Revealing Structure and Localization of Steroid Regioisomers through Predictive Fragmentation Patterns in Mass Spectrometry Imaging

Identifying and mapping steroids in tissues can provide opportunities for biomarker discovery, the interrogation of disease progression, and new therapeutics. Although separation coupled to mass spectrometry (MS) has emerged as a powerful tool for studying steroids, imaging and annotating steroid isomers remains challenging. Herein, we present a new method based on the fragmentation of silver-cationized steroids in tandem MS, which produces distinctive and consistent fragmentation patterns conferring confidence in steroid annotation at the regioisomeric level without using prior derivatization, separation, or instrumental modification. In addition to predicting the structure of the steroid with isomeric specificity, the method is simple, flexible, and inexpensive, suggesting that the wider community will easily adapt to it. We demonstrate the utility of our approach by visualizing steroids and steroid isomer distributions in mouse brain tissue using silver-doped pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging.


Figure S4
Figure S4Lithium adducts predominantly undergo loss of H2O in CID for the regioisomers 20αdihydroprogesterone and pregnenolone.10 µM of both regioisomers were directly infused into Orbitrap Velos Pro at a flow rate of 10 µl/min and fragmented at a collision energy of 35 NCE at a resolution of 100 000 (Δm/m at m/z 400)

Figure S7
Figure S7 Protonated adducts of three hydroxyprogesterone's fragment in CID to give isomeric product ions in MS 2 .a) MS2 mass spectrum of 11-hydroxyprogesterone b) MS 2 mass spectrum of 17-hydroxyprogesterone c) MS 2 mass spectrum of 21-hydroxyprogesterone.All regioisomers were directly infused into Orbitrap Velos Pro at a flow rate of 10 µl/min and fragmented at a collision energy of 20 NCE at a resolution of 100 000 (Δm/m at m/z 400).All m/z values are with ±5ppm of the theoretical value.

Figure S8 .
Figure S8.Fragmentation of 11βOHP produces a diagnostic product ion at m/z = 271.1710differentiating it from its epimer 11αOHP.All regioisomers were directly infused into Orbitrap Velos Pro at a flow rate of 10 µl/min and fragmented at a collision energy of 20 NCE at a resolution of 100 000 (Δm/m at m/z 400).All m/z values are with ±5ppm of the theoretical value.

Figure S9
Figure S9 Plausible molecular structure of neutral loss fragments upon fragmentation of argenated adduct of pregnenolone molecule with CID.

Figure S10 .
Figure S10.Fragmentation of d-ring and e-side chain by CRF only occurs by CID and not by HCD.MS 2 spectra of pregnenolone fragmented by HCD (top) and CID (inverted).

Figure S11 .Figure S12
Figure S11.Predicted fragmentation that enables separation of 11DOC and CORT.a) m/z at 365.0670 and 367.0670 corresponds to the fragment ion C17H22O2 107 Ag and C17H24O2 107 Ag of corticosterone.b) m/z at 349.0721 and 351.0878 corresponds to C17H22O2 107 Ag and C17H24O2 107 Ag of 11-deoxycortisol.

Figure S13 .
Figure S13.CID of the two regioisomers corticosterone and 11-deoxycortisol as [M+Ag] + induce c-c bond dissociation in the d-ring producing isomer specific fragment ions in MS 3 with less than 2 ppm mass difference compared to the theoretically predicted m/z values.Corticosterone produces a fragment ion at m/z = 353.0675corresponding to d1 and 11-deoxycortisol produces a fragment ion at m/z = 337.0727and 351.0878 corresponding to d1 and d2 respectively.

Figure S14
Figure S14 Extracted ion chronograms from a line scan of mouse brain tissue section: a-g) for m/z values corresponding to [M+ 107 Ag] + , h-n) for m/z values corresponding to [M+H] + , o-u) for m/z values corresponding to [M-H2O+H] + steroid adducts from line scans of mouse brain tissue section.The liquid bridge is in contact with the tissue from time 0.69 to 3.84 and 0.49 min to 3.86 min for solvents with and without silver, respectively (marked in grey on each line scan).The intensity of silver adducts increases when the liquid bridge is in contact with the tissue.

Table S1 :
Sensitivity comparison between silver cautioned steroids and protonated adducts.