Chemoproteomic Profiling of Phosphoaspartate Modifications in Prokaryotes

Article abstract of DOI:10.1002/anie.201809059

Phosphorylation at aspartic acid residues represents an abundant and critical post-translational modification (PTM) in prokaryotes. In contrast to most characterized PTMs, such as phosphorylation at serine or threonine, the phosphoaspartate moiety is intr

Full text of DOI:10.1002/anie.201809059

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Accepted Article
Title: Chemoproteomic Profiling of Phosphoaspartate Modifications in
Prokaryotes
Authors: Jae Won Chang, Gihoon Lee, Jeffrey E Montgomery, and
Raymond Moellering
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Chemoproteomic Profiling of Phosphoaspartate Modifications in
Prokaryotes
Jae Won Chang, Jeffrey E. Montgomery, Gihoon Lee, and Raymond E. Moellering*
Abstract: Phosphorylation at aspartic acid residues represents an
abundant and critical posttranslational modification (PTM) in
prokaryotes. In contrast to most characterized PTMs, such as
phosphorylation at serine or threonine, the phosphoaspartate moiety
is intrinsically labile, and therefore incompatible with common
with phospho-specific antibodies, and global identification with
LC-MS/MS proteomic approaches[6-7]. Combined, these methods
have identified thousands of phosphorylation sites in diverse cell
types and organisms. The comprehensive set of tools available to
study protein phosphorylation, which is invoked here as a
prototypical PTM, hinges on a single aspect of the modification
itself: chemical stability to biochemical and proteomic workflows.
proteomic profiling methods. Here we report
a nucleophilic,
desthiobiotin-containing hydroxylamine (DBHA) chemical probe that
covalently labels modified aspartic acid residues in native proteomes.
DBHA treatment coupled with LC-MS/MS analysis enabled detection
of known phosphoaspartate modifications, as well as novel aspartic
acid sites in the E. coli proteome. Coupled with isotopic labelling,
DBHA-dependent proteomic profiling also permitted global
quantification of changes in endogenous protein modification status,
as demonstrated here with the detection of increased E. coli OmpR
phosphorylation, but not abundance, in response to changes in
osmolarity. The DBHA probe and proteomic workflow reported herein
should be broadly useful for the discovery and quantitative
interrogation of phosphoaspartate modifications in diverse organisms
and biological contexts.
Posttranslational modifications (PTMs) have evolved as
a
selective and reversible means to alter protein function, and thus
propagate signals within and between cells. These chemical tags
are exceptionally diverse in structure and function, and are known
to target a majority of proteins in many organisms, including
mammals [1- 2]. While the prevalence and perceived importance of
PTMs is conserved across all forms of life, the relative abundance
of specific PTMs is extremely variable. Humans, for example,
encode more than 500 protein kinases that catalytically
phosphorylate the alcohol functionality in serine, threonine and
tyrosine residues [3-4]. Protein phosphorylation is recognized as a
major communication mechanism in mammalian cells, a fact that
is bolstered by high association with disease when
phosphorylation networks are deregulated[5]. An important
technical consideration when reflecting upon the vast number of
phosphorylation marks that are known in mammalian systems is
the fact that systematic, quantitative analysis of these PTMs is
made possible through several technologies, including detection
Figure 1. DBHA probe labelling, detection and quantification of modified
aspartic acids in native proteins. (a) Schematic of dynamic phosphorylation at
aspartic acids, as well as labelling with a prototypical nucleophilic probe in situ,
permitting detection and quantification. (b) Chemical structure of the
desthiobiotin-linked hydroxylamine (DBHA) probe. (c) Western blot visualization
of desthiobiotin-labelled proteins demonstrates dose-dependent and
hydroxylamine competitive labelling of E. coli proteins in DBHA-treated lysates.
[a] Dr. J.W. Chang, J.E. Montgomery, G. Lee, Prof. Dr. R. E. Moellering
Department of Chemistry
Institute for Genomics and System Biology
The University of Chicago
5735 S. Ellis Ave, Chicago, IL 60637 (USA)
E-mail: rmoellering@uchicago.edu
Prokaryotes also employ phosphorylation as a major form of
biochemical communication, but in contrast to eukaryotes the
number of serine/threonine phosphorylation sites appears to be
orders of magnitude lower[8]. Instead, these lower organisms rely
more on two-component signalling pathways that involve
phosphorylation of histidine “receiver” residues, which ultimately
phosphorylate aspartic acids to form an acylphosphate
modification[9-11]. The resulting phosphoaspartate residue (pD) is
Supporting information for this article is given via a link at the end of
the document.
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generally thought to cause changes in protein structure, which in
the case of many characterized response regulator (RR)
transcription factors can promote or inhibit DNA binding and
subsequent downstream signalling. While thought to represent
the major form of signal transduction, the widespread discovery
and dynamic quantification of these modifications has not been
possible due to the intrinsic lability of the pD group itself. As a
result, pD sites have almost exclusively been studied one-by-one
with the use of 32P-ATP as a means to radiolabel the active protein
for visualization by gel electrophoresis[12]. Even in these settings,
however, protein purification is often required and the
endogenous environment cannot be probed. Therefore, new
methods with which to study these chemically labile yet
functionally important modifications are necessary. Here we
report a chemoproteomic approach to rapidly trap the electrophilic
acylphosphate group unique to pD sites within native bacterial
proteomes (Figure 1a). We demonstrate that a desthiobiotin-
containing hydroxylamine (DBHA) probe selectively enriches and
site-specifically identifies both known and novel electrophilic
aspartic acid sites throughout the E. coli proteome. Furthermore,
we demonstrate the potential to measure signalling dynamics in
the osmolarity sensing OmpR/OmpF two-component signalling
pathway by combining isotopic proteome labelling and pD-site
enrichment from native proteomes.
We found that DBHA labelling was optimal under the denaturing
conditions of 6 M urea, which likely improves probe access to pD
modification sites. Proteome incubation with DBHA and Western
blot detection of desthiobiotin labelled proteins revealed dose-
dependent labelling of the E. coli proteome across a wide range
of concentrations (Figure 1c). These labelling events were
inhibited by pretreatment of proteome with soluble hydroxylamine
or acidic conditions (pH = 2), confirming that the proteins and sites
being visualized were dependent upon intact reactivity (Figure 1c;
Figure S2). Determining the site of labelling is particularly
important for probes such as DBHA because they could, in
principle, react with numerous modifications on proteins. For
example, hydrazine-probes have previously been shown to
modify glycosylated proteins in their open-chain form[18], as well
as capture pyruvoyl and glyoxylyl modifications in mammalian
cells[16]
.
Despite the potential for interaction with other
electrophilic moieties that are present on proteins, our probe and
workflow should differentiate these events through the detection
of the DBHA-modified aspartic acids directly on tryptic peptides
with high resolution LC-MS/MS and site-of-labelling analysis (vide
infra; Figure S1). To identify the proteins and specific sites being
modified by DBHA we developed an efficient workflow for
modified protein enrichment and processing by LC-MS/MS
analysis (Figure S3). DBHA treatments were performed at 10 mM
and 100 mM concentrations in order to maximally capture both
low and high abundance sites within the E. coli proteome.
Modified proteins were directly enriched with streptavidin-agarose
immunoprecipitation, washing to remove non-labelled proteins,
and processed for on-bead tryptic digestion. Importantly, bulk
tryptic peptides from enriched proteins could be collected in the
digest eluent, followed by elution of DBHA-modified peptides. The
combination of these two pools enables high-confidence
detection of target proteins (i.e. from many detected tryptic
peptides per protein) as well as site-specific resolution of DBHA-
modification sites. DBHA-labelled sites were filtered based on
stringent mass tolerance, decoy database false-discovery rates,
and MS2 statistics. Beyond these initial filters, we required that a
putative modification site be observed in four or more biological
replicates to be included in this dataset. Finally, in order to identify
the specific DBHA-modified residue in each detected tryptic
peptide we analyzed the “hits“ that passed our initial specificity
filters above using the LuciPHor modification site localization
algorithm[19]. Application of this analysis pipeline to LC-MS/MS
data from the 10 mM (n = eight replicates) and 100 mM (n = six
replicates) DBHA datasets permitted detection of 138 high
confidence pAsp sites from 98 proteins (site global false-
We previously reported that intrinsically labile metabolites,
such as the glycolytic metabolite 1,3-bisphosphoglycerate, could
be efficiently trapped and converted into a stable hydroxamic acid
derivative upon treatment with hydroxylamine in situ[13]. Based on
the success of this approach for acylphosphate-containing
metabolites, and precedence for mapping ADP-ribosylation[14], we
reasoned that a hydroxylamine-based chemical probe may be
useful for capturing pD sites in the proteome. Such a probe would
encompass two main components: 1) an O-alkyl hydroxylamine
warhead capable of forming a stable intermediate with pD sites in
native proteins under mild conditions; 2) a retrieval tag that
permits direct enrichment and elution of modified proteins or
peptides for LC-MS/MS or other quantitative detection methods
(Figure 1a; Figure S1). We first attempted to use a minimal probe,
O-propargyl hydroxylamine, which would label modified
aspartates with a small alkyne tag that could be used for detection
or enrichment following [3+2] Huisgen “click” chemistry with
secondary retrieval/visualization tags[15]. This approach is similar
to a recent report that employed an alkyne-containing hydrazine
probe in mammalian cells[16]. Our preliminary experiments using
O-propargyl hydroxylamine suffered from poor retrieval of tagged
species as measured by either in-gel fluorescence or LC-MS/MS,
which we attribute to decreased efficiency that comes with
additional steps involved in click chemistry sample processing.
Therefore, we sought to develop a probe that could be directly
retrieved and processed for a variety of proteomic detection
methods. We synthesized a desthiobiotin-containing O-alkyl
hydroxylamine (DBHA) probe that could bypass the need for
subsequent chemical modification prior to detection or enrichment
(Figure 1b). Desthiobiotin was specifically used in place of biotin
to permit efficient release of enriched peptides for LC-MS/MS
detection, which can be a major bottleneck in chemical proteomic
localization rate in LuciPHor at < 1%; Table S1).
The identification of >100 modified aspartic acids in the E. coli.
proteome was surprising since there are ~20-30 predicted RR’s
present in this and other typical prokaryotes[20]. Analysis of the
local sequence context of DBHA-modified aspartic acids revealed
a modest motif preference for small, hydrophilic residues around
the DBHA-modified aspartic acid, as well as preference for proline
at the +4 position (Figure 2a). This overall profile was distinct from
the motif surrounding known response regulator pD sites in E. coli.
However, a conserved preference for proline at the +4 position
was also present, confirming partial similarities between the two
motifs (Figure 2a). Bioinformatic analysis of modified proteins with
DAVID and KEGG pathway analysis revealed a dominant
signature of proteins involved in metabolism, protein folding and
workflows[17]
.
We first tested whether incubation of DBHA across assay
conditions would covalently label native proteins in K12 E. coli.
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transcriptional regulation (Figure 2b; Figure S4). These sites were
found in proteins spanning the entire range of relative abundance
in E. coli, from metabolic proteins like Pgk (estimated at ~100,000
copies/cell) at one extreme, to transcription factors like BasR
(estimated at ~20 copies/cell) on the other (Table S1; Figure
S5)[20]. Among the lower abundance proteins identified were
response regulators and related two-component signalling
proteins with known pD sites. For example, the response
regulators OmpR and BasR were identified harboring DBHA-
modifications at D55 and D51, respectively (Figure 2c-d). These
sites are known phosphorylation sites that regulate OmpR and
BasR DNA-binding activity[21-22]. Additional DBHA-modification
sites included known phosphohistidine-containing proteins, like
Asp38 in Crr. Intriguingly, the DBHA modification site in Crr was
found to be highly proximal to a known phosphohistidine site,
perhaps indicating that dynamic relay between pH and pD sites
occurs within this and other proteins (Figure S6)[23]. Taken
together, this dataset validated DBHA-labelling and enrichment
as a method to detect both known and potentially novel
phosphoaspartate sites in native proteins from whole prokaryotic
proteome.
Figure 2. DBHA-dependent proteomic profiling identifies known and novel aspartic acid modification sites in E. coli. ( ) Enriched sequence motifs derived from
a
DBHA-modified aspartic acid sites (top) compared to the motif generated by known response regulator pD sites in E. coli (bottom). (b) Gene-ontology biological
process categories (GOTERM_BP; bottom) and KEGG pathways (top) enriched among DBHA-modified proteins. Representative, statistically significant categories
are shown, with a complete list in Suppl. Fig. 4. (c-d) MS/MS spectra of DBHA-modified tryptic peptides at two known pD sites in the response regulator proteins
OmpR (c) and BasR (d). Observed b- and y-ions are labelled on each spectrum and highlighted on the tryptic peptide. The known pD sites at D55 and D51 in OmpR
and BasR, respectively, are highlighted and starred (*) in red.
DBHA-enrichment of proteins via labelling of pD sites
presents an opportunity to directly interrogate changes in
phosphorylated proteins in parallel to, and distinct from,
monitoring global changes in protein abundance. To determine
whether DBHA-profiling could identify physiologically-relevant
changes in two-component signalling, we interrogated the well-
characterized EnvZ/OmpR pathway in E. coli. This pathway
facilitates sensing of extracellular osmolarity by the histidine
kinase and sensor protein EnvZ, which phosphorylates the
response regulator OmpR at D55 in response to increased
osmolarity. Phosphorylated OmpR exhibits markedly increased
DNA-binding activity and differential regulation of transcription of
the membrane porin proteins OmpF and OmpC (Figure S7)[24-
25].To track this activation directly in cells, E. coli growing in
standard LB media were switched to either 20% sucrose-
containing NB (high osmolarity) or NB alone (mock), and grown
for 12 hr. A minor fraction of each bulk proteome was trypsinized,
isotopically labelled with unique tandem-mass tag (TMT)
channels[26], and pooled to compare protein abundance changes
resulting from high osmolarity (Figure 3a). In parallel, the bulk of
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the native proteome from each condition was labelled with DBHA
probe, enriched on streptavidin-agarose resin and typrisinized on-
bead. The resulting tryptic peptides were labelled with unique
TMT channels and pooled for LC-MS/MS quantitation of
phosphorylation changes in response to high osmolarity (Figure
3a). Abundance changes for more than 1,200 proteins were
quantified between conditions (Figure 3b; Table S2). OmpR
protein levels were increased 1.4-fold in response to high
osmolarity, which matches literature precedent showing that
OmpR levels remain relatively constant or slightly increase in
response to increased osmolarity (Figure 3b)[27]. This abundance
profile also detected significantly decreased levels of OmpF
protein, consistent with repression by active OmpR. Significant
changes in the levels of other proteins involved in osmoregulation
were also apparent in the global profile, including the
transcriptional regulator BetI (downregulated in high osmolarity)
and the target of its repression, BetB (upregulated in high
osmolarity; Fig. 3b)[28-29]
. Quantification of DBHA-enriched
proteins, in contrast, identified OmpR as the most affected protein,
with nearly 4-fold higher levels of DBHA-enriched OmpR present
in the cells grown in 20% sucrose (Figure 3c; Table S2). Other
known response regulators that harbor pD sites, including CpxR
and ArcA, were detected and quantified in the enriched profile,
but were not affected by osmolarity at either the protein
abundance (Figure 3b) or pD level (Figure 3c). Combined, these
data validate that DBHA enrichment coupled to quantitative
proteomics enables global profiling of response regulator
phosphorylation, as opposed to abundance, in response to
physiological signals in E. coli.
Figure 3. DBHA-dependent LC-MS/MS enables global profiling of pD modification dynamics. (a) Proteomic workflow to quantify global changes in protein levels
(top) and pD or other DBHA-reactive modifications (bottom) by LC-MS/MS. Lysates from high and low osmolarity samples of E. coli are split for protein level
quantification (top) or treated with DBHA probe (bottom). This workflow thus generates two isotopically labelled tryptic peptide pools that provide quantitative
information on bulk protein level changes (top) and DBHA-sensitive modification levels (bottom). (b) Global quantification of protein level changes in response to
high osmolarity in E. coli. Proteins are ranked from most down-regulated (left) to up-regulated (right) under high osmolarity conditions. Proteins discussed in the text
are labelled in red with their quantitative ratio listed. (c) Quantification of DBHA-enriched proteins in response to high osmolarity in E. coli, thus reporting on specific
changes in modification status, in contrast to changes in abundance alone. The osmoregulatory response regulatory OmpR was the most affected protein in the
dataset. All data points in (b) and (c) represent the mean ratios from n = 4 (two technical of two biological) replicate runs.
In summary, we have developed a novel chemical proteomic
workflow that offers several advantages over current approaches
to interrogate phosphoaspartate sites, and likely other
electrophilic modifications, in native proteomes. First, peptide-
based enrichment and LC-MS/MS profiling provides unequivocal
identification of modified residues. This not only enables
quantitation of pD signalling dynamics at specific sites, but also
allows for exploratory mapping in unannotated proteomes and
organisms. In line with this attribute, the dataset herein contained
many high-confidence, reproducible DBHA-modified residues that
are not previously annotated as phosphoaspartate sites. These
sites may represent heretofore unrecognized phosphoaspartate
sites that are introduced through enzymatic or non-enzymatic
means, as has been observed for other intrinsically reactive
metabolic intermediates like acetylphosphate[30-32], which is used
in vitro to chemically phosphorylate Asp sites in response
regulators[33]. Beyond phosphoaspartate, other modifications
could contribute to the profile observed here, including
methylesterification, ADP-ribosylation, and potentially other less-
characterized PTMs that occur at aspartic acids in prokaryotes.
Despite this potential for overlap, we view the labelling of
additional electrophilic modifications as a feature not a flaw of
DBHA- profiling because the use quantitative LC-MS/MS provides
site-specific resolution necessary for follow up analysis at a site-
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by-site level. The dataset produced herein represents a starting
point for these future efforts. Finally, we have demonstrated the
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Acknowledgements
We thank M. Rust and G. Pattanayak for discussions surrounding
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The authors declare no conflict of interest.
Keywords: Chemoproteomics • Two-component signalling •
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Entry for the Table of Contents
Layout 1:
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Phosphoaspartate
modifications: A desthiobiotin-
Dr. Jae Won Chang, Jeffrey E.
Montgomery, Gihoon Lee, Prof.
Dr. Raymond E. Moellering*
containing
(DBHA)
covalently
hydroxylamine
chemical
labels
probe
modified
Page No. – Page No.
aspartic acid residues in native
Chemoproteomic Profiling of
Phosphoaspartate
Modifications in Prokaryotes
proteomes.
isotopic
Coupled
labelling,
with
DBHA-
dependent proteomic profiling
enables global quantification of
changes in phosphoaspartate
modification levels and protein
levels.
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