Labeling Preferences of Diazirines with Protein Biomolecules

Article abstract of DOI:10.1021/jacs.1c02509

Diazirines are widely used in photoaffinity labeling (PAL) to trap noncovalent interactions with biomolecules. However, design and interpretation of PAL experiments is challenging without a molecular understanding of the reactivity of diazirines with protein biomolecules. Herein, we report a systematic evaluation of the labeling preferences of alkyl and aryl diazirines with individual amino acids, single proteins, and in the whole cell proteome. We find that alkyl diazirines exhibit preferential labeling of acidic amino acids in a pH-dependent manner that is characteristic of a reactive alkyl diazo intermediate, while the aryl-fluorodiazirine labeling pattern reflects reaction primarily through a carbene intermediate. From a survey of 32 alkyl diazirine probes, we use this reactivity profile to rationalize why alkyl diazirine probes preferentially enrich highly acidic proteins or those embedded in membranes and why probes with a net positive charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling experiments with diazirines.

Full text of DOI:10.1021/jacs.1c02509

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Labeling Preferences of Diazirines with Protein Biomolecules
Alexander V. West, Giovanni Muncipinto, Hung-Yi Wu, Andrew C. Huang, Matthew T. Labenski,
Lyn H. Jones, and Christina M. Woo*
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ABSTRACT: Diazirines are widely used in photoaffinity labeling
(PAL) to trap noncovalent interactions with biomolecules. However,
design and interpretation of PAL experiments is challenging without a
molecular understanding of the reactivity of diazirines with protein
biomolecules. Herein, we report a systematic evaluation of the labeling
preferences of alkyl and aryl diazirines with individual amino acids,
single proteins, and in the whole cell proteome. We find that alkyl
diazirines exhibit preferential labeling of acidic amino acids in a pH-
dependent manner that is characteristic of a reactive alkyl diazo
intermediate, while the aryl-fluorodiazirine labeling pattern reflects
reaction primarily through a carbene intermediate. From a survey of 32
alkyl diazirine probes, we use this reactivity profile to rationalize why
alkyl diazirine probes preferentially enrich highly acidic proteins or
those embedded in membranes and why probes with a net positive
charge tend to produce higher labeling yields in cells and in vitro. These results indicate that alkyl diazirines are an especially
effective chemistry for surveying the membrane proteome and will facilitate design and interpretation of biomolecular labeling
experiments with diazirines.
biomolecular conjugation,16,17 which will either rapidly form
INTRODUCTION
■
a covalent bond with a nearby biomolecule or be quenched by
Since the development of photoaffinity labeling (PAL) in
the aqueous environment (Figure 1a).18 However, the
1962,1 PAL has emerged as a key approach for measuring
generation of a reactive, electrophilic diazo intermediate
biomolecular interactions, protein structure, and the identi-
during PAL may drive protein labeling through an alternate
fication of small molecule targets in the cell. The combination
“pseudo-PAL” mechanism.18 For example, stabilized diazo
of PAL with high-throughput sequencing2 and mass spectrom-
compounds exhibit preferential reactivity with organic acids19
etry-based proteomics3,4 has significantly increased the depth
and selectively react with protein carboxylates to generate
of information measured from a single experiment and has
esters.20 Evidence for alkyl diazirine reactivity patterns that
enabled mapping of lipid,5 carbohydrate,6 and small molecule
transition through the diazo isomer to preferentially react with
interactomes,7 broadly widening the scope of binding
acidic residues has been recently noted using in vitro peptide
interactions with proteins in cells. In particular, the design of
or protein model systems.21−23 Despite the investigation of
small tags embedded with the alkyl diazirine as the PAL
reactivity preferences of other PAL functional groups (e.g., aryl
functional group and a chemical enrichment handle has
azides,24,25 benzophenones,26,27 and aryl tetrazoles28−30), the
accelerated the use of PAL within large-scale biological
chemical reactivity and resulting labeling preferences of the
measurements,8−10 and the combination of this technology
diazirine with amino acids have only been investigated via in
with isotopically encoded enrichment tools has enabled the
vitro systems and peptide digests by mass spectrometry.31
mapping of small molecule binding sites in cells.7,11,12
Systematic establishment of how alkyl diazirines exhibit
differential reactivity preferences from aryl diazirines, and the
implications this has on biomolecular labeling, would facilitate
Despite the use of diazirines as probes to measure
biomolecular interactions, the incomplete understanding of
the labeling preferences of the diazirine limits the design and
interpretation of these experiments. To overcome challenges in
Received: March 6, 2021
Published: April 20, 2021
interpretation, the development of a diazirine “backgroun-
dome”13 or methods to differentiate between selective and
nonselective labeling,14 including the use of enantioprobes,12
or introduction of a scavenger,15 have been implemented.
During photolysis, the diazirine primarily isomerizes to a diazo
intermediate that further converts to the carbene for
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a ketone (10−20%), presumably from reaction of the carbene
with oxygen (Figure S1).32 We next examined the reactivity of
the Fmoc-protected alkyl diazirine 1 with individual amino
acids (Ac-AA-OMe) under neat conditions to represent the
closest possible binding interaction between the diazirine and a
protein. Neat mixtures of the Fmoc-diazirine 1 with each Ac-
AA-OMe (4 equiv) were irradiated using a broadband lamp
(280−400 nm) and analyzed by liquid chromatography−mass
spectrometry (LC-MS) (Figure 2a). The Fmoc-diazirine 1
covalently labeled relatively acidic polar amino acids with the
highest yields (40−60% yield), followed by a subset of the
additional polar amino acids (<20%), and had no observable
labeling of the remaining polar or aliphatic amino acids (<3%).
For comparison, the aryl diazirine 2 was evaluated against
individual amino acids (4 equiv) under neat conditions by 19F
NMR. The aryl diazirine 2 produced insertion products with
all 20 amino acids and afforded the highest yield with cysteine
(Figures 2b and S2). While optimizing these reactions, we
observed a transient 19F NMR peak at −57 ppm, matching the
reported 19F NMR shift of similar aryl-trifluoromethyl diazo
compounds.33 We found the intermediate did not independ-
ently react and disappeared following longer irradiation times.
Alkyl and Aryl Diazirine Reactivity in Aqueous
Solution. We next evaluated the product distribution in
aqueous conditions (80% D2O−CD3CN) at equimolar
concentrations of the amino acid and the diazirine (1 mM).
Under these conditions, we expected that the carbene
intermediate generated by each probe would be rapidly
quenched by D2O before reacting with the amino acid residue.
Surprisingly, photoirradiation of the alkyl diazirine 3 at
equimolar amounts of the individual amino acids (1 mM)
under aqueous conditions revealed selective conjugation with
Glu and Asp (10−15% yield, Figures 2c and S3). The insertion
of the alkyl diazirine 3 to Tyr and Cys was additionally
detected in aqueous solution at elevated concentrations (10
mM, 10 equiv). In order to investigate the possibility that the
alkyl diazo intermediate 3 was inducing the observed reactivity
with acidic residues, analogous to the reactivity of bona fide
diazo reagents with acidic amino acid residues,19 we filtered the
broadband excitation wavelength to exclude wavelengths below
320 nm34 and thus slowed the transition of the diazo to
carbene at 300 nm. Indeed, filtration of the light source to a
narrower range (320−400 nm) increased reactivity of the
diazirine 3 with the weak acids, Tyr and Cys (Figure 2c).
By contrast, the aryl diazirine 2 formed no observed adducts
with any amino acid up to 10 mM (10 equiv) in aqueous
solution. To evaluate the limit of reactivity of the aryl diazirine
2 to conjugate with a molecule in aqueous solution, β-
mercaptoethanol (BME) was used as a more soluble surrogate
for the most-reactive amino acid, cysteine (Figures 2d and S4).
The aryl diazirine 2 formed conjugated products with BME
beginning at 250 mM concentrations (250 equiv). Notably, the
aryl diazirine 2 formed comparable yields with BME at 500
mM to the alkyl diazirine 3 with 1 mM Glu. These data suggest
that the aryl diazirine 2 primarily reacts through a short-lived
carbene and that the aryl diazo intermediate is not driving
alternate reaction pathways under the photolysis conditions
used here.
Figure 1. Overview of diazirine reactivity pathways. (a) Alkyl and aryl
diazirines form carbene and diazo intermediates upon irradiation.
Carbenes label nearby proteins but are rapidly quenched if no protein
substrate is nearby. Alkyl diazo intermediates react selectively with
acids, while electronically stabilized aryl diazo intermediates do not.
(b) The acid-selectivity of the alkyl diazo intermediates causes
increased labeling of membrane proteins, which have more reactive
protonated carboxylic acids, and proteins with large negative
electrostatic surfaces. Labeling of these protein surfaces increases
when the PAL probe (highlighted in green) is positively charged.
interpretation of the measurements and conclusions from PAL
experiments using diazirines.
Here, we report a systematic study of alkyl and aryl diazirine
labeling preferences with individual amino acids and single
proteins and use these data to interpret the protein binding
partners visualized by a 32-membered alkyl diazirine library
from the whole cell proteome. We demonstrate that the aryl-
trifluorodiazirine primarily reacts through a carbene inter-
mediate, while the alkyl diazirine transitions through a reactive
diazo intermediate that preferentially reacts with acidic amino
acids in a pH-dependent manner. Reaction of the alkyl
diazirine through a diazo intermediate rationalizes the
preferential labeling of membrane proteins and proteins with
negatively charged electrostatic surfaces in cells (Figure 1b).
PAL ligands that are positively charged at physiological pH,
but not ligands that are neutral or negatively charged at
physiological pH, possess affinity for, and therefore heightened
labeling of, these protein surfaces, which is reflected by global
protein profiles and binding site maps. This understanding of
diazirine labeling preferences improves the ability to design
and interpret the results from biological experiments using
diazirines.
RESULTS
■
Alkyl and Aryl Diazirine Reactivity Profile with
Individual Amino Acids under Neat Conditions. We
initiated a systematic evaluation of diazirine chemistry by
evaluating the photolysis products of the alkyl diazirine9 in
aqueous conditions (20% CD3CN−D2O) and recovered a
mixture of olefins (50%), a water-insertion product (35%), and
Alkyl Diazirine Labeling of Amino Acids is pH-
Dependent. Reaction of acidic residues with the alkyl
diazirine through the diazo intermediate requires a proton
transfer from the acid to give a cationic intermediate prior to
displacement of dinitrogen by the conjugate base (Figure 1a).
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Figure 2. Reactivity of alkyl and aryl diazirines with the 20 natural amino acids. (a) Reactivity of alkyl diazirines with the 20 N-acetyl, O-Me
protected amino acids (4 equiv) in neat conditions. Yields calculated against an internal standard by LC-MS. (b) Reactivity of aryl diazirines with
the 20 amino acids (4 equiv) in neat conditions. Yields calculated against an internal standard by 19F NMR. (c) Reactivity of alkyl diazirines with
the 20 amino acids in solution (1 mM 3 and 1 mM or 10 mM amino acid) with 280−400 nm or 320−400 nm excitation. Only reactive amino acids
1
are displayed. Yields calculated against an internal standard by H NMR. (d) Reaction of aryl diazirine 2 with β-mercapto ethanol at varying
concentrations in aqueous conditions. Yields calculated with relative integration by LC-MS. (e) Yield of alkyl diazirines with individual amino acids
as a function of pH. Yields calculated against an internal standard by LC-MS. Plotted values show the mean with error bars representing the
standard deviation with n = 3.
If the proton transfer is the rate-limiting step, the insertion
yield will be dependent on the protonation state of the amino
acid. The pH-dependence of alkyl diazirine labeling was
investigated by titrating aqueous solutions of Glu, His, Tyr,
and Lys (10 equiv) with acid or base before addition of the
diazirine 1 (1 equiv) and filtered UV irradiation (320−400
nm). Product yields were measured by LC-MS. Glu, His, and
Tyr produced pH-dependent insertion products with the alkyl
diazirine 1, while no observable insertion was observed with
the fully deprotonated carboxylate and free base His (Figure
2e). Protonated Lys was also not reactive under these
conditions, suggesting that the pKa of the conjugate acid is
too high to react with the diazo intermediate generated by the
diazirine 1. In sum, these data suggest that the alkyl diazirine 1
generates a reactive intermediate that leads to preferential
reactivity with protonated acidic residues in aqueous solution.
In the course of these studies, we additionally found that a
significant fraction of the photolyzed diazirine underwent rapid
internal rearrangement to a mixture of olefins, which may be a
major rearrangement product from the carbene intermediate.23
To minimize this elimination pathway, we drew on the kinetic
isotope effect35 and synthesized a deuterated alkyl diazirine
(Figure S5). Photolysis of the tetra-deuterated diazirine
afforded a greater yield of the ketone (42%) and reduced the
yield of the olefin products (34%) as compared to the tetra-
proton analogue. Thus, deuteration of the alkyl diazirine may
improve covalent labeling yields in PAL experiments.
Alkyl Diazirine Labeling of Single Proteins is pH-
Dependent. To translate these observations to proteins, we
next examined the labeling efficiency of alkyl and aryl
diazirines36 in vitro on an individual protein, bovine serum
albumin (BSA), over a 5.8−8.0 pH range in Tris buffer (Figure
3a). Analogous to the reactions with single amino acids,
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probes with a distribution of net positive, negative, or neutral
charges was used (representative structures in Figure 4a; for all
structures see Figure S8). The PAL probes cover a diversity of
small molecules including aromatic scaffolds (e.g., JN3, JN939,
JN849, and JN935), analogues of nucleotides (e.g., JN846,
JN847, and JN936), lipids (e.g., JN38), and other metabolites
(e.g., JN247 and JN835).
From this library, we selected representative probe
structures based on their overall charge at physiological pH
and evaluated their labeling efficiency by in-gel fluorescence.
HEK293T cells were incubated with individual probes at 10
μM and photoirradiated for 60 s to cross-link the ligands to
their protein binding partners. The cells were harvested, lysed,
and visualized by in-gel fluorescence following click chemistry
with Azide-Fluor 488. As expected, probe labeling efficiency
generally correlated with the net charge of the probe (Figure
4b). The alkyl diazirine probes with a net positive charge
exhibited higher labeling than neutral or negatively charged
probes (Figure 1b). To examine how factors such as cell
permeability affected labeling efficiency, we performed PAL
with the alkyl diazirine probes on HEK293T lysates (Figure
S10). In general, the same trend of positively charged probes
producing higher labeling yields than neutral or negatively
charged probes were observed in lysates as in whole cells, but
higher labeling from a subset of the negatively charged probes,
most notably JN33, was found in lysates than from whole cells.
These data suggest that, in general, positively charged probes
have higher labeling propensities that may reflect increased
interaction with negatively charged protein surfaces composed
of acidic residues that react with alkyl diazirines and that
overall labeling yields from a particular probe reflect a
composite of the diazirine chemistry and cellular permeability.
To directly examine the contribution of charge to the
labeling efficiency in probe design, we compared the enriched
proteome of two pairs of alkyl diazirine PAL probes with
different net charge by chemical proteomics: first, a primary
amine versus a carboxylic acid (JN26 vs JN33), and second, a
tertiary amine versus an amide (JN938 vs JN939). SK-N-SH
cells were incubated with individual PAL probes, photo-cross-
linked, harvested, and lysed. Lysates were conjugated with
biotin-azide using click chemistry, and the biotinylated
proteins were enriched with streptavidin-agarose. The enriched
proteins were digested with trypsin for quantitative proteomics
(TMT10plex). The positively charged JN26 exhibited a much
higher degree of labeling compared to the negatively charged
analogue JN33 (819 and 9 enriched proteins, respectively;
Figure 4c). The effect from probe charge state was diminished
but still noticeable when comparing neutral versus positively
charged probes with larger, more hydrophobic structures. The
neutral amide probe JN939 significantly enriched slightly more
proteins than the positively charged amine JN938 at a 3-fold
enrichment threshold (111 and 91 enriched proteins,
respectively), but the degree of enrichment was higher for
the positive amine JN938 than the neutral amide JN939 when
the enriched proteins from each probe were compared directly
(JN938 vs JN939, Figure 4d). Taken together, these data
imply that alkyl diazirine probes with a net positive charge will
yield elevated labeling of the proteome, potentially because of
increased localization to protein surfaces that are more readily
cross-linked by alkyl diazirine chemistry, and that this effect is
attenuated with larger hydrophobic probes.
Figure 3. Photolabeling of single proteins as a function of pH. (a)
Workflow schematic. Alkyl diazirine 3 or aryl-difluorodiazirine 5 was
incubated with BSA in Tris buffer with varying pH (5.8, 6.6, 7.4, and
8.0). The diazirines were cross-linked to the protein by UV irradiation
and visualized by in-gel fluorescence following click chemistry with
Azide-Fluor 488. (b) pH-dependent labeling of BSA by alkyl diazirine
3. (c) pH-independent labeling of BSA by aryl diazirine 5. Labeling
yields were calculated with fluorescence signal normalized to
coomassie blue (CB) stain signal. Plotted values show the mean
with error bars representing the standard deviation with n = 3.
Statistical significance was determined with a t test between pH 5.8
and 8.0 samples (* = p-value <0.05).
labeling from the alkyl diazirine 3 was higher at lower pH,
while labeling from the aryl-difluorodiazirine 5 was pH-
independent over the range examined (Figure 3b,c).
Interestingly, we found that performing a similar labeling
experiment of BSA with the alkyl diazirine 3 in acidic
phosphate buffer (pH 5.8) resulted in concentration-depend-
ent labeling of BSA by the alkyl diazirine 3, presumably via
competitive reactivity with the phosphate buffer (Figure S7).
Indeed, we observed formation of a phosphate adduct with 3 in
a pH-dependent manner analogous to the aqueous reactivity of
3 with the protected amino acids, suggesting that acidic
phosphate was reacting with the diazo isomer of 3. These data
demonstrate that the differential reactive intermediates from
the alkyl diazirine and the aryl diazirine predictably impact
observed labeling patterns on individual substrates.
PAL Probes with a Net Positive Charge Increase Alkyl
Diazirine Labeling in the Cell. The pH dependence of alkyl
diazirine chemistry in vitro implies that charge state will play a
significant role in the capture of protein substrates and may
explain the inherent labeling efficiency of different alkyl
diazirine probes.7,12 To evaluate the influence of alkyl diazirine
reactivity on PAL experiments in cells, a set of alkyl diazirine
PAL Probes Disproportionately Label Membrane
Proteins in Cells. In line with prior reports,5,7,11−13,37 we
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Figure 4. Whole proteome photolabeling in cells with PAL probes. (a) Representative probe structures (for a full list see Figure S8). (b)
Photolabeling yields of compounds visualized by fluorescence signal. (c) Comparison of photolabeling profiles of carboxylic acid probe JN33 and
primary amine JN26. Significantly enriched proteins are colored as membrane and nonmembrane proteins. (d) Photolabeling profiles of neutral
amide probe JN939 and positively charge amine analogue JN938. (e) Histogram showing the relative frequency of membrane and nonmembrane
protein enrichments for all data in panels c and d. (f) Percentage of membrane proteins above enrichment thresholds for all data in panels c and d.
observed that alkyl diazirine probes possess an enrichment bias
for membrane proteins (Figure 4e). While membrane proteins
made up 33% of all proteins observed in our data set, similar to
the distribution of these proteins in the global proteome
(27%),38 membrane proteins were over-represented after
filtering for protein enrichment: 46% of proteins that had at
least a 3-fold enrichment and 56% of proteins with at least a 4-
fold enrichment were membrane proteins (Figure 4f). These
membrane proteins derived from throughout the cell, including
the cell surface, ER/golgi, mitochondrion, and nuclear
membranes. The preferential labeling of membrane proteins
by alkyl diazirine probes may reflect the elevated pKa of Glu
and Asp residues in lipid bilayers.39−41 Similarly, a recent study
by Cravatt and co-workers examined the types of proteins
labeled by an assortment of alkyl diazirine probes and found
that membrane proteins were indeed enriched (∼20−40% of
protein targets).7 Analysis of the most-enriched set of proteins
in this data set revealed that membrane proteins were
disproportionately common (38−61% of proteins with a
SILAC ratio ≥20) (Figure S12).
Understanding Alkyl Diazirine Reactivity Improves
Interpretation of Binding Site Mapping Results. To
further evaluate the labeling trends in cells, the entire 32-
membered alkyl diazirine probe library was divided into groups
according to similarities in the probe charge and structure and
dosed to SK-N-SH cells for protein and binding site mapping
(Figure 5a). In brief, SK-N-SH cells were dosed in biological
duplicate with the alkyl diazirine probe library in six groups of
5−6 compounds at 10 μM concentrations of each compound
for 2 h. The treated cells were photoirradiated for 60 s,
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Figure 5. Binding site mapping with a 32 PAL probe library. (a) Cell cultures were treated simultaneously with 5 or 6 PAL probes which were
photoconjugated to protein binding partners. The conjugated proteins were enriched and the conjugated peptide representing the binding site of
the PAL probe was isolated for analysis using isotope-targeted MS. (b) Count of peptide spectral matches (PSMs) assigned to each PAL probe.
PAL probes with a net positive charge highlighted in blue, neutral probes in gray, and negatively charged probes in red. (c) Comparison of binding
site PSMs from membrane and nonmembrane proteins. (d) Amino acid frequency in the conjugated peptides relative to frequency in human
proteome. (e) Annotated mass spectra and assignment of a VDAC1 binding site. (f) Negative electrostatic maps (red) overlaid on the conjugated
peptide (blue) for unique binding interactions. (g) Negative electrostatic maps (red) overlaid on conjugated peptide (blue) for frequently
conjugated proteins.
collected, and lysed. Labeled proteins in the cellular lysates
were tagged with an acid-cleavable biotin picolyl biotin azide42
for affinity enrichment and trypsin digestion on streptavidin−
agarose beads. The labeled peptides representing binding
interactions of the PAL probe with the protein target were
subsequently eluted from the beads by acid cleavage and
recovered for analysis by LC-MS, which were analyzed by two
technical replicates. The probe-labeled peptide was assigned by
database searching against the Swissprot human proteome,
followed by filtration based on the embedded isotopic
pattern,43 and manual validation. A total of 632 binding sites
from 170 proteins were mapped in SK-N-SH cells across 4200
peptide spectral matches (PSMs).
Analysis of the mapped binding sites reflected the labeling
trends observed on the proteome level. PSMs were used to
approximate the relative abundance of labeling events with
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protein binding partners from each PAL probe (Figure 5b).
Examination of the number of PSMs per probe revealed a
bimodal distribution that clustered as a function of positively
charged probes versus neutral or negatively charged probes
(Figure 5b, inset). Positively charged probes produced on
average 418 PSMs corresponding to 50 unique binding sites
per probe, whereas neutral probes produced an average of 65
PSMs (14 unique binding sites) and negatively charge probes
produced an average of 14 PSMs (5 unique binding sites). No
statistical difference between the neutral or negatively charged
PAL probes was observed, although neutral PAL probes with a
larger hydrophobic surface area tended to yield a greater
number of PSMs (e.g., JN3, JN939, and JN38). Similar to
observations from the proteome, we found that a majority of
the binding sites were from membrane proteins (58% of
PSMs) (Figure 5c). The wider structural diversity of the 32
PAL probes suggests the increased labeling of membrane
proteins is a result of the labeling propensity of alkyl diazirine
chemistry in contrast to the probes’ individual structures.
Although the positively charged members of the library were
six of the top seven probes by PSM counting, the positively
charged probe JN942 was an outlier at rank 16 of 32 probes.
We investigated the cause of this outlier in vitro and observed
unaffected labeling efficiencies on individual amino acids and
proteins (Figure S13), suggesting that in addition to the net
charge, cellular accessibility of the small molecule to
biomolecular targets should also be considered in probe
design.
As further evidence of labeling preferences between alkyl
diazirines and acidic amino acid residues in the whole
proteome, we compared the occurrence of amino acids
found within labeled peptides to their natural occurrence in
the human proteome (Figure 5d). Glutamic acid was the most
enriched amino acid in labeled peptides and increased
specifically in peptides labeled by positively charged probes;
most of this enrichment was eliminated in binding sites
measured from negatively charged probes. The limited
enrichment of aspartate residues reflects previous trends
observed from labeling propensities with bona fide diazo
esterification reagents.20 This may be caused by differences in
steric accessibility or the generally lower pKa of Asp relative to
Glu, which would favor reactivity with Glu and disfavor
reactivity with Asp. In sum, for alkyl diazirine PAL experiments
in cells, glutamic acid has the highest propensity for labeling in
comparison to other amino acids, and the labeling frequency is
influenced by the local pKa environment and the physical
properties of the photoaffinity probe.
electrostatic potential of each protein surface was calculated
using the adaptive Poisson−Boltzmann solver (APBS) and
overlaid on the structure of the protein. For clarity, only the
negative electrostatic surface map is displayed in red. For
binding sites that were observed as labeled by only one or two
PAL probes, the mapped peptide has a minor degree of
negative electrostatic density nearby (Figure 5f). In some
cases, the mapped peptide is within 9 Å21 to a known binding
site for the natural protein ligand. For example, ADP-
ribosyltransferase protein PARP15 has a nicotinamide binding
cleft that is adjacent to the observed binding site.45 PD6-
interacting protein (PDCD6IP) binds to a natural peptide
ligand in close proximity to the observed small molecule
binding site (peptide ligand highlighted in orange, Figure 5f).46
The 14−3−3 proteins are receptors for peptides in a cleft
within 9 Å of the mapped binding site.47 Transferrin receptor 1
is a membrane-bound protein; the binding site is in the
extracellular region.48
In contrast, binding sites on proteins that were mapped to
multiple PAL probes exhibited significantly greater negative
electrostatic densities around the labeled peptide (highlighted
in dark blue, Figure 5g). Vimentin is a filamentous protein that
is frequently identified in alkyl diazirine labeling experiments,
likely because of the significant negative electrostatic density
on vimentin (net charge = −27). Unsurprisingly, the 17 unique
peptides conjugated across 19 different PAL probes identified
from vimentin colocalize with the negative electrostatic density
map. Likewise, 8−12 PAL probes conjugated to VDAC1, ER
chaperone BiP, and cathepsin B, which each display similar
negative electrostatic densities in close proximity to the
mapped peptide binding site. Taken together, these data
suggest that the subset of proteins that are labeled more
frequently by alkyl diazirine chemistry typically have negative
electrostatic regions that are characterized by a density of
acidic residues or those with a relatively high localized pKa,
resulting in an increase in the degree of protonation of the
amino acid for labeling through the diazo intermediate during
PAL experiments.
DISCUSSION
■
Despite the growing use and application of diazirine chemistry
in PAL experiments, a systematic understanding of the labeling
preferences of the diazirine in cells has yet to emerge. Here, we
report a systematic analysis of diazirines and their preferential
reactivity pattern with protein biomolecules in vitro and in
cells. We find that alkyl diazirines preferentially react with
acidic amino acid residues in a pH-dependent manner in vitro,
which can be rationalized by the formation of a long-lived
diazo intermediate that is intercepted by organic acids prior to
formation of an alkyl carbene. By contrast, the aryl-
trifluorodiazirine forms insertion products with all 20 amino
acids, is less dependent on pH, and its labeling chemistry is
readily quenched by water; these are characteristics of a
reaction pathway through a carbene intermediate. Although
reactions with individual amino acids in neat conditions are
only an approximation of the PAL probe reactivity with a
target protein in cells, the impact of the alkyl diazirine
reactivity pattern translates to a predictable enhancement of
cellular labeling with alkyl diazirine probes carrying a net
positive charge from a library of 32 alkyl diazirine probes,
which may arise from enhanced association of the probe with
the matched acidic regions on protein surfaces. In line with this
expectation, chemoproteomics and binding site data show that
Across all the mapped probe-labeled binding sites, we found
that a binding site on VDAC1 was frequently observed
(representative assignment, Figure 5e). This peptide on
VDAC1 is in close proximity to two negatively charged
residues on the protein (Y67 and E73). VDAC1 E73 is notable
for its unusually high pKa (predicted membrane pKa = 7.4) and
ability to sensitively mediate dimerization of VDAC1 in a pH-
dependent manner.44 Protonation of VDAC1 E73 would
increase the propensity for E73 to react with a diazo
intermediate. VDAC1 and VDAC2, but not VDAC3, possess
this glutamic acid residue, which explains why only VDAC1
and VDAC2 are frequently enriched targets in alkyl diazirine
studies.13
Intrigued by this analysis with VDAC1, we next visualized a
subset of the binding sites onto available protein structures
(binding sites highlighted in dark blue, Figure 5f,g). The
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these probes preferentially enrich proteins with negative
electrostatic surfaces or proteins embedded in membranes,
which may be a consequence of the predicted elevation of the
pKa of acidic residues in the hydrophobic membrane
environment.41
of a given probe in vitro versus in cells, such as what we
observed with JN942 and JN33.
CONCLUSION
■
In conclusion, the alkyl diazirine preferentially reacts with
acidic amino acid residues because of the major contribution of
a reactive diazo intermediate, while the aryl-trifluoro diazirine
possesses a reactivity pattern in line with a reactive carbene
intermediate. These reactivity patterns are consistent across
individual amino acids, single proteins, and the whole
proteome and assist with the design and interpretation of
PAL experiments using alkyl diazirine probes. Understanding
the reactivity pattern of alkyl diazirines therefore facilitates
design and interpretation of PAL experiments. Given these
reactivity considerations for the alkyl diazirine, there is
opportunity for innovation of new PAL chemistries with
different labeling preferences. The design of new chemistries
and the systematic examination of their reactivity patterns may
provide additional opportunities for generation of an “ideal”
PAL functional group or development of methods to
selectively map noncovalent interactions with certain regions
of the cell or types or proteins.
These data assist with the design and interpretation of PAL
experiments using alkyl diazirine chemistry. Design of the
probe can be tuned to enhance or reduce protein capture
simply by altering the net overall charge of the probe molecule.
Alkyl diazirine probes with a net neutral or negative charge
may selectively visualize stronger binding interactions, while
probes with a net positive charge may capture more transient
interactions in the proteome. The charge state of the molecule
is readily altered by the type of linker chemistry employed
when embedding the alkyl diazirine to the probe molecule of
interest (e.g., amide versus amine linker chemistry). Alkyl
diazirine chemistry should also be selected if membrane
proteins or those with large acidic patches are desired targets.
The preference of alkyl diazirine chemistry for these protein
types is reflected herein and in prior studies.5,7,11−13,37,42,49
This enhances the utility of diazirine chemistry in probes for
visualizing binding over a range of affinities with membrane
proteins, which are typically challenging proteins to target.
Although these results point to a significant contribution of
alkyl diazirine PAL through the diazo intermediate, dissection
of the exact ratio of labeling from the carbene and the diazo
intermediate remains to be fully elucidated. Theoretically,
products resulting from the carbene are more likely to arise
from tight binding interactions with a lower binding constant,
because the carbene is a short-lived intermediate that is rapidly
quenched in aqueous media.17,50 Conversely, products
resulting from labeling through the diazo are more likely to
represent weaker binding interactions, because the lifetime of
the diazo is above the rate of diffusion.51 There are relatively
few chemistries that have been used to selectively label
carboxylic acids in whole cells52−54 and fewer photoactivatable
chemistries,30 suggesting that this chemistry could be inten-
tionally utilized to profile carboxylic acids in cells as a method
that senses the protonation state of the acids. Additionally,
once a carbene intermediate is generated, the contribution
from singlet or triplet states is another open question that may
affect the amino acid labeling distribution, because the carbene
is formed as a singlet but can relax to the triplet state.55−59
In addition to the contribution of the chemical reactivity of
the alkyl diazirine, the overall labeling trends of diazirine
probes are also impacted by other factors, such as overall
hydrophobicity of the probe, subcellular localization, or cell
permeability, which must be additionally considered when
designing PAL probes or interpreting data from PAL
experiments. These factors likely contribute to the observed
labeling trends, by either reducing cellular permeability60 or
enhancing the cellular uptake and subcellular localization of
positively charged probes to membranous organelles.61,62
However, we observed higher labeling yields from probes
with a positive charge in cells and cell lysates, suggesting that
increased cellular uptake of positive probes does not fully
explain the increase in labeling. In addition, the increased
membrane protein enrichment is a general trend observed here
with alkyl diazirines, which is independent of the charge of the
probe. Potentially, the subcellular localization of positively
charged probes to the membrane is synergistic with the pH-
dependent reactivity of alkyl diazirines. The influence of these
factors can be distinguished by comparison of labeling trends
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c02509.
Full description of the experimental methods used in
this study; supplementary figures, synthetic protocols,
representative spectra for amino acid reactions, charac-
terization of novel compounds, and representative
binding site spectra (PDF)
Quantitative proteomics for JN26 v JN1, JN33 v JN1,
JN938 v JN1, and JN939 v JN1; binding site data for all
32 PAL probes (Tables S1−S5) (XLSX)
AUTHOR INFORMATION
Corresponding Author
■
Christina M. Woo − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States; orcid.org/0000-0001-8687-9105;
Email: cwoo@chemistry.harvard.edu
Authors
Alexander V. West − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States
Giovanni Muncipinto − Jnana Therapeutics, Boston,
Massachusetts 02210, United States
Hung-Yi Wu − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States
Andrew C. Huang − Department of Chemistry and Chemical
Biology, Harvard University, Cambridge, Massachusetts
02138, United States
Matthew T. Labenski − Jnana Therapeutics, Boston,
Massachusetts 02210, United States
Lyn H. Jones − Dana-Farber Cancer Institute, Boston,
Massachusetts 02215, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c02509
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Notes
The authors declare no competing financial interest.
All proteomics data are available within this paper, in the
Supporting Information, or in the Supplementary Tables. The
full mass spectrometry proteomics data have been deposited to
the ProteomeXchange Consortium via the PRIDE partner
repository with the data set identifier PXD025140.
The IsoStamp software used for analysis in this paper is
available online at GitHub: https://github.com/
harvardinformatics/quantproteomics/tree/master/Isostamp
ACKNOWLEDGMENTS
■
We thank H. Flaxman, N. Vallavoju, C. Chang, and A. D’Souza
for their advice and helpful discussions and M. Parsons for his
assistance. Mass spectrometry data was collected at the
Harvard University Mass Spectrometry and Proteomics
Resource Laboratory (B. Budnik), and NMR data was
collected at the Laukin-Purcell Instrumentation Center (D.
Cui). Support from NIH NIDA (DP1DA046586), Burroughs
Welcome Fund, Sloan Foundation, Camille and Henry Dreyfus
Foundation, and Harvard University are gratefully acknowl-
edged.
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