Isobaric Stable Isotope Phosphorylation Labeling For Protein Quantification And Its Application In Targets Identification For HIV Latency Activator

Stable isotope labeling technology based on high-resolution mass spectrometry has become one of the core methods in quantitative proteomics research.Among them,the isobaric stable isotope labeling method based on secondary mass spectrometry report ion analysis(MS2)has the advantage of quantitative throughput,such as the commercial kits TMT and iTRAQ,which are currently widely used in the fields of life science research and discovery of clinical diagnostic biomarker.However,for the identification and quantitative analysis of trace proteins,the isobaric labeling method has not solved the bottleneck of analytical sensitivity,which greatly limits the deep coverage of the proteome.In this paper,iSIPL 2-plex were designed and synthesized.The high-efficiency chromatographic separation preparation method for stable isotope labeled phosphorous reagents was established.The related structures were characterized and identified by nuclear magnetic resonance spectrometer and high resolution mass spectrometry.The labeling reagent is based on the structure of the characteristic phosphoramidate reporter ion with improved sensitivity.By introducing 15N atoms for the synthesis of isobaric labeling reagents,the labeled peptides will generate m/z 180.0789 and m/z 181.0758 reporter ions with high-intensity during the tandem mass spectrometry cleavage process.Systematic optimization of preparation methods and protein labeling conditions was performed by using diethoxyphosphoryl glycyl-beta-alanine ester(iSIPL0)as model compound.Compared with the commercial labeling reagent TMT0 for protein identification and analysis,it was found that the best protein identification results could be obtained with acetonitrile as the labeling solvent and 20:1(labeling reagent to peptide).Furthermore,excess labeling reagent will not affect subsequent mass spectrometry analysis.In order to compare the identification ability of iSIPL0 and TMT0,1?g HeLa cell proteolytic peptides were labeled and analyzed by using MS.It was found that the number of proteins detected by iSIPL0 and TMT0 was 1758 and 1544 respectively,indicating that iSIPL has certain advantages in the identification and analysis of proteins in low-concentration.The quantitative results of iSIPL 2-plex showed that a total of 1509 proteins have been successfully detected for 10?g of HeLa proteolytic peptides derived from HeLa cell samples.Among them,1438 proteins could be quantified with ratios focused in 0.75-1.25,accounting for 95.3%of the total number of proteins detected.This above results shown that iSIPL 2-plex was accurate in protein quantitative analysis and could be used for quantitative analysis of complex protein systems.Subsequently,based on the iSIPL2 quantitative method,the target of HIV-1 latent activator#6 protein was quantitatively identified and analyzed.Nine potential protein targets have been determined.In summary,the synthesis and application of iSIPL labeling reagents based on organophosphorus chemistry provide a new labeling strategy for protein quantitative analysis.It laid the foundation for the development of new reagents with more superior and efficient quantitative analysis.