Tandem LC-MS Identification of Antitubercular Compounds in Zones of Growth Inhibition Produced by South African Filamentous Actinobacteria

Novel antitubercular compounds are urgently needed to combat drug-resistant Mycobacterium tuberculosis (Mtb). Filamentous actinobacteria have historically been an excellent source of antitubercular drugs. Despite this, drug discovery from these microorganisms has fallen out of favour due to the continual rediscovery of known compounds. To increase the chance of discovering novel antibiotics, biodiverse and rare strains should be prioritised. Subsequently, active samples need to be dereplicated as early as possible to focus efforts on truly novel compounds. In this study, 42 South African filamentous actinobacteria were screened for antimycobacterial activity using the agar overlay method against the Mtb indicator Mycolicibacterium aurum under six different nutrient growth conditions. Known compounds were subsequently identified through extraction and high-resolution mass spectrometric analysis of the zones of growth inhibition produced by active strains. This allowed the dereplication of 15 hits from six strains that were found to be producing puromycin, actinomycin D and valinomycin. The remaining active strains were grown in liquid cultures, extracted and submitted for screening against Mtb in vitro. Actinomadura napierensis B60T was the most active sample and was selected for bioassay-guided purification. This resulted in the identification of tetromadurin, a known compound, but which we show for the first time to have potent antitubercular activity, with the MIC90s within the range of 73.7–151.6 nM against M. tuberculosis H37RvT in vitro under different test conditions. This shows that South African actinobacteria are a good source of novel antitubercular compounds and warrant further screening. It is also revealed that active hits can be dereplicated by HPLC-MS/MS analysis of the zones of growth inhibition produced by the agar overlay technique.


Introduction
Novel antitubercular compounds are urgently needed to combat Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Despite an array of treatment options, TB was estimated to be responsible for 1.6 million deaths in 2021, representing the first increase in deaths since 2005 [1]. While the COVID-19 pandemic is partially responsible, a major cause of this increase is the rapid spread of drug-resistant strains, which are neutralising our first-line treatments. The number of drug-resistant cases of TB is on the rise, with an increase of 450,000 cases of rifampicin-resistant infections observed between 2020 and 2021. The second-line treatments, used to treat drug-resistant cases, see patient non-adherence to the drug regimen due to the high pill burden, long treatment times and toxic side effects, which further complicates treatment [1,2]. This necessitates the discovery and development of improved chemotherapeutics to treat drug-resistant M. tuberculosis strains. Furthermore, new treatments should ideally be more efficient (shorter treatment times) and have fewer side effects.
Natural products from the filamentous actinobacteria (actinomycetes) have historically been a rich source of novel antitubercular compounds. Drugs such as streptomycin, the rifamycins, kanamycin and capreomycin were all isolated from filamentous actinobacteria and developed as clinical antitubercular drugs [3][4][5]. However, natural product drug discovery is fraught with difficulties, such as long and expensive studies, which tend to result in the re-isolation of known compounds and have low new molecule hit rates. While these challenges are arduous and intricate, they are not insurmountable and actinobacterial natural products are still a good source of drugs. The advent of new technology and new knowledge regarding the filamentous actinobacteria allow us to develop improved workflows to overcome these challenges and screen these microorganisms for novel antitubercular molecules.
One of the key components in improving natural product screening programs is to dereplicate active samples as soon as possible to prevent the re-isolation of known compounds [6]. This can be achieved using tools such as the Global Natural Product Social (GNPS) molecular networking platform, which has been used globally to identify known and novel compounds [7]. Another crucial aspect is strain selection. Multiple studies have revealed that genetically diverse strains should be targeted as they are more likely to have greater chemical diversity, which increases the likelihood of identifying novel antimicrobial compounds [8][9][10]. These strains include microorganisms isolated from biodiverse environments or that represent rare genera that are difficult to isolate under laboratory conditions [11].
South Africa is the third most biodiverse country in the world, with unique biomes such as the fynbos region [12]. This biodiversity is reflected in the microorganisms isolated from these environments [13,14]. However, few compounds have ever been identified or elucidated from South African filamentous actinobacteria, despite their potential, with most of the literature focusing on the isolation of South African actinobacteria and descriptions of crude extract screening. There are a small number of examples demonstrating the biosynthetic ability of these strains, such as platensimycin and natamycin (pimaricin). However, more work needs to be done on these strains [15][16][17]. Therefore, the aim of this project was to screen South African filamentous actinobacteria for novel antitubercular compounds and isolate the compounds from the most active strains.

Agar Overlay Screening against Mycolicibacterium aurum
The agar overlay technique was used for primary antimycobacterial screening, as it is a cheap and fast semi-quantitative method that allows for the screening of antibacterial activity under a wide range of nutrient growth conditions [18]. Activity is recorded when a zone of growth inhibition is seen after a bacterial indicator is grown over a stab-inoculated filamentous actinobacterium.
Forty-two environmental strains of filamentous actinobacteria from eight genera, namely Actinomadura, Amycolatopsis, Kribbella, Microbispora, Micromonospora, Nocardia, Nonomuraea and Streptomyces, were screened for antimycobacterial activity using the agar overlay assay against Mycolicibacterium aurum strain A+. M. aurum was used as an indicator for M. tuberculosis as they have a comparable drug susceptibility profile, but M. aurum is non-pathogenic and has a faster growth rate [19]. Each filamentous actinobacterial strain was grown on six diverse growth media with the aim of inducing the production of a wide range of secondary metabolites [20].
Thirty-one strains showed activity on at least one type of growth medium, with a total of 79 hits in the primary screen (Table S1). These results were confirmed by stab-inoculating each active strain individually in the same growth media as those on which activity was recorded and repeating the overlay assays. Previous studies have shown that the zones of growth inhibition can provide a window into the compounds produced by an active strain. For example, Boya P. et al. (2017) identified several actinomycins and macrolides at the site of interaction on agar between two Streptomyces strains and the pathogenic fungus Escovopsis TZ49 using MALDI imaging mass spectrometry [21]. Therefore, any antimycobacterial compound detected here would likely be responsible for the activity observed. To analyse these zones, their diameters were measured, and the agar within each zone was excised and extracted before analysis by high-pressure liquid chromatography coupled to a quadrupole time of flight instrument (HPLC-qTOF). The raw spectra were then processed using MSConvert and GNPS molecular networking to identify known antibiotics [7,22]. These hits were subsequently confirmed by manual annotation using MZMine 3 to build chromatograms and PubChem and Kyoto Encyclopedia of Genes and Genomes (KEGG) database searches to support the GNPS identification. This workflow is summarised in Figure 1. inoculating each active strain individually in the same growth media as those on which activity was recorded and repeating the overlay assays. Previous studies have shown that the zones of growth inhibition can provide a window into the compounds produced by an active strain. For example, Boya P. et al. (2017) identified several actinomycins and macrolides at the site of interaction on agar between two Streptomyces strains and the pathogenic fungus Escovopsis TZ49 using MALDI imaging mass spectrometry [21]. Therefore, any antimycobacterial compound detected here would likely be responsible for the activity observed. To analyse these zones, their diameters were measured, and the agar within each zone was excised and extracted before analysis by high-pressure liquid chromatography coupled to a quadrupole time of flight instrument (HPLC-qTOF). The raw spectra were then processed using MSConvert and GNPS molecular networking to identify known antibiotics [7,22]. These hits were subsequently confirmed by manual annotation using MZMine 3 to build chromatograms and PubChem and Kyoto Encyclopedia of Genes and Genomes (KEGG) database searches to support the GNPS identification. This workflow is summarised in Figure 1. These additional steps allowed the identification and elimination of 15 hits from six different strains, which were found to be known antimycobacterial compounds. These were puromycin, actinomycin D and valinomycin; example chromatograms and annotated product ion spectra of each compound obtained from agar extracts are shown in Figures  2-7). These antibiotics are commonly produced by the filamentous actinobacteria and are known to have antimycobacterial activity [23][24][25]. Interestingly, Kribbella strain 1B8Tu was found to produce actinomycin D. While actinomycin D is commonly produced by members of the Streptomyces genus [4], it has never been reported in members of the genus Kribbella. These additional steps allowed the identification and elimination of 15 hits from six different strains, which were found to be known antimycobacterial compounds. These were puromycin, actinomycin D and valinomycin; example chromatograms and annotated product ion spectra of each compound obtained from agar extracts are shown in Figures 2-7). These antibiotics are commonly produced by the filamentous actinobacteria and are known to have antimycobacterial activity [23][24][25]. Interestingly, Kribbella strain 1B8Tu was found to produce actinomycin D. While actinomycin D is commonly produced by members of the Streptomyces genus [4], it has never been reported in members of the genus Kribbella.               To investigate whether the antibiotic production observed in the zones of inhibition matched that in liquid cultures, the six strains producing known compounds were grown in small-scale (25 mL) liquid cultures in their respective media for antibiotic production, extracted and analysed in a similar manner to the zones of inhibition. There was a strong correlation between the molecules identified in the traditional liquid culture extraction method and the zone of inhibition method (Table S2). This suggested that the analysis of the zones of inhibition from agar plates was a suitable method for the rapid identification of strains producing known compounds. Importantly, strains for which the active compounds could not be identified through analysis of their zones of inhibition could possibly be producing novel antimycobacterial compounds, which warrant further study against M. tuberculosis.

Mycobacterium tuberculosis In Vitro Screening
Strains for which the active compound could not be identified through HPLC-MS/MS analysis of their zones of inhibition were grown in small-scale liquid cultures, extracted and submitted for testing against M. tuberculosis H37Rv T in vitro.
Thirty-four samples showed antitubercular activity in vitro in at least one of the growth media (Table S3). Only samples that showed minimum concentrations required to inhibit the growth of 90% of the M. tuberculosis H37Rv T culture (MIC 90 ) < 20 µg/mL in at least two growth media were selected for further screening. Eight samples (from seven actinobacterial strains) met these conditions and were retested to confirm the in vitro antitubercular activity and screened for in vitro cytotoxicity against the Chinese Hamster Ovary (CHO) cell line to determine selectivity (Table 1). Streptomyces fractus MV32 T and Streptomyces strains CW5 and Y10 all exhibited potent cytotoxicity against the Chinese Hamster Ovary cell line, excluding them from further study [26].
Streptomyces africanus CPJVR-H T , Streptomyces strain HMC5 and Actinomadura napierensis B60 T all displayed no cytotoxicity up to 100 µg/mL. A. napierensis B60 T was the most active strain, with low microgram/mL activity in all three M. tuberculosis test media conditions. Therefore, it was selected for bioassay-guided fractionation studies to identify the active compound that it was producing.

Purification and Identification of Tetromadurin
Eight litres of A. napierensis B60 T were cultured in ISP-2 and the cell mass and broth were extracted with ethyl acetate and methanol. The crude extracts were pooled and separated by reverse-phase solid-phase extraction on C18 cartridges before fractionation by high-pressure liquid chromatography coupled to a diode array detector (HPLC-DAD). One active fraction was identified and analysed by HPLC-qTOF to identify a major peak with a m/z of 778.4747.
Analysis of the mass spectrum of the peak at m/z 778.4747 revealed it to be an ammonium adduct [M + NH 4 + ] with a mass of 760.4403 Da. Mass searches using Antibase 2017 [27] matched to A-80577 (tetromadurin), a linear tetronate. This identification was corroborated by comparison of the published ultraviolet absorption spectrum and NMR spectra, which showed identical results (Table S4 and Figures S1-S5) [28,29]. The same feature (m/z 778.4738, [M + NH 4 + ]) was detected in the agar extract from A. napierensis' zone of growth inhibition during initial screening, providing additional evidence to corroborate its identification as the active antimycobacterial compound. The chromatogram and accurate masses of both tetromadurin features are displayed in Figure 8 and Table 2. The mass spectrum and annotated product ion spectrum of tetromadurin are displayed in Figures 9 and 10.      Tetromadurin is a type I polyketide polyether tetronate originally isolated from Actinomadura verrucosospora strain SF2487 (NRRL B-18236) [28,29]. Tetronates are compounds consisting of a linear fatty acid or polyketide chain and a tetronic acid ring [30]. Tetromadurin is structurally related to tetronasin and tetronomycin, all of which are characterised by possessing a cyclohexane ring, a tetrahydropyran ring and at least one tetrahydrofuran ring [28]. Tetromadurin is an ionophore that acts by disrupting chemical ion gradients, which inhibits primary cellular functions [31,32]. As electrochemical gradients are conserved in all organisms, ionophores tend to display a wide range of antimicrobial activity [24,33,34]. Tetromadurin is no exception and possesses antibacterial activity against  Tetromadurin is a type I polyketide polyether tetronate originally isolated from Actinomadura verrucosospora strain SF2487 (NRRL B-18236) [28,29]. Tetronates are compounds consisting of a linear fatty acid or polyketide chain and a tetronic acid ring [30]. Tetromadurin is structurally related to tetronasin and tetronomycin, all of which are characterised by possessing a cyclohexane ring, a tetrahydropyran ring and at least one tetrahydrofuran ring [28]. Tetromadurin is an ionophore that acts by disrupting chemical ion gradients, which inhibits primary cellular functions [31,32]. As electrochemical gradients are conserved in all organisms, ionophores tend to display a wide range of antimicrobial activity [24,33,34]. Tetromadurin is no exception and possesses antibacterial activity against Figure 10. The annotated high-resolution product ion spectrum of tetromadurin from liquid cultures of Actinomadura napierensis B60 T following bioassay-guided fractionation.
Tetromadurin is structurally related to tetronasin and tetronomycin, all of which are characterised by possessing a cyclohexane ring, a tetrahydropyran ring and at least one tetrahydrofuran ring [28]. Tetromadurin is an ionophore that acts by disrupting chemical ion gradients, which inhibits primary cellular functions [31,32]. As electrochemical gradients are conserved in all organisms, ionophores tend to display a wide range of antimicrobial activity [24,33,34]. Tetromadurin is no exception and possesses antibacterial activity against Gram-positive bacteria, antiplasmodial activity and antiretroviral activity [28,29,32]. As with many other ionophores, it has no effect against Gram-negative bacteria [24,29]. To our knowledge, tetromadurin has never been screened for antitubercular activity. Screening against M. tuberculosis H37Rv T in vitro revealed that tetromadurin has potent antitubercular activity, with MIC 90 s ranging from 73.7 to 151.6 nM (Table 3) under the different growth conditions. However, when screened for cytotoxicity, it displayed an IC 50 of 1.94 µM against the CHO cell line. The low selectivity indices suggest that tetromadurin is not selective enough for further study [26]. This is also commonly seen with ionophores, as few make suitable chemotherapeutics due to their broad activity profiles and indiscriminate mechanisms of action [31].

Conclusions
In conclusion, we were able to demonstrate that the agar overlay method combined with HPLC-MS/MS analysis is an effective tool to rapidly screen filamentous actinobacteria under different nutrient conditions. With a few additional steps, the zones of inhibition produced by active strains can be used to identify known compounds, streamlining the discovery process and prioritising producers of novel compounds. In the literature, the isolation of novel antitubercular compounds from filamentous actinobacteria is usually done by traditional bioassay-guided fractionation and elucidation by high-resolution mass spectrometry and NMR analysis. For example, dumulmycin was recently isolated from a river sediment Streptomyces strain and was shown to have strong antitubercular activity in vitro, with an MIC 50 of 27.1 µM [35]. However, few studies have utilised zones of growth inhibition to identify known compounds, and, to our knowledge, none have done so for antimycobacterial compounds or to the extent discussed in this investigation [21,36]. This method is limited by the databases used for dereplication and the size of the zone of growth inhibition. Smaller zones of inhibition may contain known antibacterial agents, which could go undetected if insufficient material is extracted. Excitingly, tetromadurin has been shown to be a novel antitubercular compound, and while in vitro screens suggest that it is not suitable for further study, it does represent a unique scaffold for derivation and medicinal chemistry studies. Ionophores show potential as novel chemotherapeutics; if they can be altered or dosed with other molecules to be more selective, they may represent a new frontier in drug development [31,37].
Finally, this study provides further evidence that South African filamentous actinobacteria are sources of potent antibacterial compounds. Further study should focus on additional screening programs and the isolation and identification of the active compounds in the other top strains from this investigation, namely S. africanus CPJVR-H T and Streptomyces strain HMC5.

Agar Overlay Assay and Zone of Inhibition Extraction
All filamentous actinobacteria were obtained from the Meyers Culture Collection in the Department of Molecular and Cell Biology, Faculty of Science, University of Cape Town, South Africa. These strains were isolated from a variety of terrestrial and aquatic environments in South Africa by members of Dr Meyers' research group.
One day prior to overlay, 4-5 loopfuls of Mycolicibacterium aurum strain A+ were inoculated into 10 mL of Middlebrook 7H9 supplemented with 0.05% glycerol and 0.02% Tween 80 (Merck) in a sterile universal tube and incubated at 30 • C with shaking at 100 rpm for 24 h. On the day of overlay, the M. aurum culture was streaked for single colonies on Middlebrook 7H9 and Gram-stained to ensure purity [42]. The optical density at 600 nm (OD 600 ) was measured and the culture volume required for each plate was determined using the following empirical formula: Volume × OD 600 = 160.
The calculated volume of M. aurum culture was then added to tubes of 6 mL 2 × yeast extract tryptone (2YT) [40] sloppy agar (0.7% agar), mixed and gently poured over each stab-inoculated plate to cover the entire plate, but not the actinobacterial colonies. The overlaid agar was allowed to set and the plates were incubated at 37 • C for 3 days. Activity was recorded if a zone of inhibition of the growth of M. aurum A+ was observed around an actinobacterial colony. For the initial screening, four strains were inoculated per plate. Any strains that showed activity were then stab-inoculated individually in the centre of an agar plate of the same medium, incubated and subjected to the overlay assay to confirm activity.
The agar within the zone of inhibition was then excised and added to a 50 mL Falcon tube before freezing at −20 • C. Two millilitres of methanol (MeOH) (>99%, Kimix Chemical and Lab Supplies, Cape Town, South Africa) and 40 mL of ethyl acetate (EtOAc) (≥98%, Merck) were added to each frozen tube and the agar pieces were allowed to thaw at room temperature while shaking at 90 rpm. The agar pieces were filtered from the solvent using two coffee filters (size 1 × 4, House of Coffees, Johannesburg, South Africa) and the solvent layer was washed with 40 mL of Millipore water in a separating funnel. Once the layers had separated, the solvent layer was collected and dried down in a fume hood.

Bacterial Cell Cultivation and Extraction
Strains that showed antimycobacterial activity by the overlay assay (and for which the active compound could not be identified) were grown in the growth media in which activity had been detected and extracted. Frozen actinomycete stock cultures (15% v/v glycerol) were thawed at room temperature and inoculated into 30 mL of liquid medium in a 250 mL Erlenmeyer flask and incubated for 7-10 days at 30 • C with shaking at 100 rpm.
Once sufficient cell mass had been produced, the culture was Gram-stained [42] and streaked for single colonies to determine purity. Whole culture extraction was achieved with the addition of 5 mL MeOH and 50 mL EtOAc. The culture and solvent were agitated for 2 h at 90 rpm at room temperature to assist extraction, before filtering the cell mass through two coffee filters and separating the organic and aqueous layers in a separating funnel. The organic layer was collected and dried down. The cell mass and aqueous layer were discarded.
The M. tuberculosis H37Rv T cultures were grown until an OD 600 of 0.6-0.7 was reached and then diluted 1:500. Next, 50 µL of the respective medium was added to each well in a 96-well microtitre plate. Test samples were diluted in dimethyl sulfoxide (DMSO) and 50 µL was added to Row 1. Two-fold serial dilutions were performed in 50 µL volumes down the rest of the plate to Row 10. Next, 50 µL of the diluted M. tuberculosis culture was added to each well. This generated a concentration test range of 62.5-0.12 µg/mL. Isoniazid and moxifloxacin were used as positive controls for antimycobacterial activity. The microtitre plate was sealed in a secondary container and incubated at 37 • C with 5% CO 2 and humidification for 7 days. Alamar Blue reagent was added to each well of the assay plate 24 h prior to the assay end, after which incubation of the microtitre plate was continued. Relative fluorescence (excitation 540 nm; emission 590 nm) was measured using a SpectraMax i3x Plate Reader at day 7. The raw fluorescent data (Relative Fluorescent Units) were acquired using a SpectraMax i3x Plate reader (Molecular Devices Corporation 1311 Orleans Drive Sunnyvale, CA 94089, USA). The Softmax ® Pro 6, 4-parameter curve fit protocol was used to calculate the MIC 90 s.

Cytotoxicity Testing
Cytotoxicity assays were conducted against the Chinese Hamster Ovary (CHO) cell line (ATCC CCL-61, strain CHO-K1) by the H3D Parasitology Unit at the University of Cape Town, South Africa. CHO cells were cultured in a medium consisting of 10% foetal calf serum (FCS, Celtic Molecular Diagnostics, Mowbray, South Africa), 45% Dulbecco's Modified Eagle's Medium (DMEM, Highveld Biologicals, Lyndhurst, South Africa) and 45% HAMSF12 medium (1:1; Sigma, St. Louis, MO, USA). Cells were grown at 37 • C in a humidified atmosphere of 5% CO 2 and maintained by passage. Cell lines were seeded in 96-well microtitre plates at 104 cells/well in cell medium. Once seeded, cells were incubated at 37 • C for 24 h before the test samples were added. Each sample was tested in triplicate at six concentration points (100, 10, 1, 0.1, 0.01 and 0.001 µg/mL). Emetine was used as the reference standard for cytotoxicity. Cell viability was determined using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described by Mosmann [46]. One hundred microliters of dimethyl sulfoxide (DMSO, Sigma-Aldrich, Johannesburg, South Africa) was added to dissolve the MTT formazan derivatives, and the plates were gently agitated for 2 min to ensure homogenous mixtures. Absorbance was measured at 540 nm using a ModulusTM II Microplate Reader (Turner BioSystems, Inc. Sunnyvale, CA, USA). The data were analysed by nonlinear regression analysis using the GraphPad PRISM version 4.00 program to determine the IC 50 of each compound.

Large-Scale Cultivation
Actinomadura napierensis B60 T [47] was cultured from frozen glycerol stocks (15% v/v) in 15 mL of ISP-2 liquid medium in a 250 mL Erlenmeyer flask. After 3 days of incubation at 30 • C with shaking, this seed culture was used to inoculate 100 mL ISP-2 in a 1 L Erlenmeyer flask. After 3 days of incubation at 30 • C with shaking, this second seed culture was used to inoculate 1 L ISP-2 in a 5 L Erlenmeyer flask. This culture was incubated for 14 days at 30 • C with shaking. The cell mass was filtered from the broth by vacuum filtration through glass wool and extracted with 350 mL of MeOH, followed by repeated extraction with 350 mL of EtOAc. The broth was extracted thrice with 350 mL EtOAc in a separating funnel. The organic layers were pooled and dried down in a fume hood.
Crude extracts were reconstituted in water/MeOH (50%) and fractioned using Phenomenex ® StrataTM X-33 µm Reverse Phase C18 SPE cartridges (200 mg, 3 mL) (Separations, Johannesburg, South Africa). A SPEEDISK ® 48 manifold was used to wash solvents and dissolved crude extract samples through the C18 cartridges under pressure. The C18 cartridges were equilibrated before use by washing 2 mL MeOH (99.9%, Honeywell, Johannesburg, South Africa) through each cartridge, followed by 2 mL Millipore water under pressure. The crude extracts were washed onto the cartridges, followed by MeOH and water in different concentrations, starting at 50% and increasing by 10% MeOH, with a final wash step of 100% MeOH. The second, third and fourth fractions (60-80%) were found to be active and were collected, pooled and dried under nitrogen at 37 • C.
The active compound of A. napierensis B60 T was identified following high-pressure liquid chromatography using a Shimadzu 20-A LC system couped to a CBM-40 diode array detector and FRC-10 fraction collector with a Kinetex ® C18 column (5 µm, 100 Å, 150 mm × 6 mm). The mobile phases that were used were 1 mM ammonium formate in water as the aqueous mobile phase and MeOH with 0.05% formic acid as the organic mobile phase. The flow rate used was 0.7 mL/min, with a total runtime of 60 min, starting at 30% organic and increasing to 95% over 45 min; this was held for 2 min and then decreased back to 30% organic to equilibrate the column for the next run. The column oven was set to 30 • C and the scanning range of the DAD was 190-400 nm. The peak was collected from 36.5 to 37 min.

HPLC-MS Analysis and Molecular Networking
High-pressure liquid chromatography linked to quadrupole time of flight (HPLC-qTOF) high-resolution mass spectrometry was performed using an AB Sciex ® X500R QTOF coupled to an AB Sciex ® Exion LC system. Spectral data were obtained using informationdependent acquisition (IDA) at a mass range of 50-2000 Da. All methods, batches and data were processed using OS Sciex ® v1.2. The declustering potential was 80 V, the curtain gas (N 2 ) was at 25 pounds per square inch (psi), the ion spray voltage was 5500 V, and the source temperature was 450 • C. Ion source gases 1 and 2 were at 45 and 55 psi, respectively. The collision energy was 10 eV for the MS scans and 20-50 eV for MS/MS scans. The IDA intensity threshold was 50 cycles per second. The aqueous mobile phase used was 1 mM ammonium formate in water, and the organic mobile phase was MeOH/0.5% formic acid. The method used a gradient starting at 2% organic and ending at 98% organic phase, with a flow rate of 700 µL/min and a run time of 35 min. A Kinetex ® C18 column (5 µm, 100 Å, 150 mm × 6 mm) with a column protector was used. All solvents were sonicated for 10 min before use to remove bubbles.