Analytical Sciences

Observation of Water Molecules Bound to a Protein Using Cold-Spray Ionization Mass Spectrometry

Yoshihisa SEI,*^1,*² Sakurako SHIMOTAKAHARA,^*3 Juri ISHII,*³ Heisaburo SHINDO,*³ Hiroko SEKI,*⁴ Kentaro YAMAGUCHI,*^1,*² and Mitsuru TASHIRO*⁵

*1 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, Shido, Sanuki-city, Kagawa 769-2193, Japan
*2 CREST, Japan Science and Technology Agency (JST), Japan
*3 School of Pharmacy, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan
*4 Chemical Analysis Center, Chiba University, Yayoi, Inage, Chiba 263-8522, Japan
*5 Department of Chemistry, Faculty of Science, Tokyo Metropolitan University, Minami-Osawa, Hachioji, Tokyo 192-0367, Japan

The characterization of water molecules bound to ribonuclease T₁ (RNase T₁) was carried out using cold-spray ionization mass spectrometry (CSI-MS). CSI-MS is a variant of electrospray ionization mass spectrometry (ESI-MS) operating at low temperature, and is particularly suitable for investigating the weaker molecular associations, since the temperature at the spray interface is much lower than that in the conventional ESI-MS. In this approach, ion peaks due to the addition of nine water molecules were identified at a spray temperature of 48°C. This result showed good agreement with that inferred by the combinational analysis of NMR and X-ray crystallography, indicating that CSI-MS is capable of rapidly providing reliable information to characterize the number of water molecules bound to a macromolecule.

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