Combination of biomolecular interaction analysis and mass spectrometric amino acid sequencing.
We describe an approach for the combination of biomolecular interaction analysis (BIA) and electrospray tandem mass spectrometry (ESI/MS/MS) to obtain sequence information on the affinity-bound proteins on the sensor chip of BIA. The procedure is illustrated with stable and unstable interactions of recombinant proteins, i.e., histidine-tagged protein-Ni2+/NTA and 1,4,5-inositol trisphosphate receptor-ligand interactions. The E. coli lysates expressing the recombinant proteins were passed through the sensor chips, and biomolecular interactions were monitored in real time. The molecules detected on the sensor chip were digested by delivering proteolytic enzyme to the sensing flow cells. The resulting on-chip digested peptide mixture at the mid- to low-femtomole level was recovered on a microcapillary reversed-phase precolumn by an on-line system and analyzed using HPLC-MS/MS. In both cases, unambiguous sequence information on the recombinant proteins isolated on the sensor chip was obtained from only a single run of analysis. The combined BIA-MS/MS may prove to be a general and versatile system to discover novel biomolecular interactions and to analyze protein complexes.
Natsume
T
T
Calciosignal Net Project, Exploratory Research for Advanced Technology, JST, RIKEN Komagome Branch, Tokyo, Japan. natsume@comp.metro-u.ac.jp
Nakayama
H
H
Jansson
O
O
Isobe
T
T
Takio
K
K
Mikoshiba
K
K
D015220:Calcium Channels / Q000032:analysis; D004926:Escherichia coli; D053496:Inositol 1,4,5-Trisphosphate Receptors; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis; D020539:Sequence Analysis, Protein / Q000379:methods*
D015220:Calcium Channels; D053496:Inositol 1,4,5-Trisphosphate Receptors; D011506:Proteins; D018160:Receptors, Cytoplasmic and Nuclear