<p> Supplementary Information for:</p><p>A cyclic-olefin-copolymer microfluidic immobilised-enzyme reactor for</p><p> rapid digestion of proteins from dried blood spots</p><p>Bert Wouters1,2,+,*, Irena Dapic1,+, Thalassa S.E. Valkenburg3, Sam Wouters4, Leon Niezen1,</p><p>Sebastiaan Eeltink4, Garry L. Corthals1 and Peter J. Schoenmakers1</p><p>1University of Amsterdam, van 't Hoff Institute for Molecular Sciences, Analytical-Chemistry</p><p>Group, Science Park 904, 1098 XH Amsterdam, The Netherlands</p><p>2TI-COAST, Science Park 904, 1098 XH Amsterdam, The Netherlands</p><p>3University of Leicester, Department of Chemistry, University Road, Leicester LE1 7RH,</p><p>United Kingdom</p><p>4Vrije Universiteit Brussel, Department of Chemical Engineering, Pleinlaan 2, B-1050</p><p>Brussels, Belgium</p><p>(+) equal contribution</p><p>(*) corresponding author</p><p>Science Park 904, 1098 XH Amsterdam, The Netherlands</p><p>Tel.: +31 (0) 205256416, E-mail: [email protected]</p><p>Supplementary information 1 Table I. Sequence coverage and number of identified peptides of α-S1 obtained for different</p><p>α-casein concentrations using IMER-facilitated or in-solution digestion. IMER-facilitated digestion was performed without protein pre-treatment, for 59 s at room temperature. In- solution digestion was performed with protein pre-treatment, for 18 hours at 37 °C. </p><p>IMER digestion (room temperature, 59 s) In-solution digestion (37 °C, 18 h) Concentration Sequence coverage *Number of identified Sequence coverage *Number of identified (mg/L) (%) peptides (%) peptides 1 56.5 39 0 0 5 64.0 49 0 0 10 67.8 74 19.2 8 25 80.4 100 72.0 48 50 77.1 142 75.7 144 75 77.1 146 75.7 199 100 75.2 182 76.2 184 * Identified at 95% confidence</p><p>Table II: Sequence coverage and number of identified peptides of α-S1 obtained for different</p><p>IMER residence times, for α-casein concentration of 100 mg/L. . IMER-facilitated digestion was performed without protein pre-treatment, at room temperature. In-solution digestion was performed with protein pre-treatment, for 18 hours at 37 °C. </p><p>IMER residence time (s) Sequence coverage (%) *Number of identified peptides 39 81.3 196 47 75.2 184</p><p>59 76.2 171 78 75.7 179</p><p>117 89.3 212 167 80.8 177 293 81.3 174 * Identified at 95% confidence</p><p>Table III: Sequence coverage, number of identified peptides, and missed cleaved peptides of</p><p>α-S1 obtained for 9 repeated measurements. IMER-facilitated digestion was performed</p><p>Supplementary information 2 without protein pre-treatment, for 59 s at room temperature. α-casein concentration was 100 mg/L.</p><p>IMER digestion (room temperature, 59 s) In-solution digestion (37 °C, 18 h) # Sequence *Number of Missed cleaved Sequence *Number of Missed cleaved coverage (%) identified peptides peptides (%) coverage (%) identified peptides peptides (%) 1 80.4 179 17.3 79.4 233 23.7 2 83.2 227 26.5 75.7 212 23.1 3 80.4 172 25.4 79.9 196 24.6 4 90.2 198 27.0 76.2 170 24.7 5 79.4 180 22.7 80.8 189 23.0 6 80.4 177 21.0 79.9 189 22.5 7 80.4 169 27.4 80.8 193 23.0 8 89.3 253 27.0 77.1 198 26.8 9 93.0 196 19.4 72.6 153 23.2 * Identified at 95% confidence</p><p>Supplementary information 3 Table IV: Sequence coverage, number of identified peptides, and missed cleaved peptides obtained for proteins with different molecular weights using IMER-facilitated or in-solution digestion. Proteins were cytochrome c (CYC, 11.7 kDa), myoglobin (MYO, 17 kDa), α-S1 casein (CAS, 23 kDa), ovalbumin (OVA, 42.8 kDa), bovine serum albumin (BSA, 66.5 kDa), transferrin (THS, 75.2 kDa), and catalase (CAT, 240 kDa). IMER-facilitated digestion was performed without protein pre-treatment, for 293 s at room temperature. In-solution digestion was performed with protein pre-treatment, for 18 hours at 37 °C. Protein concentrations were 100 mg/L.</p><p>IMER digestion (room temperature, 293 s) In-solution digestion (37 °C, 18 h) Molecular Sequence *Number of Missed Sequence *Number of Missed Protein weight coverage identified cleaved coverage identified cleaved (kDa) (%) peptides peptides (%) (%) peptides peptides (%) CYC 11.7 70.5 39 22.5 46.7 19 47.4 MYO 17 99.4 158 27.4 92.9 194 12.8 CAS 23 81.3 175 25.9 76.2 184 25.7 OVA 42.8 64.3 117 5.3 49.7 57 7.0 BSA 66.5 55.2 113 N/A 94.9 285 30.3 THS 75.2 56.0 103 N/A 77.9 268 21.5 CAT 240 75.3 186 8.5 88.4 230 14.0 * Identified at 95% confidence</p><p>Supplementary information 4</p>