Detection of phosphopeptides using Fe(III)-nitrilotriacetate complexes immobilized on a MALDI plate.
Metal affinity complexes were chemically grafted onto the surface of gold matrix-assisted laser desorption/ionization (MALDI) plates by coupling a derivative of nitrilotriacetate (NTA) to immobilized poly(acrylic acid) (PAA) and subsequently forming the Fe(III)-NTA complex. The immobilized complexes can adsorb phosphorylated peptides preferentially from protein digests; deposition of digests on these surface-modified plates, followed by rinsing with an acetic acid solution, addition of matrix, and subsequent analysis by MALDI MS, resulted in mass spectra dominated by peaks corresponding to phosphopeptides. In the case of analyzing a tryptic digest of beta-casein, conventional MALDI MS revealed only one monophosphopeptide, while use of the Fe(III)-NTA-PAA-modified plate resulted in strong signals due to two additional tetraphosphorylated species. The diminution or elimination of signals due to nonphosphorylated species also greatly simplified the identification of phosphopeptides during analysis of ovalbumin digests and myoglobin digests spiked with an equimolar mixture of angiotensin and phosphoangiotensin. The matrix 2',4',6'-trihydroxyacetophenone mixed with diammonium hydrogen citrate proved to be much better than alpha-cyano-4-hydroxycinnamic acid for the detection of phosphorylated peptides from digests of beta-casein and ovalbumin.
Dunn
Jamie D
JD
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Watson
J Throck
JT
Bruening
Merlin L
ML
D000180:Acrylic Resins; D005290:Ferric Compounds; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D010446:Peptide Fragments; D010748:Phosphopeptides / Q000032:analysis*; D011506:Proteins / Q000032:analysis / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000180:Acrylic Resins; D005290:Ferric Compounds; D010446:Peptide Fragments; D010748:Phosphopeptides; D011506:Proteins; C006903:carbopol 940; D014357:Trypsin; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate