BIA/MS of epitope-tagged peptides directly from E. coli lysate: multiplex detection and protein identification at low-femtomole to subfemtomole levels.
The use of biomolecular interaction analysis mass spectrometry to selectively isolate, detect, and characterize epitope-tagged peptides present in total cell lysates is demonstrated. Epitope-tagged tryptic peptides were captured via affinity interactions with either chelated Ni2+ or monoclonal antibodies and detected using surface plasmon resonance biomolecular interaction analysis (SPR-BIA). After SPR-BIA the tagged peptides were either eluted from the biosensor chips for mass spectrometric analysis or analyzed directly from the biosensor chip using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). Protein database searches were performed using the masses of the tagged tryptic peptides, resulting in identification of the protein into which the epitope tag was inserted. Detection limits for both SPR-BIA and MALDI-TOF were at the low-femtomole to subfemtomole level. The approach represents a (multiplexed) high-sensitivity chip-based technique capable of identifying epitope-tagged proteins as they are present in complex mixtures.
Nelson
R W
RW
Intrinsic Bioprobes, Inc., Tempe, Arizona 85281, USA.
Jarvik
J W
JW
Taillon
B E
BE
Tubbs
K A
KA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D017931:DNA Primers; D000939:Epitopes / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000032:analysis*; D012680:Sensitivity and Specificity
D001426:Bacterial Proteins; D017931:DNA Primers; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments