Abstract
To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni2+-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot β-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni2+-IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per microliter.
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Published online March 24, 2007
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Jalili, P.R., Sharma, D. & Ball, H.L. Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag. J Am Soc Mass Spectrom 18, 1007–1017 (2007). https://doi.org/10.1016/j.jasms.2007.02.010
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DOI: https://doi.org/10.1016/j.jasms.2007.02.010