Comparison of metal and metal oxide media for phosphopeptide enrichment prior to mass spectrometric analyses.
Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC) resins using manufacturer's and/or published protocols were compared and evaluated for the most efficient and selective method that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest of casein proteins, it was determined that in our hands, two of the resins out-performed the others based on a variety of criteria, including the number of phosphorylation sites identified during MS analyses, the lower numbers of nonspecifically bound peptides observed, and the limits of detection. Applicability of these enrichment resins to a complex biological mixture was investigated. For this work, a mixture of avian histones was digested, subjected to titanium dioxide phosphopeptide enrichment, and analyzed by mass spectrometry. Eight phosphorylated tryptic peptides were observed following enrichment and subsequent LC/MS/MS analyses. Of note, seven of the eight phosphopeptides were not observed without titanium dioxide enrichment. From these analyses, four sites of phosphorylation were unequivocally determined, two of which have not been reported previously. Four additional phosphopeptides were observed; however, the site of phosphorylation could not be distinguished but was localized to one of two possible amino acids. These methods should aid in the investigation of proteins post-translationally modified with phosphate, especially those present at low concentrations as was demonstrated by successful enrichment at the femtomole level.
Gates
Matthew B
MB
Laboratory of Structural Biology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Tomer
Kenneth B
KB
Deterding
Leesa J
LJ
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D002645:Chickens; D002846:Chromatography, Affinity / Q000379:methods*; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D058185:Magnetite Nanoparticles; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
D002364:Caseins; D006657:Histones; D058185:Magnetite Nanoparticles; D010446:Peptide Fragments; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium; D014357:Trypsin