Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag.
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 Ni(2+)-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot beta-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni(2+)-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.
Jalili
Pegah R
PR
Protein Chemistry Technology Center, University of Texas, Southwestern Medical Center, Dallas, Texas 75390-8816, USA.
Sharma
Deepti
D
Ball
Haydn L
HL
D045424:Complex Mixtures / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D045424:Complex Mixtures; D010455:Peptides; D011506:Proteins; C033223:polyhistidine; D006639:Histidine