An optimized magnetite microparticle-based phosphopeptide enrichment strategy for identifying multiple phosphorylation sites in an immunoprecipitated protein.
To further improve the selectivity and throughput of phosphopeptide analysis for the samples from real-time cell lysates, here we demonstrate a highly efficient method for phosphopeptide enrichment via newly synthesized magnetite microparticles and the concurrent mass spectrometric analysis. The magnetite microparticles show excellent magnetic responsivity and redispersibility for a quick enrichment of those phosphopeptides in solution. The selectivity and sensitivity of magnetite microparticles in phosphopeptide enrichment are first evaluated by a known mixture containing both phosphorylated and nonphosphorylated proteins. Compared with the titanium dioxide-coated magnetic beads commercially available, our magnetite microparticles show a better specificity toward phosphopeptides. The selectively-enriched phosphopeptides from tryptic digests of β-casein can be detected down to 0.4 fmol μl⁻¹, whereas the recovery efficiency is approximately 90% for monophosphopeptides. This magnetite microparticle-based affinity technology with optimized enrichment conditions is then immediately applied to identify all possible phosphorylation sites on a signal protein isolated in real time from a stress-stimulated mammalian cell culture. A large fraction of peptides eluted from the magnetic particle enrichment step were identified and characterized as either single- or multiphosphorylated species by tandem mass spectrometry. With their high efficiency and utility for phosphopeptide enrichment, the magnetite microparticles hold great potential in the phosphoproteomic studies on real-time samples from cell lysates.
Huang
Yi
Y
Department of Chemistry, Institutes of Biomedical Sciences, Fudan University, Shanghai, People's Republic of China.
Shi
Qihui
Q
Tsung
Chia-Kuang
CK
Gunawardena
Harsha P
HP
Xie
Ling
L
Yu
Yanbao
Y
Liang
Hongjun
H
Yang
Pengyuan
P
Stucky
Galen D
GD
Chen
Xian
X
D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D052203:Ferrosoferric Oxide / Q000737:chemistry; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D008280:Magnetics; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry* / Q000302:isolation & purification; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014025:Titanium / Q000737:chemistry; D051197:Toll-Like Receptor 4 / Q000737:chemistry / Q000378:metabolism
D002364:Caseins; D010748:Phosphopeptides; D010750:Phosphoproteins; D051197:Toll-Like Receptor 4; C009495:titanium dioxide; D014025:Titanium; D052203:Ferrosoferric Oxide