Supplemental Section

A New Matrix Assisted Ionization Method for the

Analysis of Volatile and Nonvolatile Compounds by

Atmospheric Probe Mass Spectrometry

Shubhashis Chakrabarty,1* Vincent S. Pagnotti,1* Ellen Inutan,2

Sarah Trimpin,2 Charles N. McEwen1

1. University of the Sciences, Philadelphia, PA, 2. Wayne State University, Detroit, MI

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.

1 a 100 530.790 +2 MH2 e c n a d n u b A

e v i t a l e R

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

e v i t 561.246 a l e R

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

e v i t +2 a l MH2 e

R 530.790

0 500 1000 m/z

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.

2 100 432.902 [M + 3H]3+ e c n a d n u b A

e v i t a l e R [M + 2H]2+ 648.849

0 400 600 800 1000 1200 m/z

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.

3 100 +4 1147.528 +3 1434.159 e c n a d n u b A

e v i t +2 a l

e 1911.875 R

1183.077

0 1100 1300 1500 1700 1900 m/z

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.

4 813.648 100 [M + H]+ e c n a d n u b A

e v i t a l [M + H]+ e

R 731.573 662.136 984.056 579.131 878.020 1076.320

0 500 600 700 800 900 1000 1100 m/z

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.

5 455.269 100 [M+H]+ e c n a d n u b A

e v i t a l e R

288.276 316.306 498.347 0 150 200 300 400 500 600 m/z

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.

6 338.326 100 e c n a

d 161.064 n u b A

e v i t a l e

R 363.200 [M+H]+

0 100 200 300 400 500 600 700 m/z

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.

7 100 362.135 [M+H]+ e c n a d n u b A e v i t a l e R

318.147 344.125

0 100 200 300 400 500 m/z

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.

8 a 648.849 100 530.791 MH 2+ 2+ 2 MH2 e c n a d n u b A

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

R 1134.563 744.284 662.169 1059.335 0 500 m/z 1000

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.

9 100 663.427 +8 +9 1071.417 952.594 e

c +7 n

a 1224.477

d +10 n

u 857.336 b A e v i t +6 a l

e 1428.389 R

+5 1713.867 +4 1954.749 0 600 1000 1400 1800 m/z

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.

10 100 1231.328 e c n a d n u b 12+ A

e 11+ v

i 13+ 1413.570 t

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+

0 1000 1200 1400 1600 1800 900 m/z

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.

11 a 1931.992 70 304.295 100 +3 e c n a d n u b A

e

v 288.285 i t e a l c e n a R 316.316 759.875 d

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

e +4 +3

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

279.155 477.239 c n a d n u b A

e v i t e c a l n e a R d n u

b 0 A 1200 1400 1600 1800 m/z e 363.287 v i t a l

e 607.557 R 883.763 577.511

855.731 833.748

0 200 400 600 800 1000 m/z

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.

12 100 530.62 + MH2 e c n a d n u b A

e v i t a l e R

0 500 600 700 800 900 1000 m/z

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.

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