Enrichment of phosphopeptides by Fe3+-immobilized magnetic nanoparticles for phosphoproteome analysis of the plasma membrane of mouse liver.
Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe3+-immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein alpha-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDI-TOFMS analysis. The lowest detectable concentration is 5 x 10(-11) M for 100 microL of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis.
Tan
Feng
F
State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing, China.
Zhang
Yangjun
Y
Mi
Wei
W
Wang
Jinglan
J
Wei
Junying
J
Cai
Yun
Y
Qian
Xiaohong
X
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D005290:Ferric Compounds / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D008280:Magnetics; D053768:Metal Nanoparticles; D051379:Mice; D010748:Phosphopeptides / Q000737:chemistry*; D010766:Phosphorylation; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005290:Ferric Compounds; D010748:Phosphopeptides; D020543:Proteome