Si 2017
Si 2017
Si 2017
pubs.acs.org/JACS
■ INTRODUCTION
Mass spectrometry (MS) has become an invaluable analytical
reactions, such as enzyme complex formation, temporal and
stoichiometric control to avoid intermediate build-up, and the
technique, in part because it offers label-free analyses of target need to fine-tune reaction conditions to be compatible with
molecules. Matrix-assisted laser desorption/ionization time-of- multiple enzymes. This may explain the limited use of MSI-
flight (MALDI-ToF) MS is particularly well suited for the rapid based screening of multistep biochemical reactions. To
inspection of a large number of biological samples because of its engineer a multistep reaction, modified intermediates must be
simple sample preparation, high salt tolerance, and wide accepted at each step of the catalytic sequence to obtain a final
coverage of diverse biomolecules.1,2 Accordingly, MALDI mass product. Engineering an individual step in isolation ignores
spectrometry imaging (MSI) has been increasingly applied for possible downstream effects, and MSI screening platforms
the rapid profiling of enzymatic reactions.3−8 However, MALDI primarily designed for single-step enzymatic reactions may be
MSI uses fixed raster steps for sampling, which requires high- ill-suited for engineering multistep pathways. Multistep biosyn-
density deposition of reaction components in a regular array.6−8 thesis is vital for the production of many important molecules,
Although up to three enzymes with no cross-activities have including fuels, fine chemicals, and pharmaceuticals,12,13 driving
been multiplexed in MSI screening,7,9−11 it is technically the need to develop improved methods to screen multistep
demanding to colocalize multiple enzymes, requiring advanced enzymatic reactions.
liquid handling, such as acoustic deposition,7 to increase the
throughput. Compared with multiplexed enzyme assays, there Received: May 8, 2017
are further challenges associated with screening multistep Published: August 9, 2017
Recombinant variants of a multienzymatic pathway are In both cases, high-dimensional data are reduced to low-
constructed as plasmid DNA libraries, which are used to dimensional variates to reflect a target phenotype, such as
transform a production host such as Escherichia coli (Figure spectral classifications or congener ratios. The reduced data sets
1A). The transformants are plated on a filter membrane for are then visualized by overlaying optical images with false color
straightforward manipulation of many colonies simultane- markers to produce straightforward readouts similar to
ously,36 such as exchanging culture media or imprinting onto colorimetric assays (Figure 1D). Notably, as m/z values in
MALDI targets. Microbial cells are initially cultivated on the MALDI-ToF mass spectra are utilized in the computational
noninducing agar media to obtain individual colonies and then analyses to link mass spectral signals to target compounds,
transferred onto inducing plates to initiate enzyme expression analyte assignments are further confirmed using high-resolution
and target molecule production (Figure 1A). Each clonal MS and tandem MS (MS/MS). These analyses can be
population contains a single variant of the multistep pathway. performed subsequent to high-throughput profiling, as more
Separation of the induction phase from the growth phase allows than 60% of the analytes remain on the target even after
accumulation of sufficient biomass before cellular resources are exhaustive MALDI-ToF MS measurements.40 This two-tiered
diverted to target enzyme production, and minimizes uneven approach allows rapid survey of the whole library, which helps
growth among mutant colonies if the final products affect to identify colonies of interest for slower but more informative
cellular fitness. MS analyses. Finally, select colonies are recovered for further
For MALDI-ToF MS analyses, colonies are imprinted on characterization, such as DNA sequencing or liquid fermenta-
conductive, indium tin oxide (ITO)-coated glass slides (Figure tion (Figure 1D).
1B). Relative to agar bacterial cultures mounted directly onto a Substrate Libraries of a Peptidic NP. We first applied the
MALDI target, profiling from imprinted biomass on a uniform workflow to survey the structural diversity of PZN 1 analogues,
target surface obtains better ion signal for cell-associated created using a substrate library. Ribosomally synthesized and
compounds.37 The use of transparent MALDI targets allows post-translationally modified peptides (RiPPs) form a major
acquisition of optical images prior to matrix application using class of NPs.41,42 As the product is synthesized from a
an artist’s airbrush (Figure 1C). To aid automated colony ribosomal peptide, product variants can be generated by
finding, whole-slide images are acquired to locate the etched mutagenesis of the precursor gene.43,44 PZN 1 is a member of
fiducials and imprinted colonies in bright-field and autofluor- one RiPP subclass termed the linear azol(in)e-containing
escence images (Figure 1C). At least 16 fiducial markers were peptides. During biosynthesis of this subclass, a trimeric
etched directly on the ITO-coated slide in the area surrounding heterocycle synthetase converts select cysteine (C), serine
the imprint region (Figure 1B) using a diamond-tipped pen. A (S), and threonine (T) residues in the C-terminal (core) region
target accuracy of ∼20 μm during MALDI-ToF MS profiling of the precursor peptide to thiazol(in)e and (methyl)oxazol-
can be achieved following fiducial training.38 A Python platform (in)e heterocycles (Scheme S1). PZN 1 is naturally produced
for image-guided MS analyses, microMS,38 is used to generate by Bacillus velezensis FZB4245 and exhibits remarkable
MALDI laser coordinates for automatic colony profiling antibacterial selectivity against Bacillus anthracis,46 the causative
(Figure 1C). Laser shots are patterned around the peripheries agent of anthrax. We previously achieved heterologous
of the imprinted colonies for optimal sensitivity, as described production of PZN 1 in E. coli using a fosmid bearing the
below. corresponding biosynthetic gene cluster.47 Analogues of PZN 1
The resulting mass spectra are processed using multivariate were also created by site-directed mutagenesis of the precursor
statistical analyses. For a strain library producing analogues of a peptide gene, followed by liquid cultivation and methanol
target compound, t-distributed stochastic neighbor embedding extraction before MS analyses.47 For successful synthesis of an
(t-SNE)39 was utilized to visualize spectral similarity and analogue of PZN 1, a mutant precursor peptide must be
identify nonisobaric variants (Figure 1D). t-SNE is a accepted as a substrate by multiple enzymes of the biosynthetic
dimensionality reduction method, similar to principle compo- pathway, including the cyclodehydratase, dehydrogenase, leader
nent analysis (PCA), for the visualization of high-dimensional peptidase, and methyltransferase (Scheme S1).47
data sets.39 While PCA is commonly utilized to separate To apply optically guided MALDI-ToF MS screening to
dissimilar data within a low-dimensional map, t-SNE also keeps E. coli colonies producing PZN 1 analogues, we targeted two
similar data close together. As such, we found clearer grouping noncyclized positions, I7 and I8 (Scheme 1, red; the numbering
of spectra within a t-SNE plot than a PCA score plot. The for original core residues is given in Scheme S1), where
observed grouping is advantageous for spectral data with mutations are relatively tolerated by the native biosynthetic
multiple classes, such as those acquired from a compound machinery.47 Site-saturation mutagenesis was performed using
analogue library. Alternatively, when screening a strain library degenerate codon (NNK)-containing primers. Polyclonal
producing the same set of products at different ratios, ion plasmid DNA was used to transform competent E. coli cells
intensities at select m/z values are extracted to calculate the harboring a refactored version of the PZN cluster, where native
total and relative abundances. Bacillus promoters were replaced with a strong T7 promoter to
12468 DOI: 10.1021/jacs.7b04641
J. Am. Chem. Soc. 2017, 139, 12466−12473
Journal of the American Chemical Society Article
enhance production. Isopropyl β-D-1-thiogalactopyranoside (Figure S1), respectively. However, further analyses are
(IPTG) was used to induce production of PZN 1 on M9 required to differentiate the single-residual mutations between
medium containing acetate as the sole carbon source. glutamine (Q) and lysine (K), as well as between leucine (L)
For I7 and I8 libraries, 352 and 393 colonies were screened, and isoleucine (I). It is challenging to distinguish the 0.036 Da
respectively, achieving a > 99.9% probability of full coverage of mass difference between the Q and K mutations, as an m/z
the NNK libraries.48 Following the analysis workflow, we first tolerance of ±0.25 Da was employed to assign residue
performed t-SNE analyses for unsupervised clustering of the substitutions of PZN analogues from the MALDI-ToF MS
resulting mass spectra, utilizing each binned m/z value to data. Such mass accuracy results from the limited mass
evaluate population heterogeneity and variance. We manually resolution of a ToF mass analyzer (∼10 000 in a m/z window
examined each spectral class for tentative PZN peaks, and of 1100−1600 for PZN molecules), and uneven sample heights
found all the base peaks (Figure S1) were consistent with of imprinted biomass that affect the time-of-flight of target ions
single-residual-mutation analogues with “wild-type-like” mod- and reduce mass accuracy. As noted above, after MALDI-ToF
ifications: nine azole rings, one azoline ring, leader peptidolysis MS profiling, enough of the analytes remain on the targets,40
N-terminal to arginine R1, and N-terminal dimethylation allowing follow-up analyses. High-resolution MALDI-Fourier
(Scheme 1).47 A targeted clustering (Figure 2) was then transform ion cyclotron resonance (FT-ICR) MS analyses were
performed using the predicted m/z values of these analogues performed on selected colonies of interest, and unambiguously
differentiated between Q and K mutations, revealing both K
(see SI “Multivariate data analysis” for details) (Figure S1).
and Q substitutions at I7, but only Q at I8 (Figure S1). The
From the MALDI-ToF MS data alone, we observed 12 and 9
high-resolution MS data also corroborated the predicted
variant classes of PZN 1 for the libraries of I7 (Figure 2) and I8
molecular formulas of other PZN analogues with <1 ppm
mass errors (Figure S1)
On the other hand, as the PZN analogues with I and L
substitutions are isomers with no mass differences, DNA
sequencing is necessary to differentiate these two mutants
(Figure S2). Colonies belonging to each class were inoculated
in liquid cultures for plasmid isolation and DNA sequencing.
Each colony sequenced presented mutations consistent with
predicted and observed mass shifts in the base peaks assuming
full maturation (Figure S1). In this study, all 13 previously
isolated PZN analogues with single residue mutations at I7 or
I8 were detected,47 as well as 10 previously unreported variants
(Figure S1). Select analogues with sufficient residual analytes
on the sample target were subjected to in situ ion identification
with MS/MS (Figures S2−S5). The tandem mass spectra
suggested “wild-type-like” modifications (Scheme 1) for all
examined base peaks (Figures S2−S5). The detection of
unreported PZN analogues in this study validates the improved
methodology. Production of PZN 1 was increased through
pathway refactoring and growth medium optimization, enabling
observation of PZN variants not previously detected due to
insufficient amounts. Also, enhanced production allowed
detection of PZN 1 analogues directly from single colonies.
This improvement was leveraged by optically guided MALDI-
ToF MS to substantially increase analytical throughput,
allowing more comprehensive codons (NNK versus NNC47)
for mutagenesis while retaining high probabilities of full library
coverage.
In addition to spectral classes containing base peaks
matching predicted m/z values of PZN analogues, a class
exhibiting a low signal-to-noise ratio or chemical background
was also observed (Figure 2). This spectral class likely resulted
either from (1) mutations not tolerated by the biosynthetic
machinery or reducing analogue production below our
detection limit, (2) the UAG stop codon contained in the
degenerate NNK codon, (3) artifacts during optical image
acquisition such as dust, and/or (4) problems targeting
Figure 2. Multivariate analysis of PZN analogues. (A) Visualization irregularly shaped colony imprints (Figure 3B). The first two
with targeted t-SNE clustering of the I7 library from a single
possibilities were not further investigated given the consistency
experiment. Each point corresponds to a single mass spectrum, with
each cluster surrounded by a 95% confidence ellipsoid. The N/A between current and previous results.47 The latter two can be
cluster contains spectra without observable peptide signals. The alleviated by more vigilant colony finding and target patterning.
position of (B) each mutant or (C) N/A colony is mapped onto the In particular, we found the best sensitivity was obtained when
optical image to aid mutant recovery. The three colonies highlighted in directing the MALDI laser to the peripheries of the imprinted
panel B are displayed in more detail in Figure 3. biomass (Figure 3). Direct sampling on the imprinted biomass
12469 DOI: 10.1021/jacs.7b04641
J. Am. Chem. Soc. 2017, 139, 12466−12473
Journal of the American Chemical Society Article
Scheme 2. Biosynthesis of RL 5
to specific target analyte classes. For example, matrix Huimin Zhao: 0000-0002-9069-6739
application by sublimation offers a solvent-free preparation to Jonathan V. Sweedler: 0000-0003-3107-9922
minimize detection of intracellular metabolites. Alternatively, Author Contributions
the geometry of MALDI laser shots is easily modified to ¶
T.S., B.L., and T.J.C. contributed equally.
examine the interior regions of imprinted biomass to enhance
detection of intracellular metabolites (provided there is Notes
sufficient solvent extraction and matrix crystallization on top The authors declare no competing financial interest.
of the biomass). Finally, colonies grown on thin agar may be
mounted directly on a MALDI target to acquire mass spectra
from areas surrounding colonies to improve detection of
■ ACKNOWLEDGMENTS
We gratefully acknowledge financial support from the National
secreted compounds.49
■
Institutes of Health (GM077596 to H.Z., AI113219 to J.V.S.,
and GM097142 to D.A.M.). J.V.S. also acknowledges NSF
CONCLUSIONS CHE 16-067915. T.S. acknowledges postdoctoral fellowship
We have developed an integrated workflow for high-throughput support from the Carl R. Woese Institute for Genomic Biology
screening of multistep enzymatic reactions in bacterial colonies. (UIUC). T.J.C. acknowledges support from an NSF Graduate
Traditional screening methods are either limited to photo- Research Fellowship Program, the Springborn Fellowship, and
metrically active molecules and labeled surrogates, or require the Training Program at the Chemistry-Biology Interface (T32
low-throughput chromatographic separation.12 MS provides a GM070421). We thank Prof. Joshua D. Shrout at the
label-free, highly sensitive platform for monitoring products, University of Notre Dame for kindly providing P. aeruginosa
reactants, and byproducts with high accuracy. Incorporating genomic DNA. We thank Elizabeth Neumann for the help with
machine vision and automatic target patterning greatly microscopy and FT-ICR analyses, Sage J.B. Dunham for
improves MS acquisition efficiency over traditional MSI assays, initiating the rhamnolipid engineering project, and Adam J.
especially for randomly distributed colonies. The resulting mass DiCaprio with the construction of PZN analogue libraries. We
spectra data sets may be subjected to multivariate clustering or also thank Dr. Benjamin Bowen at the Lawrence Berkeley
reduced into univariate plots to quickly assess and select National Lab for the help with mass spectrum deposition at
mutants with desirable phenotypes. Moreover, the capacity to OpenMSI.
rapidly survey the molecular contents of a whole library
provides new biological insights, such as the overall substrate
tolerance of a biosynthetic pathway and the trade-off between
■ REFERENCES
(1) Bothner, B.; Chavez, R.; Wei, J.; Strupp, C.; Phung, Q.;
phenotypic gain and further evolvability during the course of Schneemann, A.; Siuzdak, G. J. Biol. Chem. 2000, 275, 13455−13459.
directed protein evolution. Additional improvements may be (2) Greis, K. D. Mass Spectrom. Rev. 2007, 26, 324−339.
achieved through increased throughput, automatic imprinting/ (3) Northen, T. R.; Lee, J.-C.; Hoang, L.; Raymond, J.; Hwang, D.-R.;
matrix coating to enhance sample uniformity, and derivatization Yannone, S. M.; Wong, C.-H.; Siuzdak, G. Proc. Natl. Acad. Sci. U. S. A.
of analytes with poor native MALDI-ToF MS sensitivity. The 2008, 105, 3678−3683.
optically guided approach may also be incorporated when (4) Ban, L.; Pettit, N.; Li, L.; Stuparu, A. D.; Cai, L.; Chen, W.; Guan,
profiling randomly distributed colonies using other surface W.; Han, W.; Wang, P. G.; Mrksich, M. Nat. Chem. Biol. 2012, 8, 769−
773.
analysis approaches, such as DESI,15−17 which provide
(5) Heins, R. A.; Cheng, X.; Nath, S.; Deng, K.; Bowen, B. P.;
complementary molecular coverage and analytical capabilities Chivian, D. C.; Datta, S.; Friedland, G. D.; D’Haeseleer, P.; Wu, D.;
relative to MALDI-ToF MS, as in real-time monitoring of living Tran-Gyamfi, M.; Scullin, C. S.; Singh, S.; Shi, W.; Hamilton, M. G.;
cells. Given its simplicity and effectiveness, this workflow Bendall, M. L.; Sczyrba, A.; Thompson, J.; Feldman, T.; Guenther, J.
should be applicable to a wide range of multistep enzyme M.; Gladden, J. M.; Cheng, J. F.; Adams, P. D.; Rubin, E. M.;
reactions and facilitate high-throughput screening of microbial Simmons, B. A.; Sale, K. L.; Northen, T. R.; Deutsch, S. ACS Chem.
libraries. Biol. 2014, 9, 2082−2091.
■
(6) Gurard-Levin, Z. A.; Scholle, M. D.; Eisenberg, A. H.; Mrksich,
ASSOCIATED CONTENT M. ACS Comb. Sci. 2011, 13, 347−350.
(7) Greving, M.; Cheng, X.; Reindl, W.; Bowen, B.; Deng, K.; Louie,
*
S Supporting Information
K.; Nyman, M.; Cohen, J.; Singh, A.; Simmons, B.; Adams, P.; Siuzdak,
The Supporting Information is available free of charge on the G.; Northen, T. Anal. Bioanal. Chem. 2012, 403, 707−711.
ACS Publications website at DOI: 10.1021/jacs.7b04641. (8) de Rond, T.; Danielewicz, M.; Northen, T. Curr. Opin. Biotechnol.
Experimental details on the DNA and strain con- 2015, 31, 1−9.
(9) Smith, A. M. E.; Brennan, J. D. ChemBioChem 2014, 15, 587−
struction, the acquisition, processing and visualization 594.
of MALDI-ToF MS screening data sets, Table S1, (10) Rathore, R.; Pribil, P.; Corr, J. J.; Seibel, W. L.; Evdokimov, A.;
Scheme S1, and Figures S1−S8 (PDF) Greis, K. D. J. Biomol. Screening 2010, 15, 1001−1007.
■
(11) Reindl, W.; Deng, K.; Gladden, J. M.; Cheng, G.; Wong, A.;
AUTHOR INFORMATION Singer, S. W.; Singh, S.; Lee, J. C.; Yao, C. H.; Hazen, T. C.; Singh, A.
K.; Simmons, B. A.; Adams, P. D.; Northen, T. R. Energy Environ. Sci.
Corresponding Authors 2011, 4, 2884−2893.
*[email protected] (12) Dietrich, J. A.; McKee, A. E.; Keasling, J. D. Annu. Rev. Biochem.
*[email protected] 2010, 79, 563−590.
ORCID (13) Du, J.; Shao, Z.; Zhao, H. J. Ind. Microbiol. Biotechnol. 2011, 38,
873−890.
Tong Si: 0000-0003-2985-9014 (14) Yan, C.; Parmeggiani, F.; Jones, E. A.; Claude, E.; Hussain, S. A.;
Troy J. Comi: 0000-0002-3215-4026 Turner, N. J.; Flitsch, S. L.; Barran, P. E. J. Am. Chem. Soc. 2017, 139,
Douglas A. Mitchell: 0000-0002-9564-0953 1408−1411.
(15) Song, Y.; Talaty, N.; Tao, W. A.; Pan, Z.; Cooks, R. G. Chem. Patchett, M. L.; Piel, J.; Reaney, M. J.; Rebuffat, S.; Ross, R. P.; Sahl, H.
Commun. (Cambridge, U. K.) 2007, 61−63. G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen,
(16) Song, Y.; Talaty, N.; Datsenko, K.; Wanner, B. L.; Cooks, R. G. K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang, G. L.;
Analyst 2009, 134, 838−841. Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S.
(17) Roach, P. J.; Laskin, J.; Laskin, A. Analyst 2010, 135, 2233− C.; Willey, J. M.; van der Donk, W. A. Nat. Prod. Rep. 2013, 30, 108−
2236. 160.
(18) Masyuko, R. N.; Lanni, E. J.; Driscoll, C. M.; Shrout, J. D.; (43) Young, T. S.; Dorrestein, P. C.; Walsh, C. T. Chem. Biol. 2012,
Sweedler, J. V.; Bohn, P. W. Analyst 2014, 139, 5700−5708. 19, 1600−1610.
(19) Lanni, E. J.; Masyuko, R. N.; Driscoll, C. M.; Aerts, J. T.; Shrout, (44) Ruffner, D. E.; Schmidt, E. W.; Heemstra, J. R. ACS Synth. Biol.
J. D.; Bohn, P. W.; Sweedler, J. V. Anal. Chem. 2014, 86, 9139−9145. 2015, 4, 482−492.
(20) Si, T.; Li, B.; Zhang, K.; Xu, Y.; Zhao, H.; Sweedler, J. V. J. (45) Scholz, R.; Molohon, K. J.; Nachtigall, J.; Vater, J.; Markley, A.
Proteome Res. 2016, 15, 1955−1962. L.; Sussmuth, R. D.; Mitchell, D. A.; Borriss, R. J. Bacteriol. 2011, 193,
(21) Yang, Y. L.; Xu, Y.; Straight, P.; Dorrestein, P. C. Nat. Chem. 215−224.
Biol. 2009, 5, 885−887. (46) Molohon, K. J.; Blair, P. M.; Park, S.; Doroghazi, J. R.; Maxson,
(22) Dunham, S. J.; Ellis, J. F.; Li, B.; Sweedler, J. V. Acc. Chem. Res. T.; Hershfield, J. R.; Flatt, K. M.; Schroeder, N. E.; Ha, T.; Mitchell, D.
2017, 50, 96−104. A. ACS Infect. Dis. 2016, 2, 207−220.
(23) Watrous, J. D.; Dorrestein, P. C. Nat. Rev. Microbiol. 2011, 9, (47) Deane, C. D.; Melby, J. O.; Molohon, K. J.; Susarrey, A. R.;
683−694. Mitchell, D. A. ACS Chem. Biol. 2013, 8, 1998−2008.
(24) Wakeman, C. A.; Moore, J. L.; Noto, M. J.; Zhang, Y.; Singleton, (48) Nov, Y. Appl. Environ. Microbiol. 2012, 78, 258−262.
M. D.; Prentice, B. M.; Gilston, B. A.; Doster, R. S.; Gaddy, J. A.; (49) Yang, J. Y.; Phelan, V. V.; Simkovsky, R.; Watrous, J. D.; Trial, R.
Chazin, W. J.; Caprioli, R. M.; Skaar, E. P. Nat. Commun. 2016, 7, M.; Fleming, T. C.; Wenter, R.; Moore, B. S.; Golden, S. S.; Pogliano,
11951. K.; Dorrestein, P. C. J. Bacteriol. 2012, 194, 6023−6028.
(25) Zackular, J. P.; Moore, J. L.; Jordan, A. T.; Juttukonda, L. J.; (50) Howe, J.; Bauer, J.; Andra, J.; Schromm, A. B.; Ernst, M.; Rossle,
Noto, M. J.; Nicholson, M. R.; Crews, J. D.; Semler, M. W.; Zhang, Y.; M.; Zahringer, U.; Rademann, J.; Brandenburg, K. FEBS J. 2006, 273,
Ware, L. B.; Washington, M. K.; Chazin, W. J.; Caprioli, R. M.; Skaar, 5101−5112.
E. P. Nat. Med. 2016, 22, 1330−1334. (51) Dobler, L.; Vilela, L. F.; Almeida, R. V.; Neves, B. C. New
(26) Romero, P. A.; Arnold, F. H. Nat. Rev. Mol. Cell Biol. 2009, 10, Biotechnol. 2016, 33, 123−135.
866−876. (52) Muller, M. M.; Kugler, J. H.; Henkel, M.; Gerlitzki, M.;
(27) Macarron, R.; Banks, M. N.; Bojanic, D.; Burns, D. J.; Cirovic, Hormann, B.; Pohnlein, M.; Syldatk, C.; Hausmann, R. J. Biotechnol.
D. A.; Garyantes, T.; Green, D. V.; Hertzberg, R. P.; Janzen, W. P.; 2012, 162, 366−380.
Paslay, J. W.; Schopfer, U.; Sittampalam, G. S. Nat. Rev. Drug Discovery (53) Zhu, K.; Rock, C. O. J. Bacteriol. 2008, 190, 3147−3154.
2011, 10, 188−195. (54) Abdel-Mawgoud, A. M.; Lepine, F.; Deziel, E. Appl. Microbiol.
(28) Ong, T. H.; Kissick, D. J.; Jansson, E. T.; Comi, T. J.; Biotechnol. 2010, 86, 1323−1336.
Romanova, E. V.; Rubakhin, S. S.; Sweedler, J. V. Anal. Chem. 2015, (55) Han, L.; Liu, P.; Peng, Y.; Lin, J.; Wang, Q.; Ma, Y. J. Appl.
87, 7036−7042. Microbiol. 2014, 117, 139−150.
(29) Jansson, E. T.; Comi, T. J.; Rubakhin, S. S.; Sweedler, J. V. ACS (56) Cabrera-Valladares, N.; Richardson, A. P.; Olvera, C.; Trevino,
Chem. Biol. 2016, 11, 2588−2595. L. G.; Deziel, E.; Lepine, F.; Soberon-Chavez, G. Appl. Microbiol.
(30) Do, T. D.; Comi, T. J.; Dunham, S. J.; Rubakhin, S. S.; Sweedler, Biotechnol. 2006, 73, 187−194.
J. V. Anal. Chem. 2017, 89, 3078−3086. (57) Zhao, H.; Chockalingam, K.; Chen, Z. Curr. Opin. Biotechnol.
(31) Comi, T. J.; Do, T. D.; Rubakhin, S. S.; Sweedler, J. V. J. Am. 2002, 13, 104−110.
Chem. Soc. 2017, 139, 3920−3929. (58) Wang, M.; Si, T.; Zhao, H. Bioresour. Technol. 2012, 115, 117−
(32) Rodrigues, T.; Reker, D.; Schneider, P.; Schneider, G. Nat. 125.
Chem. 2016, 8, 531−541. (59) Turner, N. J. In Enzyme Assays; Wiley-VCH Verlag GmbH &
(33) Newman, D. J.; Cragg, G. M. J. Nat. Prod. 2012, 75, 311−335. Co. KGaA, 2006; pp 137−161.
(34) Kim, E.; Moore, B. S.; Yoon, Y. J. Nat. Chem. Biol. 2015, 11,
649−659.
(35) Evans, B. S.; Chen, Y.; Metcalf, W. W.; Zhao, H.; Kelleher, N. L.
Chem. Biol. 2011, 18, 601−607.
(36) Cornvik, T.; Dahlroth, S. L.; Magnusdottir, A.; Herman, M. D.;
Knaust, R.; Ekberg, M.; Nordlund, P. Nat. Methods 2005, 2, 507−509.
(37) Watrous, J.; Hendricks, N.; Meehan, M.; Dorrestein, P. C. Anal.
Chem. 2010, 82, 1598−1600.
(38) Comi, T. J.; Neumann, E. K.; Do, T. D.; Sweedler, J. V. J. Am.
Soc. Mass Spectrom. 2017, 28, 1919.
(39) van der Maaten, L.; Hinton, G. J. Mach. Learn. Res. 2008, 9,
2579−2605.
(40) Page, J. S.; Sweedler, J. V. Anal. Chem. 2002, 74, 6200−6204.
(41) Skinnider, M. A.; Johnston, C. W.; Edgar, R. E.; Dejong, C. A.;
Merwin, N. J.; Rees, P. N.; Magarvey, N. A. Proc. Natl. Acad. Sci. U. S.
A. 2016, 113, E6343−E6351.
(42) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.; Bugni,
T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G. L.;
Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.;
Donadio, S.; Dorrestein, P. C.; Entian, K. D.; Fischbach, M. A.;
Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.;
Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars,
M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.;
Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller, R.;
Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.;