Gallium metal affinity capture tandem mass spectrometry for the selective detection of phosphopeptides in complex mixtures.
Metal affinity capture tandem mass spectrometry (MAC-MSMS) is evaluated in a comparative study of a lysine-derived nitrilotriacetic acid (N(alpha), N(alpha)-bis-(carboxymethyl)lysine, LysNTA) and an aspartic-acid-related iminodiacetic acid (N-(4-aminobutyl)aspartic acid, AspIDA) as selective phosphopeptide detection reagents. Both LysNTA and AspIDA spontaneously form ternary complexes with Ga(III) and phosphorylated amino acids and phosphopeptides upon mixing in solution. Collision-induced dissociation of positive complex ions produced by electrospray produces common fragments (LysNTA + H)(+) or (AspIDA + H)(+) at m/z 263 and 205, respectively. MSMS precursor scans using these fragments as reporter ions allow one to selectively detect multiple charge states of phosphopeptides in mixtures. It follows from this comparative study that LysNTA is superior to AspIDA in detecting phosphopeptides, possibly because of the higher coordination number and greater stability constant for Ga(III)-phosphopeptide complexation of the former reagent. In a continuing development of MAC-MSMS for proteomics applications, we demonstrate its utility in a post-column reaction format. Using a simple post-column-reaction 'T' and syringe pump to deliver our chelating reagents, alpha-casein tryptic phosphopeptides can be selectively analyzed from a solution containing a twofold molar excess of bovine serum albumin. The MAC-MSMS method is shown to be superior to the commonly used neutral loss scan for the common loss of phosphoric acid.
Blacken
Grady R
GR
Department of Chemistry, Bagley Hall, University of Washington, Seattle, WA 98195-1700, USA.
Sadílek
Martin
M
Turecek
Frantisek
F
D001224:Aspartic Acid / Q000737:chemistry; D002614:Chelating Agents / Q000737:chemistry; D045424:Complex Mixtures / Q000737:chemistry*; D005708:Gallium / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000737:chemistry; D010748:Phosphopeptides / Q000032:analysis*; D040901:Proteomics; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002614:Chelating Agents; D045424:Complex Mixtures; D010748:Phosphopeptides; D001224:Aspartic Acid; D005708:Gallium; D008239:Lysine; D009571:Nitrilotriacetic Acid