This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phosphopeptides in complex mixtures can be enriched by immobilized metal-ion affinity chromatography (IMAC) thereby increasing detection sensitivity in mass spectrometry (MS) analysis. Methyl esterification of acidic groups has been utilized to increase the specificity of IMAC. This study focuses on gaining better understanding of the methyl esterification reaction of by: 1) determining the optimal time frame for complete incorporation of methyl esters and, 2) identifying side products formed during the progression of the reaction. The resulting decrease in heterogeneity will maximize phosphopeptide signal intensity in MS. Additionally, knowledge of unavoidable side products will aid interpretation and database searching of MS data. Purified peptides and enzymatic digests of known proteins were dried in a centrifugal vacuum concentrator. Dried samples were resuspended and incubated for variable time periods in the presence of anhydrous 1.75 M methanolic HCl at room temperature. Samples were dried and resuspended in water and stored at -20 degrees C. Samples were diluted into the appropriate solvents for subsequent mass spectrometry analysis. Samples were analyzed by MS (Bruker Reflex IV MALDI-TOF, Applied Biosystems Pulsar i qoTOF with oMALDI 1 and Protana nanoelectrospray sources) to determine the extent of methyl esterification of acidic groups as well as to identify side products. Peptides were treated for 0.5, 1.0, 2.0, and 3.0 hours with anhydrous methanolic HCl. Reaction products were analyzed by MALDI-TOF and nanoelectrospray quadrupole orthogonal TOF mass spectrometry. A manuscript reporting the results of this study is in preparation.
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