Isobaric Stable Isotope N-Phosphorylation Labeling For Mass Spectrometry-based Quantitative Proteomics

With the development of proteomics techniques,a large number of functional proteins and potential protein biomarkers have been discovered and identified.Disease progression or transition between different physiological states is often associated with global changes in protein abundance.Therefore,it is inevitable that proteome research will shift from qualitative identification to quantitative analysis.Mass spectrometry-based quantitative proteomics using stable isotope labeling play an important role in revealing the chemical nature of life processes.In this project,six-plex stable isotope phosphorylation labeling reagents(iSIPL)have been designed and synthesized based on the synthetic organophosphorus chemistry,which can be used to quantitatively analyze proteomedynamic of biological process in combination with mass spectrometry.The chemical structures of phosphorus reagents are systematically characterized and identified by using NMR,HPLC,and MS.Based on a novel structure of the characteristic phosphoramide as reporter ion,stable isotopes,such as 0-18,N-15,and C-13 have been successfully incorporated into the above mentioned organophosphorus molecular probes with high-yield.Firstly,diethoxyphosphoryl alanyl-glycine dipeptide(iSIPLzero)without stable isotopes was synthesized and subsequently used to determine the standard proteins with different concentrations as compared with the commercially available labeling reagent TMTzero under the same conditions.It was found that 77 peptides could be identified with 88%protein coverage at 2 ng/?L concentration.Furthermore,comparing the qualitative results of iSIPL0 and TMT0 labeling for whole lysate samples of HeLa cells,the number of proteins identified by the iSIPL0 labeling from 10-fold diluted sample was comparable to that of the TMT0 labeling without further dilution,indicating that iSIPLzero reagent have certain advantages in the identification and analysis of proteins at very low concentrations.The number of identified proteins was 2634 for TMTzero and 2433 for iSIPLzero,of which 554 were new proteins,indicating the complementary usage of the two labeling reagents.Finally,the accuracy and stability of the six-plex iSIPL reagents in the quantitative analysis of proteins was validated.The quantification of the standard protein BSA could be easily obtained by the abundance ratios of characteristic reporter ion at m/z 180,181,182.183,184 and 185 in MS/MS spectrum.The relative intensities of the reporter ions produced by the standard peptides were approximately 3:3:3:1:1:1,which is consistent with the protein mixing ratio,indicating that the quantitative analysis of the six-plex iSIPL is accurate and applicable.In conclusion,the synthesis and application of iSIPL labeling reagents based on organophosphorus chemistry provides a new isobaric stable isotope labeling strategy for quantitative protein analysis,which lays a solid foundation for the development of more superior and efficient quantitative reagents in the future.