The highly selective capture of phosphopeptides by zirconium phosphonate-modified magnetic nanoparticles for phosphoproteome analysis.
The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe(3)O(4)/SiO(2) core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol beta-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe(3)O(4) nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of alpha-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.
Zhao
Liang
L
National Chromatographic R and A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China.
Wu
Ren'an
R
Han
Guanghui
G
Zhou
Houjiang
H
Ren
Lianbing
L
Tian
Ruijun
R
Zou
Hanfa
H
D000818:Animals; D002364:Caseins / Q000737:chemistry; D003627:Data Interpretation, Statistical; D030562:Databases, Protein; D005296:Ferrous Compounds / Q000737:chemistry; D005898:Glass; D006868:Hydrolysis; D007202:Indicators and Reagents; D008099:Liver / Q000737:chemistry; D008280:Magnetics; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D063065:Organophosphonates / Q000737:chemistry*; D010047:Ovalbumin / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry*; D020543:Proteome / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin; D015040:Zirconium / Q000737:chemistry*
D002364:Caseins; D005296:Ferrous Compounds; D007202:Indicators and Reagents; D063065:Organophosphonates; D010750:Phosphoproteins; D020543:Proteome; D010047:Ovalbumin; D015040:Zirconium; D014357:Trypsin; C034236:ferrous oxide