TY - JOUR
T1 - Protein-metal ion interactions, stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry
AU - Benson, Linda M.
AU - Kumar, Rajiv
AU - Cavanagh, John
AU - Naylor, Stephen
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2003
Y1 - 2003
N2 - The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (μESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/μESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D28K. Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.
AB - The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (μESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/μESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D28K. Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.
UR - http://www.scopus.com/inward/record.url?scp=12244297743&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=12244297743&partnerID=8YFLogxK
U2 - 10.1002/rcm.903
DO - 10.1002/rcm.903
M3 - Article
C2 - 12569434
AN - SCOPUS:12244297743
SN - 0951-4198
VL - 17
SP - 267
EP - 271
JO - Rapid Communications in Mass Spectrometry
JF - Rapid Communications in Mass Spectrometry
IS - 4
ER -