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J Biomol Tech. 2007 February; 18(1): 28.
PMCID: PMC2291830

P82-S MALDI-MS Analysis of Phosphopeptides Complexed with Metal Ions

Abstract

Objective: To develop a procedure that can serve to identify phosphopeptides in complex mixtures.

The identification and characterization of phosphopeptides by mass spectrometry is an active area of investigation due to the importance of the modification in a variety of regulatory processes. Phosphopeptides exhibit high binding affinity for a number of metal ions, including iron(III) and gallium(III). We have investigated whether MALDI-MS allows the detection of cationized phosphopeptides. A number of metal ions, including Fe(III), Ga(III) and Ca(II), were tested in a variety of matrices. It was found that Fe(III) binds preferentially to phosphopeptides in the presence of non-phosphorylated peptides under acidic conditions.

Preliminary results: When incubating phosphopeptides with millimolar concentrations of metal salts (FeCl3, Ga(NO3)3, CaCl2), cationized species were observed in MALDI-MS spectra with all matrices tested. The major cationized species carried a single positive charge, e.g., [M –2H +Fe]+. The cationized species typically exhibited a signal intensity 10–20% that of the protonated one ([M+H]+). To test the specificity of the metal ion binding, a model phosphopeptide (beta-casein 48-63) was mixed with a BSA tryptic digest and incubated with metal salts on the MALDI target in the presence of matrix. Cationization was observed to a varying extent in all cases, but only one metal ion/matrix combination exhibited specificity for the phosphopeptide. Iron(III) bound to the phosphopeptide exclusively when employing ACHC as the matrix. A variety of phosphopeptides containing phosphorylated serine, threonine, and tyrosine residues were analyzed in the same manner with similar results. We were able to obtain TOF/ TOF fragmentation spectra when selecting the [M+H]+ ion corresponding with the phosphopeptides but not for the cationized precursor. Currently, conditions are being tested that increase the specificity of Fe(III) for phosphopeptides as opposed to other acidic and potentially chelating peptides.


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