Nanoprobe-based Immobilized Metal Affinity Chromatography (NB-IMAC) for Qualitative and Quantitative Profiling of Phosphoproteome
Date Issued
2009
Date
2009
Author(s)
Hsu, Chuan-Chih
Abstract
Despite the significant regulatory role of protein phosphorylation in cellular signaling, comprehensive characterization and quantitation of site-specific phosphorylation still remained challenges, especially for multiply phosphorylated peptides. Due to the unique features of surface area-to-volume ratio, magnetic separation and flexible surface functionalization, magnetic nanoparticles have been widely fabricated as biological probes. Here we demonstrate a surface-blocked nanoprobe-based immobilized metal affinity chromatography (NB-IMAC) for high performance phosphopeptide enrichment. This approach show unbiased extraction of both mono- and multiply phosphorylated peptides from highly diluted β-casein solution at 2×10-10 M. By surface blocking with polyethylene glycol, controlling the concentration of acetic acid and pH value, and adjusting MNP-to-protein ratio, 1283 phosphopeptides (false discovery rate is 2.9%) could be identified 400 microgram Raji B cell with nearly 80% purification specificity. Notably, two-fold increment of multiply phophorylated peptides was achieved by NB-IMAC compared to the micro-scale IMAC. For further quantitative phosphoproteomics analysis, we integrated the NB-IMAC protocol with LC-MS/MS to develop a label-free quantitation method. Using replicate cell lysate from human embryonic stem cells, the quantitation strategy provides narrow distribution of log2 ratio -0.05 (2SD = 95% confidence), in consistent with expected peptide ratio of 1. uman embryonic stem cells (hESCs) have generated an enormous interest because of its pluripotent ability to differentiate into various types of cells. We applied NB-IMAC protocol to quantitatively analyze the differentiation-mediated phosphoproteomic alteration during embryonic differentiation. After cell proteolysis, phosphorylated internal standard was spiked into the undifferentiated hESCs or differentiated hESCs and subjected to gel-assisted digestion, NBIMAC purification and LC-MS/MS analysis. Without any fractionation procedure, 402 phosphopeptides and 203 phosphoproteins were quantified. The preliminary data revealed 132 up-regulated and 245 down-regulated phosphorylated sites, indicating dramatic change in phosphoproteome during the process of embryonic differentiation. Among these phosphorylation sites, activated ERK 1/2 by phosphorylation at Thr 202 and Thr 184 site in differentiated state of embryoid bodies (EBs) has been previously reported to play an important role in cell differentiation. We expect that this newly developed phosphopeptides purification and quantitation strategy will offer a generic platform to investigate differential phosphorylation levels.
Subjects
Magnetic Nanoparticle
Immobilized metal ion affinity chromatography
Mass spectrometry
Phosphoproteomics
Label-free Quantitative method
Human embryonic stem cell
Type
thesis
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