the dominant factor in determining protein solubilization. In combination with the experimental data, 1-dodecyl-3-methylimidazolium chloride (C12Im-Cl) is on the shortlist for the suitable candidates from comprehensive aspects. Inspired by the advantages of C12Im-Cl, an ionic liquid-based filter-aided sample preparation (i-FASP) method was developed. Using this strategy, over 3,300 proteins were confidently identified from 103 HeLa cells (~100 ng proteins) in a single run, an improvement of 53% over the conventional FASP method. Then the i-FASP method was further successfully applied to the label-free relative quantitation of human liver cancer and para-carcinoma tissues with obviously improved accuracy, reproducibility and coverage than the commonly used urea-based FASP method. The above results demonstrated that the i-FASP method could be performed as a versatile tool for the in-depth coverage proteomic analysis of biological samples.

Urinary proteomics investigations into contrast-induced acute kidney injury

“…The urine samples were prepared using the FASP method following the procedures described previously with minor modifications [ 8 ]. Briefly, we mixed the 4 CI-AKI samples and 20 non-CI-AKI samples.…”

“…High confidence peptide identification was obtained by setting a false discovery rate of <1% at the PSM and protein level with the target-decoy based strategy. At least one unique peptide per protein group was required for identification of proteins and unique and razor peptides were used for quantification of proteins [ 8 ]. Matching between runs with a retention time window of 1 min and the label-free quantification (LFQ) algorithm were performed.…”

“…Matching between runs with a retention time window of 1 min and the label-free quantification (LFQ) algorithm were performed. Then the proteins quantified in triplicates were remained for subsequent analysis, and a two-tailed t test applied with correction for multiple testing (Benjamini-Hochberg) [ 8 ]. DEPs were defined as having a p-value <0.05 based on a t-test and a ratio(CI-AKI/non-CI-AKI) >2.…”

Advances of ionic liquids-based novel methods for proteomic sample preparation

“…图 4 [24] 基于 C12Im-Cl 的滤膜原位样品处理方法(i-FASP)的流程图 (A) 及其与 FASP 方法的比较 (B) Figure 4 [24] . Flowchart of C12Im-Cl based filter-aided sample preparation (i-FASP) (A) and comparison with FASP method (B).…”

“…Analysis method of plant tissue phosphorylated protein based on C12Im-Cl assisted extraction (A) and comparison of extraction efficiency of tobacco proteins with existing extraction systems (B). [24] 细菌视紫红质(BR)与含不同阳离子基团的 ILs 的相互作用能 Table1 [24] Interaction energies between the bacteriorhodopsin (BR) and various ionic liquids with different cation groups a) MTBA-Cl(methyltributylammonium chloride):甲基三丁基氯化铵; MTBP-Cl(methyltributylphosphonium chloride):甲基三丁基氯化磷;…”