Encoding substrates with mass tags to resolve stereospecific reactions using Nimzyme

Article abstract of DOI:10.1002/rcm.6134

RATIONALE The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve stereospecific reactions. METHODS We utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric analysis was conducted using conventional MALDI-TOF instrumentation. RESULTS Stereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and products. Not only purified enzymes, but also crude cell lysates can be used in this assay. CONCLUSIONS The Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional annotation of a wide range of glycan-modifying enzymes. Copyright

Full text of DOI:10.1002/rcm.6134

Research Article
Received: 31 October 2011
Revised: 13 December 2011
Accepted: 18 December 2011
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2012, 26, 611–615
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6134
Encoding substrates with mass tags to resolve stereospecific
reactions using Nimzyme
Kai Deng1,2, Kevin W. George1, Wolfgang Reindl1,3, Jay D. Keasling1,4,5, Paul D. Adams1,5,
1,3
Taek Soon Lee1, Anup K. Singh1,2 and Trent R. Northen
*
*
1Joint BioEnergy Institute, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Emeryville, CA 94608, USA
2Biotechnology and Bioengineering Department, Sandia National Laboratories, Livermore, CA 94551, USA
3Department of Bioenergy/GTL & Structural Biology, Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley,
CA 94704, USA
4Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA
5Department of Bioengineering, University of California, Berkeley, CA 94720, USA
RATIONALE: The nanostructure-initiator mass spectrometry based enzyme assay (Nimzyme) provides a rapid method
for screening glycan modifying reactions. However, this approach cannot resolve stereospecific reactions which are
common in glycobiology and are typically assayed using lower-throughput methods (gas chromatography/mass
spectrometry (GC/MS) or liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis) often in conjunction
with stable isotopically labeled reactants. However, in many applications, library size necessitates the development of
higher-throughput screening approaches of stereospecific reactions from crude sample preparations. Therefore, here we test
the approach of utilizing Nimzyme linkers with unique masses to encode substrate identity such that this assay can resolve
stereospecific reactions.
METHODS: We utilize the nanostructure-initiator mass spectrometry (NIMS) enzyme assay in conjuction with an
accurate mass tagging approach where each reactant is tagged with a unique perfluoronated tail. Mass spectrometric
analysis was conducted using conventional MALDI-TOF instrumentation.
RESULTS: Stereospecific reaction pathways of three stereoisomers (maltose, lactose and cellobiose) to afford the same
product glucose were resolved simutaneously due to the presence of unique fluorous tags on both reactants and
products. Not only purified enzymes, but also crude cell lysates can be used in this assay.
CONCLUSIONS: The Nimzyme assay with accurate mass tagging provides a rapid method for screening for targeted
stereospecific reactions using mass spectrometry and may be useful for high-throughput screening and functional
annotation of a wide range of glycan-modifying enzymes. Copyright © 2012 John Wiley & Sons, Ltd.
In recent years accurate mass tagging strategy has grown in
popularity for the quantification of both small molecules[1]
and biological molecules, particularly proteins.[2] For
example, a mass tag is added to encode a peptide allowing
multiple samples to be pooled and analyzed simultaneously
to quantify relative expression of the peptide in various
samples (e.g. iTRAQ[3]). Previously, we reported that fluorous
tags can be utilized to perform enzyme assays on perfluori-
nated nanostructure-initiator mass spectrometry (NIMS)[4]
surfaces to distinguish between products with different
masses.[5] However, the highly degenerate mass of glycans
(primarily hexoses and pentoses) and structural diversity
due to glycosidic linkages, and range of branch points
severely constrain application of Nimzyme to glycobiology.[6]
Indeed, it is common to have two products with exactly the
same mass but from different structures, for example, the
beta-linked cellobiose vs. alpha-linked maltose. While tandem
mass spectrometry (MS/MS) can be used to distinguish
these substrates,[7] the products are identical and, hence,
one cannot use glucose abundance to determine conversion
of maltose vs. cellobiose. Clasically, stable isotopomers are
used to differentiate stereoselective enzyme reactions[8]
and enzyme inhibition.[9] More recently, isotope tagging of
glycans has been applied for quantitative and comparative
glycomis. For example, Bowman and Zaia[10] showed the
generation of tags that vary only in their isotope content
and the incorporation of these tags into glycans to study
multiple samples using mass spectrometry. Withers and
co-workers[11] developed an activity-based isotope-coded
affinity tagging strategy for studying activities of glycosi-
dases in biological systems. While similar approaches could
be used to construct stable isotopically labeled Nimzyme
substrates, a much more direct approach is to simply encode
the idendity of the substrate using the unique mass of the
fluorous linker.
Here we describe the use of accurate mass tags to encode
the identity of the substrate such that the mass spectrometry
imaging (MSI) characterization for the product mixture can
be used to simultaneously resolve the conversion of multiple
glycoside hydrolase reactions resulting in the same product
* Correspondence to: A. K. Singh or T. R. Northen, Joint
BioEnergy Institute, Physical Biosciences Division, Lawrence
Berkeley National Laboratory, Emeryville, CA 94608, USA.
E-mail: aksingh@sandia.gov; trnorthen@lbl.gov
Rapid Commun. Mass Spectrom. 2012, 26, 611–615
Copyright © 2012 John Wiley & Sons, Ltd.

K. Deng et al.
(glucose), as shown in Fig. 1. This approach is amenable to
high throughput taking advantage of the wide range of
commercially available fluorous linkers with unique mass.
Synthesis of maltose substrate S1
N,N’-Diisopropylcarbodiimide (10.2 mL, 0.066 mmol) and
1-hydroxybenzotriazole (10.1 mg, 0.066 mmol) were added
to a stirred solution of 5-aminopentyl-b-D-maltoside (28
mg, 0.066 mmol) and (S)-5-(3,3-dimethylguanidino)-2-((((4-
(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)benzyl)
oxy)carbonyl)amino)pentanoic acid (43 mg, 0.055 mmol) in
dimethylformamide (DMF) (2.0 mL) at room temperature, and
the reaction was kept stirring overnight. The mixture was
concentrated under reduced pressure, and the residue was
purified by flash column chromatography to provide maltose
EXPERIMENTAL
Organic synthesis
All chemicals were purchased as reagent grade and used
without further purification. Flash column chromatography
steps were performed on a CombiFlash Rf chromatography
system from Teledyne ISCO (Lincoln, NE, USA). Reactions
were monitored using analytical thin-layer chromatography
(TLC) in EM silica gel 60 F254 plates and/or by staining with
acidic ceric ammonium molybdate or ninhydrin. 1H NMR
and 13 C NMR spectra were recorded on a Bruker AVB-400
or AV-600 instrument.
The synthesis of maltose substrate S1 is shown below as a
representive example. Details about the synthesis of fluorous
mass tags, disaccharide-C5-NH2 and their coupling reactions
can be found in the Supporting Information.
1
substrate S1 (40 mg, 62% yield). H NMR (600 MHz, CD3OD)
d (ppm) 7.35–7.25 (m, 4H), 5.15 (d, J = 4.2 Hz, 1H), 5.07 (AB,
J = 12.6 Hz, 2H), 4.26 (d, J = 7.8 Hz, 1H), 4.10–4.05 (m, 1H),
3.92–3.86 (m, 2H), 3.85–3.75 (m, 4H), 3.72–3.58 (m, 4H), 3.57–3.49
(m, 2H), 3.44 (dd, J=9.6, 3.6 Hz, 1H), 3.38–3.34 (m, 1H),
3.28–3.17 (m, 4H), 3.02 (s, 6H), 2.93 (t, 8.4 Hz, 2H), 2.52–2.40
(m, 2H), 1.85–1.79 (m, 1H), 1.74–1.57 (m, 5H), 1.50–1.48
(m, 2H), 1.46–1.36 (2H). LRMS (NIMS) m/z calcd for
C43H58F17N5O14, 1191.37, found 1191.06.
For enzyme assay
Synthesis of mass tag-1 (S)-5-(3,3-dimethylguanidino)-2-
((((4-(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)-
benzyl)oxy)carbonyl)amino)pentanoic acid
a-1,4-Glucosidase and b-1,4-galactosidase were purchased
from Sigma and b-1,4-glucosidase was kindly provided by
Joshua Park (Joint Bioenergy Institute, Lawrence Berkeley
National Laboratory, Berkeley, CA, USA). Enzyme cocktail
(NS50012) was purchased from Novozymes.
An equimolar mixture of the three subtrates: maltose S1,
lactose S2 and cellobiose S3, were incubated with a-1,4-
glucosidase, b-1,4-galactosidase, b-1,4-glucosidase and an
enzyme cocktail. Assays were performed in triplicate with
one representative spectrum shown. Error bars represent the
standard deviation of three independent experiments.
Triethylamine (0.19 mL, 1.35 mmol) was added to
a
stirred solution of asymmetric dimethyl-arginine HCl salt
(125 mg, 0.450 mmol) and 2,5-dioxopyrrolidin-1-yl-
(3,3,4,4,5,5,6,6,7,7,8,8,9,9,10,10,10-heptadecafluorodecyl)benzyl
carbonate (380 mg, 0.550 mmol) in DMF (11.0 mL) under nitro-
ꢀ
gen at 0 C. Subsequently, the ice-bath was removed and the
reaction mixture was stirred at room temperature overnight.
The solvent was removed under reduced pressure and the
residue was purified by column chromatography (MeOH/
EtOAc/H2O = 5/3/0.1 to 5/3/1) to provide the desired mass
tag-1 (120 mg, 34% yield) as a white solid substance. 1H NMR
(600 MHz, CD3OD) d (ppm) 7.33 (d, J = 7.8 Hz, 2H), 7.27 (d,
J = 7.8 Hz, 2H), 5.06 (AB, J = 12.6 Hz, 2H), 4.07 (t, J = 6.0 Hz,
1H), 3.32–3.27 (m, 2H), 3.04 (s, 6H), 2.95–2.89 (m, 2H), 2.52–2.40
(m, 2H), 1.91–1.84 (m, 1H), 1.75–1.63 (m, 3H). LRMS (NIMS) m/z
calcd for C26H27F17N4O4, 782.18, found 782.21.
Strain construction
E. coli DH10B was used as a host for cloning and for
disaccharide-cleavage assays. All plasmids were prepared
based on the BglBrick standard.[12] Briefly, the BglBrick
cloning strategy uses four standardized restriction sites,
EcoRI and BglII as a ’prefix’ and BamHI and XhoI as a ’suffix’,
Figure 1. Resolving three stereospecific reactions with the same product using
fluorous mass tags.
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Rapid Commun. Mass Spectrom. 2012, 26, 611–615

Encoding substrates with mass tags using Nimzyme
exploiting the compatibility of BglII and BamHI for multi-
gene assemblies. The plasmid pBbE1k, containing a Ptrc
promoter, ColE1 ori, and kanamycin resistance (KmR)
cassette, served as a backbone for all constructs. a-1,4-
Glucosidase was synthesized from the coding sequence of
Geobacillus stearothermophilus ATCC12016 exo-a-1,4-glucosi-
dase (Accession No. D84648) and appended with the BglBrick
prefix and a suffix. b-1,4-Galactosidase was PCR-amplified
from E. coli DH1. b-1,4-Glucosidase was synthesized based
on the coding sequence of Cel3A from Cellvibrio japonicus
Ueda107 (Accession No. CP000934). Plasmid maps of one
(pKG001), two (pKG002), and three (pKG003) gene constructs
are provided below.
20 min in the case of pMaltase and pML. For pMLC, the
reaction mixture was incubated for 4 h at 37 ꢀC. After
cooling to room temperature, 1 mL of cold methanol was
added to quench the reaction. Subsequently, 0.25 mL of
the reaction mixture was spotted onto a NIMS surface
and analyzed.
Mass spectrometry experiments
All mass spectrometry experiments were performed on
MALDI mass spectrometer (4800 MALDI TOF/TOF mass
analyzer from Applied Biosystems; Foster City, CA, USA).
Details about the procedures can be found in a protocol paper
by Reindl et al.[14]
For disaccharide cleavage assays, cultures of DH10B[13]
containing appropriate plasmids were grown overnight in
LB containing 50 mg/L kanamycin. The following day,
cultures were diluted 1:100 into fresh LB Kan media and
induced with 500 mM IPTG at an optical density (OD) of 0.6.
After 4 h of induction, 5 mL of culture was harvested by
centrifugation (10 000 g for 10 min, 4 ꢀC) and resuspended
in 1.5 mL of cold 30 mM potassium phosphate buffer.
Suspensions were kept on ice and sonicated to achieve cell
lysis. Supernatants were harvested following centrifugation
(14 000 g for 20 min, 4 ꢀC) and used in cleavage assays. More
specifically, for pMaltase, pML and pMLC, 1.0 mL of
substrates mixture (containing 10 mM S1, S2 and S3) was
diluted with 19 mL of crude cell lysates prepared as shown
above. The resulting mixture was incubated at 37 ꢀC for
RESULTS AND DISCUSSIONS
Synthetic design of substrates
Two considerations guided the design of tags – (1) Tags must
have unique masses and (2) the tag should make the substrate
compatible with the perfluorinated surface used in NIMS.
The tags also include dimethylargine (DMA) to increase
ionization efficiency and a C5 spacer was used between the
fluorous tag and the glycan substrate to minimize steric
hindrance to enzyme binding. As shown in Fig. 2, three
stereoisomeric reactants were selected: a (1 ! 4) diglucose
Figure 2. Synthesis of substrates S1, S2, S3 by peptide coupling reaction.
Rapid Commun. Mass Spectrom. 2012, 26, 611–615
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K. Deng et al.
(maltose), b (1 ! 4) diglucose (cellobiose), and b (1 ! 4)
galactoglucose (note galactose and glucose differ by the
orientation of the hydroxy group at the C4 position of the
glucose). Although these three disaccharides have exactly
the same molecular weight, different mass tags were chosen
to distinguish them from each other. Unique substrates (S1,
S2, S3) were prepared by peptide coupling reaction of the
disaccharide-C5-NH2 with the corresponding mass tags.
Enzyme reactions using purified enzymes
Stereospecific enzymes were used to test the single-pot
specificity of the accurate fluorous mass tag strategy. In total
four enzymatic reactions were used to characterize specific-
ity. An equimolar mixture of the three subtrates: maltose
S1, lactose S2 and cellobiose S3 (Fig. 3), were incubated with
a-1,4-glucosidase, b-1,4-galactosidase, b-1,4-glucosidase and
Figure 3. Nimzyme assay for the parallel analysis of a-glucosidase, b-galactosidase, b-glucosidase
and an enzyme cocktail.
Figure 4. Nimzyme assay of crude cell lysates from plasmid-containing E. coli DH10B strains. Plasmid con-
structs (pKG001–003) contained a-glucosidase (blue), b-galactosidase (red), and b-glucosidase (green).
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Rapid Commun. Mass Spectrom. 2012, 26, 611–615

Encoding substrates with mass tags using Nimzyme
an enzyme mix. Upon incubation with a-1,4-glucosidase,
only the maltose substrate S1 was converted into the glucose
product with the corresponding fluorous tag F1 (Fig. 3(a)).
As expected, for the b-1,4-galactosidase and b-1,4-glucosidase
reactions, only lactose S2 or cellobiose S3 was hydrolyzed
to products with the corresponding unique fluorous tags
F2 or F3 (Figs. 3(b) and 3(c)). Treatment with the enzyme
mixture converted all reactants into three different glucose
products easily resolvable by mass spectroscopy (Fig. 3(d)).
Note that the data is collected simultaneously for the three
reactions, typically using 25 laser shots accumulated in
<1 s.
Acknowledgements
This work was part of the DOE Joint BioEnergy Institute
(http://www.jbei.org) supported by the U. S. Department
of Energy, Office of Science, Office of Biological and Environ-
mental Research, through contract DE-AC02-05CH11231
between Lawrence Berkeley National Laboratory and the U.
S. Department of Energy. Sandia is a multiprogram labora-
tory operated by Sandia Corp., a Lockheed Martin company,
for the U.S. Department of Energy’s Nuclear Security Admin-
istration under Contract DE-AC04-94AL85000.
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To demonstrate the utility of this strategy for in vivo ana-
lyses, plasmids which coded for a-1,4-glucosidase (maltase),
b-1,4-galactosidase (lactase), and b-1,4-glucosidase (cellulase)
were constructed and transformed into E. coli DH10B. Speci-
fically, three BglBrick plasmids (pKG001–003) encoding for
one, two, or three of the hydrolytic enzymes under a single
inducible promoter were utilized. Following induction,
whole-cell lysates were generated by sonication and incu-
bated with equimolar concentrations of maltose S1, lactose
S2, and cellobiose S3 as described above. As expected, each
reaction resulted in the conversion of the tagged glycans into
their corresponding fluorous products (Fig. 4). Whereas the
lysates from strains harboring pKG001 and pKG002 cata-
lyzed the complete and rapid hydrolysis of the tagged sub-
strates within 20 min, the activities from pKG003-containing
strains were generally weaker, requiring substantially longer
incubation times to completely hydrolyze the substrates.
Indeed, even after 4 h, residual amounts of lactose (S2) were
present, despite its rapid cleavage in pKG002-containing
strains.
In conclusion, we have developed a method based on the
application of mass tags to differentiate between stereospecific
reactions. This method is broadly applicable for the analysis
of both purified enzymes and crude cell lysates. Since the
possibility of changing the chemical structure, spacer length,
and fluorous content of the mass tag cover a large chemical
space, this method could provide for a generic method for
the construction of an enzyme substrate (glycans) library,
which would aid in the screening and functional annotation
of a wide range of glycan-modifying enzymes. While the
focus of this study is on glucosyl hydrolytic reactions, in
principle it should be applicable to a wide range of
reactions, particularly those where fluorous phase chemistry
is already used.
SUPPORTING INFORMATION
Additional supporting information may be found in the
online version of this article.
Rapid Commun. Mass Spectrom. 2012, 26, 611–615
Copyright © 2012 John Wiley & Sons, Ltd.
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