Phosphopeptide enrichment using MALDI plates modified with high-capacity polymer brushes.
Matrix-assisted laser desorption/ionization plates coated with poly(2-hydroxyethyl methacrylate) (PHEMA) brushes that are derivatized with Fe(III)-nitrilotriacetate (NTA) complexes allow selective, efficient phosphopeptide enrichment prior to analysis by mass spectrometry (MS). Fe(III)-NTA-PHEMA brushes (60 nm thick) have a phosphopeptide binding capacity of 0.6 microg/cm(2) and exhibit phosphopeptide recoveries of over 70%, whereas much thinner polymer films containing Fe(III)-NTA afford a recovery of only 20%, and a monolayer of Fe(III)-NTA shows a recovery of just 10%. Recoveries are determined by comparing signals from enriched unlabeled phosphopeptides with those of their deuterium-labeled analogues that were added to the plate just prior to addition of matrix. Mass spectra of phosphopeptide-containing samples enriched using Fe(III)-NTA-PHEMA-modified plates also demonstrate higher recoveries or fewer interfering peaks than corresponding spectra obtained with enrichment using several commercially available Fe(III)-containing films and resins or metal oxide materials. When analyzing tryptic digests of beta-casein, the Fe(III)-NTA-PHEMA brushes allow detection of as little as 15 fmol of phosphopeptide. Moreover, with both ovalbumin and beta-casein digests, phosphopeptide signals dominate the mass spectra obtained using these modified plates.
Dunn
Jamie D
JD
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Igrisan
Elizabeth A
EA
Palumbo
Amanda M
AM
Reid
Gavin E
GE
Bruening
Merlin L
ML
D002364:Caseins / Q000737:chemistry; D004867:Equipment Design; D010748:Phosphopeptides / Q000302:isolation & purification*; D011102:Polyhydroxyethyl Methacrylate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation*
D002364:Caseins; D010748:Phosphopeptides; D011102:Polyhydroxyethyl Methacrylate