Magnetic bead processor for rapid evaluation and optimization of parameters for phosphopeptide enrichment.
Qualitative and quantitative analysis of phosphorylation continues to be both an important and a challenging experimental paradigm in proteomics-based research. Unfortunately researchers face difficulties inherent to the optimization of complex, multivariable methods and their application to the analysis of rare and often experimentally intractable phosphorylated peptides. Here we describe a platform based on manipulation of magnetic beads in a 96-well format that facilitates rapid evaluation of experimental parameters required for enrichment of phosphopeptides. Optimized methods provided for automated enrichment and subsequent LC-MS/MS detection of over 1000 unique phosphopeptides (approximately 1% FDR) from 50 microg of cell lysates. In addition we demonstrate use of this platform for identification of phosphopeptides derived from proteins separated by SDS-PAGE and visualized near the detection limit of silver staining.
Ficarro
Scott B
SB
Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute.
Adelmant
Guillaume
G
Tomar
Maria N
MN
Zhang
Yi
Y
Cheng
Vincent J
VJ
Marto
Jarrod A
JA
D000595:Amino Acid Sequence; D002364:Caseins / Q000032:analysis / Q000302:isolation & purification; D045744:Cell Line, Tumor; D002614:Chelating Agents; D005591:Chemical Fractionation / Q000295:instrumentation* / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008280:Magnetics; D008670:Metals / Q000378:metabolism; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002364:Caseins; D002614:Chelating Agents; D008670:Metals; D010748:Phosphopeptides