A proteomic approach for plasma biomarker discovery with 8-plex iTRAQ labeling and SCX-LC-MS/MS

Abstract

Plasma is recognized as a promising source of disease-related biomarkers, and proteomic approaches for identifying novel plasma biomarkers are in great demand. However, the complexity and dynamic protein concentration range of plasma remain the main obstacles for current research in this field. In this study, plasma proteins were prefractioned by immunodepletion and Protein Equalizer Technology to remove high abundant proteins, then labeled with an 8-plex isobaric tags for relative and absolute quantitation (iTRAQ) to improve the peptide ionization, and analyzed by strong-cation-exchange(SCX) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results showed that both prefraction methods were complementary, with regard to the number of identified proteins. Good chromatographic technique is important to further fractionate the iTRAQ labeling peptides, which allowed 320 and 248 different proteins to be characterized from two prefraction methods, respectively, encompassing a wide array of biological functions and a broad dynamic range of 10(7). Furthermore, the accuracy of iTRAQ relative quantitation for differentially expressed proteins is associated with the number of peptides hits per protein.

Investigation of Plasma Biomarkers in HIV-1/HCV Mono- and Coinfected Individuals by Multiplex iTRAQ Quantitative Proteomics

“…In order to ascertain protein identification as well as their differential regulation, the results were reproduced in three biological replicates (Ye et al, 2010). Although we identified several plasma proteins that are reported in the literature, the identification of relatively a low total number of proteins in our analysis may be due to the lack of multidimensional fractionation approach (SCX fractionation followed by reversed-phase) (Ye et al, 2010). Furthermore, we used the equalizer bead protein enrichment strategy to reduce the complexity of the plasma proteome of HIV-1/ HCV mono-and coinfected patients (Supplementary Table S1).…”

“…Furthermore, we used the equalizer bead protein enrichment strategy to reduce the complexity of the plasma proteome of HIV-1/ HCV mono-and coinfected patients (Supplementary Table S1). Equalizer bead (ProteoMiner) technology is an established method to equalize/enrich medium to low abundant proteins in various biological samples (Guerrier et al, 2008;Ye et al, 2010). This technology has been applied successfully for the investigation of low abundant plasma and serum proteome (Ye et al, 2010).…”

“…Equalizer bead (ProteoMiner) technology is an established method to equalize/enrich medium to low abundant proteins in various biological samples (Guerrier et al, 2008;Ye et al, 2010). This technology has been applied successfully for the investigation of low abundant plasma and serum proteome (Ye et al, 2010). ProteoMiner equalization was also proved to be a complementary method for quantitative proteomics analysis (Dwivedi et al, 2010) and it was successfully applied to plasma biomarker discovery (Ernoult et al, 2010) and clinical proteomics (Hartwig et al, 2009).…”

MicroRNA-122 Influences the Development of Sperm Abnormalities from Human Induced Pluripotent Stem Cells by Regulating TNP2 Expression

“…Cell protein extraction, digestion, and labeling with iTRAQ reagents and data analysis All steps of protein extraction and labeling were according to the previously described [34][35][36][37][38][39][40][41][42][43]. Protein identification and quantification for iTRAQ samples were carried out using ProteinPilot software (version 3.0; Applied Biosystems, MDS-Sciex).…”

“…Only those proteins identified with at least 95% confidence were taken into account. All results were then exported into Excel for manual data interpretation [34][35][36][37][38][39][40][41][42][43].…”