HPLC Coupling with ESI, MALDI and SALD ICP MS – the Detection Platform for Metallothionein
                                         Characterization

 Kateřina Jägerová^1, Iva Benešová^ 1,2, Kristýna Dlabková^1, Viktor Kanický^1.2, Jan Preisler^1,2

                ^1 Department of Chemistry, Faculty of Science, Masaryk University,

                                       Brno, Czech Republic

             ^2 Central European Institute of Technology (CEITEC), Masaryk University,

                                       Brno, Czech Republic


We demonstrate the use of a detection platform that complements conventional LC-ESI MS with two
detection techniques: matrix-assisted laser desorption/ionization (MALDI) and substrate-assisted
laser desorption inductively coupled plasma (SALD ICP) mass spectrometry (MS) analyses. As a model
system, metallothionein 1, a low molecular protein enabling binding wide range of heavy metals (Zn,
Cu, Cd, Hg) was chosen.

Presentujeme použití detekční platformu která spojuje konvenční LC-ESI MS se dvěma detekčními
technikami: matrix-assisted laser desorption/ionization (MALDI) a substrate-assisted laser
desorption inductively coupled plasma (SALD ICP) mass spectrometry (MS). Jako modelový systém byl
zvolen metallothionein 1, nízkomolekulární protein umožňující vazbu širokého množství kovů (Zn, Cu,
Cd, Hg).

Methods

LC separations were performed using a Vydac C8 (150 x 4.6 mm x 5 µm) column. For the separation of
MT1 isoforms (MT1a, MT2d, MT2e), an effluent coming out of the HPLC column was split. One part of
the flow was introduced on-line for ESI oTOF MS detection, second part of the effluent was
deposited on MALDI plastic targets (Prespotted AnchorChip 96). Dry fractions were overlaid with a
0.5 µL of saturated solution of CHCA in 20% ACN and 1% TFA with In as internal standard. Off-line
MALDI TOF and SALD ICP quadrupole MS detection was performed from the same target.

LC separace byla provedena použitím použitím Vydac C8 (150 x 4.6 mm x 5 µm) kolony. Pro separaci
MT1 isoformy (MT1a, MT2d, MT2e), effluent vycházející z HPLC kolony byl rozdělen. Jedna část toku
byla vedena primo on-line do ESI MS oTOF MS detekce, druhá část eluátu byla nanesena na MALDI
plastovou destičku (Prespotted AnchorChip 96). Suché frakce byly převrstveny 0.5 µl nasyceného
roztoku CHCA v 20% ACN a 1% TFA s

Results

Simultaneous detection of the MT1 isoforms (complexes with metals), apoforms (forms without metals)
and metal quantification were obtained using off-line and on-line approach. On-line ESI MS provides
information about the MT1 complexes and their metal stoichiometry. MALDI MS provides additional
information about apoforms and thus confirms ESI MS identification. SALD ICP MS provides
quantitative data on metals in MT1 complexes.

Innovative aspects

·   HPLC separation coupled with on-line ESI MS and off-line MALDI MS and ICP MS detection
techniques

·   The multiple detection platform for MALDI and SALD ICP MS allows both qualitative and
quantitative analysis of MT1 complexes

·   Direct comparison of ESI and MALDI analysis of MT1 isofoms

References

[1] Iva Tomalová; Pavla Foltýnová ; Viktor Kanický ; Jan Preisler. “MALDI MS and ICP MS Detection
of a Single CE Separation Record: A Tool for Metalloproteomics” Anal. Chem. 2013, 8, 6448–6456.


Acknowledgements

We gratefully acknowledge the financial support of the Czech Science Foundation (GA15-05387S) and
the project CEITEC 2020 (LQ1601) of the Ministry of Education, Youth and Sports of the Czech
Republic.