Evaluation of Quantitative Phosphoproteomic Performance of Sequential Enrichment of Metal Oxide Affinity Chromatography for Phosphopeptides Using TMT 10-plex Isobaric Labeling

doi:10.7503/cjcu20210406

Abstract

Abstract:

A rapid， simple and high-flux strategy for quantitative phosphorylated proteomics was explored and developed， which was a kind of sequential enrichment method for complementary phosphopeptides labeled with Tandem mass tag（TMT） reagents. We cultured HepG2 cells treated with alkannin for 24 h and normal HepG2 cells for phosphorylation modification analysis. All the proteins were digested fully with Trypsin and labeled with TMT reagents， followed by desalting and vacuum drying. We implemented a SMOAC（Sequential enrichment of metal oxide affinity chromatography） strategy. We used firstly titanium dioxide（TiO₂） chromatography for phosphopeptides enrichment. Flow-through and wash fractions from TiO₂ were pooled and enriched secondly by ferrie nitrilotriacetate（Fe-NTA） chromatography. The platform equipped with EASY-nLC1200 and Orbitrap Exploris 480 mass spectrometry was applied to acquiring the phosphopeptides data of the HepG2 cells. We implemented two parallel sets of SMOAC strategy for 600 μg hybrid peptides labeled with TMT reagents. One of the sets， we pooled two kinds of eluate from SMOAC strategy for mass spectrometry analysis. Another of the sets， we analyzed the two kinds of eluate with mass spectrometry， respectively. Altogether we identified 4263 proteins and 16335 phosphopeptides， including 13347 phosphopeptides and 13851 phosphosites of 3848 proteins common to all channels from pooled eluate of SMOAC strategy. The specificity of the enrichment strategy was larger than 97% and 90% of the phosphoproteins identified were quantified. The study reveals SMOAC effectively improves the identification efficiency of phosphorylated peptides and also can quantify phosphorylation protein of a small amount of samples， combining with TMT reagents.

Key words: Phosphorylated proteomics, Sequential enrichment of metal oxide affinity chromatography, Tandem mass tag technology, Mass spectrometry

CLC Number:

O657

TrendMD:

ZHAO Huanhuan, LI Cunyu, JIN Hong. Evaluation of Quantitative Phosphoproteomic Performance of Sequential Enrichment of Metal Oxide Affinity Chromatography for Phosphopeptides Using TMT 10-plex Isobaric Labeling[J]. Chem. J. Chinese Universities, 2021, 42(12): 3624.

Figures/Tables 7

Table 1 TMT reagents of the corresponding samples

TMT reagent	126	127N	127C	128N	128C	131
No.1	C1	C2	C3	T1	T2	T3
No.2	C4	C5	C6	T4	T5	T6

Fig.1 Flow chart of analysis

Fig.2 Analysis results of the amount of peptides identified from TiO2 eluate and Fe eluate by LC?MS

Fig.3 Analysis results of the overlap of peptides and proteins identified from TiO2 eluate and Fe?NTA eluate by LC?MS

Fig.4 Comparison of the amount of phosphopeptides identified from two types of analysis

Fig.5 Scatterplots reflecting correlation of protein abundances in technical replicates

Fig.6 Scatter plot of enriched KEGG pathways statisticsNotes: The color and size of the dots represent the range of the -lgP and the number of the differentially expressed genes mapped to the indicated pathways, respectively.

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