Dataset Information

Comparative evaluation of small molecular additives and their effects on peptide/protein identification

ABSTRACT: Detergents and salts are widely used in lysis buffer to enhance protein extraction from biological samples, facilitating in-depth proteome analysis, however, to efficiently remove these detergent and salt additives from the digested peptides is essential for generating high quality mass spectra in LC-MS/MS analysis. The sample preparation methods, such as Filter-Aided Sample Preparation (FASP), acetone precipitation followed by in-solution digestion (AP), strong cation exchange-based centrifugal proteomic reactor (CPR), are commonly used for proteomic sample process, but the additives removal efficiency and the application scope of these methods need to be further investigated. In this study, we demonstrated an integrative work flow for systematical small molecular additives quantification as well as peptide/protein identification to provide a comprehensive evaluation for additive cleanup effect of proteomic sample preparation pipelines. The multiple reaction monitoring (MRM)-based LC-MS approach was developed for six additives (i.e., Tris, Urea, CHAPS, SDS, SDC and Triton X-100) quantification. For the peptide and protein identification, although FASP outperformed the other two methods, these three methods were complementary to each other, in terms of peptide/ protein identification, as well as GRAVY index and amino acids distributions. By integrating analysis of small molecular quantification and protein identification datasets, we first bridged the quantitative influence of additive concentration to the peptide analysis performance. Our results provide a valuable dataset for appropriate sample preparation method selection and a guideline for evaluation of small molecular additives cleanup efficiency in the sample preparation procedures.

INSTRUMENT(S): TSQ Quantiva, Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Chengli Yu

LAB HEAD: Hu Zhou

PROVIDER: PXD005405 | Pride | 2017-08-11

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	20151127_ZSY_BLANK_02.raw	Raw
	20151127_ZSY_BLANK_03.raw	Raw
	20151127_ZSY_BLANK_04.raw	Raw
	20151127_ZSY_BLANK_05.raw	Raw
	20151127_ZSY_BLANK_06.raw	Raw

Items per page:

1 - 5 of 596

Publications

Comparative Evaluation of Small Molecular Additives and Their Effects on Peptide/Protein Identification.

Gao Jing J Zhong Shaoyun S Zhou Yanting Y He Han H Peng Shuying S Zhu Zhenyun Z Liu Xing X Zheng Jing J Xu Bin B Zhou Hu H

Analytical chemistry 20170522 11

Detergents and salts are widely used in lysis buffers to enhance protein extraction from biological samples, facilitating in-depth proteomic analysis. However, these detergents and salt additives must be efficiently removed from the digested samples prior to LC-MS/MS analysis to obtain high-quality mass spectra. Although filter-aided sample preparation (FASP), acetone precipitation (AP), followed by in-solution digestion, and strong cation exchange-based centrifugal proteomic reactors (CPRs) are ...[more]

PMID: 28530406

Similar Datasets

Project description:Raw expression values (CHP data) for transcriptional profiling of the response of Saccharomyces cerevisiae to challenges with lactic acid at pH 3 and pH 5. Experiment Overall Design: The laboratory reference strain CEN.PK 113-7D (MATa) was grown at 30 Â°C in 1.5-L chemostat fermentors (Applikon, Schiedam, The Netherlands) with a working volume of 1-L using an electronic level sensor to maintain a constant volume. All cultures, including the reference, were fed with minimal medium as described by Verduyn et al. (1992) with 25 g L-1 glucose as the limiting nutrient and 0.15 ml L-1 silicone antifoam (BDH, Poole, England) to prevent excessive foaming. The dilution rate was set to 0.10 h-1 and the pH was controlled at 5.0 with the automatic addition (ADI 1031 bio controller, Applikon) of 2 M KOH. The stirrer speed was set at 800 RPM and anaerobicity was maintained by sparging the fermentor with N2 gas at 500 ml min-1. To prevent diffusion of oxygen, the fermentor was equipped with Norprene tubing and Viton O-rings and the medium vessel was also flushed with N2 gas. A comparable degree of weak acid uncoupling was ensured by decreasing the biomass yield to approximately 50% of the reference condition (no organic acids added) with the addition of the appropriate concentration of lactic acid to the reservoir media. Experiment Overall Design: Sampling of chemostat cultures, probe preparation and hybridization to Affymetrix GeneChip microarrays was performed as described previously (Piper et al., 2002), but with the following modifications. Double-stranded cDNA synthesis was carried out using 15 Î¼g of total RNA and the components of the One Cycle cDNA Synthesis Kit (Affymetrix). The double-stranded cDNA was purified (Genechip Sample Cleanup Module, Qiagen) before in vitro transcription and labeling (GeneChip IVT Labeling Kit, Affymetrix). Finally, labeled cRNA was purified (GeneChip Sample Cleanup Module) prior to fragmentation and hybridization of 15 Î¼g of biotinylated cRNA. Experiment Overall Design: Data acquisition was performed using the Affymetrix scanner 3000, quantification of array images and data filtering were performed with the Affymetrix software packages Microarray Suite v5.0, MicroDB v3.0 and Data Mining Tool v3.0.