<p> Supplemental Section</p><p>A New Matrix Assisted Ionization Method for the</p><p>Analysis of Volatile and Nonvolatile Compounds by</p><p>Atmospheric Probe Mass Spectrometry</p><p>Shubhashis Chakrabarty,1* Vincent S. Pagnotti,1* Ellen Inutan,2 </p><p>Sarah Trimpin,2 Charles N. McEwen1</p><p>1. University of the Sciences, Philadelphia, PA, 2. Wayne State University, Detroit, MI</p><p>The figures below show the mass spectra obtained by introduction of various samples in the matrices 3-nitrobenzonitrile (3-NBN) or 2,5-dihydroxyacetophenone (2,5-DHAP) dried on a melting point tube and inserted near the ion entrance aperture of a mass spectrometer with heated gas flowing over the sample. Figure 1 – 11 were obtained on a Thermo Scientific Orbitrap Exactive using the HESI probe for introducing the heated gas and the M&M Mass Spec ASAP probe for introducing the sample for analysis. Figure 12 shows a proof of principle mass spectrum of bradykinin using the matrix 2,5- DHAP on a Waters Xevo alpha unit obtained by hand holding a melting point tube, with sample applied, near the entrance skimmer and blowing heated air over the sample.</p><p>1 a 100 530.790 +2 MH2 e c n a d n u b A</p><p> e v i t a l e R</p><p>MH+ 642.338 710.364 904.471 1060.572 0 600 700 800 900 1000 1100 b 100 m/z 530.789 +2 MH2 e c n a d n u b A</p><p> e v i t 561.246 a l e R</p><p>MH+ 803.545 1060.571 664.254 904.470 0 500 1000 m/z c 100 550.630 e c n a d n u b A</p><p> e v i t +2 a l MH2 e</p><p>R 530.790</p><p>0 500 1000 m/z</p><p>Figure S1: Mass spectrum of 1 pmol of bradykinin using matrix assisted probe introduction (a) 3-NBN, (b) 2,5-DHAP, and (c) 2,5-DHB as matrices. </p><p>2 100 432.902 [M + 3H]3+ e c n a d n u b A</p><p> e v i t a l e R [M + 2H]2+ 648.849</p><p>0 400 600 800 1000 1200 m/z</p><p>Figure S2: Mass spectrum of 100 fmol of angiotensin 1 in 3-NBN matrix using the ASAP probe. The mass spectrometer inlet temperature was 200 °C and the gas from the HESI probe was at 70 °C.</p><p>3 100 +4 1147.528 +3 1434.159 e c n a d n u b A</p><p> e v i t +2 a l</p><p> e 1911.875 R</p><p>1183.077</p><p>0 1100 1300 1500 1700 1900 m/z</p><p>Figure S3: Mass spectrum of 1 pmol of bovine insulin in 2,5-DHAP matrix introduced into the ionization region using the ASAP probe. The inlet temperature was 200 °C and the gas temperature was 95 °C.</p><p>4 813.648 100 [M + H]+ e c n a d n u b A</p><p> e v i t a l [M + H]+ e</p><p>R 731.573 662.136 984.056 579.131 878.020 1076.320</p><p>0 500 600 700 800 900 1000 1100 m/z</p><p>Figure S4: Mass spectrum of 2 μ L of a 100 ppb solution of bovine brain sphingomyelin in 3- NBN matrix applied to a melting point tube and introduced into the Orbitrap Exactive ion source using an ASAP probe. The ions at e.g. m/z 813 and 731 represent the [M + H]+ ions of sphingomyelins with the long chain base 18:1 and the fatty acid of m/z 813 being 24:1 and of m/z 731 being 18:0.</p><p>5 455.269 100 [M+H]+ e c n a d n u b A</p><p> e v i t a l e R</p><p>288.276 316.306 498.347 0 150 200 300 400 500 600 m/z</p><p>Figure S5: Mass spectrum of the drug verapamil loading 1 μ L of a 100 ppb solution in the matrix 3-NBN using the ASAP probe. Inlet temperature 50 °C and gas temperature 60 °C.</p><p>6 338.326 100 e c n a</p><p> d 161.064 n u b A</p><p> e v i t a l e</p><p>R 363.200 [M+H]+</p><p>0 100 200 300 400 500 600 700 m/z</p><p>Figure S6: Mass spectrum of 1 μ L of a 100 ppb solution of hydroxycortisone in 3-NBN matrix introduced into the ion source using the ASAP probe. Inlet temperature 200 °C and gas temperature 60 °C.</p><p>7 100 362.135 [M+H]+ e c n a d n u b A e v i t a l e R</p><p>318.147 344.125</p><p>0 100 200 300 400 500 m/z</p><p>Figure S7: Mass spectrum of 1 μ L of a 100 ppb solution of the drug levofloxacin in 3-NBN matrix introduced to the ionization region using the ASAP probe. Inlet temperature 200 °C, gas temperature 70 °C and 1 kV place on the HESI probe. </p><p>8 a 648.849 100 530.791 MH 2+ 2+ 2 MH2 e c n a d n u b A</p><p>432.902 e 2+ v i MH2 t a l e R 679.306 MH+ 549.764 768.211 1296.691 824.343 MH+ 1060.574 0 500 700 900 1100 1300 m/z b 100 530.791 2+ MH2 e c n a d n u b A 549.764 e v i t a l 768.211 1028.520 e</p><p>R 1134.563 744.284 662.169 1059.335 0 500 m/z 1000</p><p>Figure S8: Mass spectrum of ca. 2 µL of (a) 600 μM angiotensin I and 240 µM bradykinin in a 1:1 acetonitrile:water solution of 3-NBN, and (b) 120 nM bradykinin acquired directly after the higher concentration sample in (a) using matrix assisted probe introduction; gas temp. 60 °C. </p><p>9 100 663.427 +8 +9 1071.417 952.594 e</p><p> c +7 n</p><p> a 1224.477</p><p> d +10 n</p><p> u 857.336 b A e v i t +6 a l</p><p> e 1428.389 R</p><p>+5 1713.867 +4 1954.749 0 600 1000 1400 1800 m/z</p><p>Figure S9: Mass spectrum of 1 pmol of ubiquitin in 3-NBN introduced to the ionization region using the ASAP probe. The inlet temperature was set at 300 °C, the gas flow from the HESI probe was heated to 70 °C and 3.5 kV was placed on the HESI probe.</p><p>10 100 1231.328 e c n a d n u b 12+ A</p><p> e 11+ v</p><p> i 13+ 1413.570 t</p><p> a 1542.167 l 1304.910 e 14+ R 10+ 15+ 1131.123 1696.081 16+ 9+ 1060.491 17+ 1884.312 998.109 18+</p><p>0 1000 1200 1400 1600 1800 900 m/z</p><p>Figure S10: Mass spectrum of 1 pmol of myoglobin in 3-NBN introduced to the ionization region using the ASAP probe. The inlet temperature was set at 250 °C, the gas flow from the HESI probe was heated to 90 °C and 3 kV was placed on the HESI probe.</p><p>11 a 1931.992 70 304.295 100 +3 e c n a d n u b A</p><p> e</p><p> v 288.285 i t e a l c e n a R 316.316 759.875 d</p><p> n 429.234 663.444 u m/z b 0 A 200 300 400 500 600 700 1458.483 e v i t a l</p><p> e +4 +3</p><p>R +5 1166.987 1700.517 +4 +4 +6 1093.536 1275.390 875.031 979.318 1727.225 0 800 1000 1200 1400 1600 1800 m/z : b 257.244 100 02 391.279 181.062 e</p><p>279.155 477.239 c n a d n u b A</p><p> e v i t e c a l n e a R d n u</p><p> b 0 A 1200 1400 1600 1800 m/z e 363.287 v i t a l</p><p> e 607.557 R 883.763 577.511</p><p>855.731 833.748</p><p>0 200 400 600 800 1000 m/z</p><p>Figure S11: Mass spectra of human saliva (a) mixed 1:1 with a water:acetonitrile saturated solution of 3- NBN using matrix assisted probe introduction: inlet temperature 250 °C, gas temperature 90 °C, and 3kV applied to the HESI probe, and (b) ASAP of saliva diluted 10X with water and 2 μL loaded onto a melting point tube: inlet temperature 250 °C, gas temperature 100-400 °C, and 3.5 kV applied to discharge needle. Insets show (a) low-mass ions with matrix assistance and (b) lack of high-mass ions with ASAP. Proteins are observed with average molecular weights of 4370, 5098, 5793, 5830, and 5870.</p><p>12 100 530.62 + MH2 e c n a d n u b A</p><p> e v i t a l e R</p><p>0 500 600 700 800 900 1000 m/z</p><p>Figure S12: Mass spectrum of 1 pmol bradykinin acquired on a Waters Xevo TQ-S (alpha unit) using 3-NBN as matrix and holding a melting point tube with the sample near the ion entrance skimmer while blowing warn air over the sample using a heat gun.</p><p>13</p>