Identification and affinity- quantification of ß-amyloid and α-synuclein polypeptides using online SAW-biosensor- mass spectrometry

Stefan Slamnoiu1, Camelia Vlad1, 2, Mihaela Stumbaum1, 2, Adrian Moise1, Kathrin Lindner1, Nicole Engel1,3, Mar Vilanova6, Mireia Diaz6, Christiaan Karreman4, Marcel Leist4, Thomas Ciossek5, Bastian Hengerer5 , Marta Vilaseca6, and Michael Przybylski1*

1 Laboratory of Analytical Chemistry and Biopolymer Structure Analysis,

Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany

2 Present address: SAW- Instruments, Schwertberger Strasse 16, 53177 Bonn, Germany

3 Present address: Department of Chemical Technology, Technical University

of Vienna, A- 1060 Vienna, Austria

4 Department of Biology, University of Konstanz, 78457 Konstanz, Germany

5 Boehringer Ingelheim Pharma, CNS Research Division, Biberach, Germany

6 IRB Barcelona - Institute for Research in Biomedicine, Barcelona, Spain

Supplementary Figures

Figure S 1

Event time line of SAW-MS interface. Three horizontal bars along the time axis show the operation time periods of SAW-MS interface and transfer to MS: Biosensor, point pattern; interface, diagonal line pattern; transfer and mass spectrometer, cross- line pattern. Perpendicular lines on the time axis delineate the different operation steps. The stages of operation are described in Figure S 2.

Figure S 2

Stages of interface operation during online affinity-MS. The positions of electrical valves and sample path through the interface are as follows: (a), Interface is monitoring the SAW biosensor for the elution injection; incoming flow from biosensor is sent to waste, while the desalting column is rinsed with solvent. Valve 1 is in position (i.) and valve 2 in position (ii). (b), The elution injection is detected, and the sample flow from the biosensor is directed to the guard column; valve 1 is in position (ii) and valve 2 is in position (i). (c), following completion of the elution injection, salts contained in the sample are washed out from the column; valve 1 is in position (i) and valve 2 is in position (ii). (d), The sample trapped on the guard column is eluted with a high acetonitrile- containing solvent and transferred to the ESI source; valve 1 is in position (i) and valve 2 is in position (i). €, The guard column is cleaned of remaining contamination with a solvent containing 90 % acetonitrile, which is sent to waste; valve 1 is in position (i) and valve 2 is in position (ii). (f), After cleaning, the guard column is equilibrated with water, being set ready for a new experiment; valve 1 is in position (i) and valve 2 is in position (ii).

Figure S 3

FTICR-MS negative control experiment of online SAW-MS. The sum of all spectra from TIC shows a background signal of very low intensity.

Figure S 4

Online FTICR- MS analysis of α-synuclein, mutant αSyn(A30P). (a), SAW binding and elution curve from immobilized anti-αSyn-antibody; (b), TIC of the biosensor eluate transferred to the ion source; (c), ESI-FTICR- MS of the eluted αSyn(A30P).

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