ABSTRACT
In the past decade, relative proteomic quantification using isobaric labeling technology has developed into a key tool for comparing the expression of proteins in biological samples. Although its multiplexing capacity and flexibility make this a valuable technology for addressing various biological questions, its quantitative accuracy and precision still pose significant challenges to the reliability of its quantification results. Here, we give a detailed overview of the different kinds of isobaric mass tags and the advantages and disadvantages of the isobaric labeling method. We also discuss which precautions should be taken at each step of the isobaric labeling workflow, to obtain reliable quantification results in large-scale quantitative proteomics experiments. In the last section, we discuss the broad applications of the isobaric labeling technology in biological and clinical studies, with an emphasis on thermal proteome profiling and proteogenomics.
Subject(s)
Proteome/metabolism , Proteomics/methods , Reproducibility of Results , Tandem Mass Spectrometry/methodsABSTRACT
Objective To screen for the differential expression proteins in brain tissues of SD rat after diffuse axonal injury (DAI) by isobaric tag for relative and absolute quantification-liquid chromatographmass spectrometer/mass spectrometer (iTRAQ-LC-MS/MS),and to explore potential biomarkers available for the diagnosis of DAI.Methods Animal models of DAI were established with the Marmarou method as reference,and the subjects were divided into blank control group (n=4),sham strike group (n=4) and fatal strike group (n=4),respectively.The proteins in rat brain tissues were detected by iTRAQ-LC-MS/MS,and bioinformatics analysis and verification were performed on the results and screened for the differential expression proteins.Results A total of 2 016 proteins were identified and quantified.The bioinformatics analysis revealed that the proteins had wide distribution and function,and participated in different biological processes.There were 16 proteins showed differential expression in fatal strike group,including one up-regulated expression protein and 15 down-regulated expression proteins.The results of iTRAQLC-MS/MS were confirmed by Western blotting method.Conclusion Multiple differential expression proteins in rat brain tissues after DAI can be screened by iTRAQ-LC-MS/MS.This not only indicates a research direction for exploring the pathogenesis of DAI,but also provides potential biomarkers available for the diagnosis of DAI.
ABSTRACT
Isobaric peptide termini labeling ( IPTL) is a technology which uses light and heavy isotopes to label C-terminus and N-terminus of peptides. As the masses of labeled peptides are equivalent, the complexity of sample is low when analyzing MS data produced by this technology. Besides, paired b and y ions are helpful while analyzing MS/MS data in this kind of experiments. On the basis of this, a novel scoring algorithm, all ions scoring algorithm (AISA), has been designed for IPTL experiments. The information of quantification and qualification can be acquired at the same time using AISA. On Q-Exactive HeLa 2D RPLC dataset, peptide spectrum matches ( PSMs) , distinct peptides and protein groups identified by AISA are 15%, 26%and 22% higher than Morpheus. On human-HCC-HL dataset, PSMs, distinct peptides and protein groups identified by AISA are 24%, 39% and 27% higher than Morpheus. Quantification ratio on Q-Exactive HeLa and human-HCC-HL datasets are 1. 18 and 0. 90, respectively, which are very close to 1. Besides, quantification ratios between 0. 5 and 2. 0 are 91% and 94%, respectively.
ABSTRACT
Isobaric peptide termini labeling ( IPTL) is a technology which uses light and heavy isotopes to label C-terminus and N-terminus of peptides. As the masses of labeled peptides are equivalent, the complexity of sample is low when analyzing MS data produced by this technology. Besides, paired b and y ions are helpful while analyzing MS/MS data in this kind of experiments. On the basis of this, a novel scoring algorithm, all ions scoring algorithm (AISA), has been designed for IPTL experiments. The information of quantification and qualification can be acquired at the same time using AISA. On Q-Exactive HeLa 2D RPLC dataset, peptide spectrum matches ( PSMs) , distinct peptides and protein groups identified by AISA are 15%, 26%and 22% higher than Morpheus. On human-HCC-HL dataset, PSMs, distinct peptides and protein groups identified by AISA are 24%, 39% and 27% higher than Morpheus. Quantification ratio on Q-Exactive HeLa and human-HCC-HL datasets are 1. 18 and 0. 90, respectively, which are very close to 1. Besides, quantification ratios between 0. 5 and 2. 0 are 91% and 94%, respectively.