Specific enrichment of phosphoproteins using functionalized multivalent nanoparticles

Article abstract of DOI:10.1021/ja511833y

Analysis of protein phosphorylation remains a significant challenge due to the low abundance of phosphoproteins and the low stoichiometry of phosphorylation, which requires effective enrichment of phosphoproteins. Here we have developed superparamagnetic

Full text of DOI:10.1021/ja511833y

Communication
pubs.acs.org/JACS
Specific Enrichment of Phosphoproteins Using Functionalized
Multivalent Nanoparticles
Leekyoung Hwang, Serife Ayaz-Guner, Zachery R. Gregorich, Wenxuan Cai,‡,§ Santosh G. Valeja,‡
†
‡
‡,§
,†
,†,‡,§,∥
Song Jin,* and Ying Ge*
†
‡
§
Department of Chemistry, Department of Cell and Regenerative Biology, Molecular and Cellular Pharmacology Program, and
∥
Human Proteomics Program, University of Wisconsin−Madison, Madison, Wisconsin 53719, United States
*
S Supporting Information
ABSTRACT: Analysis of protein phosphorylation re-
mains a significant challenge due to the low abundance
of phosphoproteins and the low stoichiometry of
phosphorylation, which requires effective enrichment of
phosphoproteins. Here we have developed superparamag-
netic nanoparticles (NPs) whose surface is functionalized
by multivalent ligand molecules that specifically bind to
the phosphate groups on any phosphoproteins. These NPs
enrich phosphoproteins from complex cell and tissue
lysates with high specificity as confirmed by SDS-PAGE
analysis with a phosphoprotein-specific stain and mass
spectrometry analysis of the enriched phosphoproteins.
This method enables universal and effective capture,
enrichment, and detection of intact phosphoproteins
toward a comprehensive analysis of the phosphoproteome.
rotein phosphorylation, one of the most common and
P
important post-translational modifications, plays a pivotal
role in the control of many biological processes, including cell
1
growth, division, and signaling; and aberrant phosphorylation
2
has been implicated in the pathogenesis of human diseases.
Figure 1. (A) Schematic illustration of the synthesis and functionaliza-
Therefore, a comprehensive analysis of phosphoproteins is
essential for understanding cellular biology and disease
mechanisms. However, analysis of protein phosphorylation is
challenging mainly due to the low abundance of phosphopro-
tion of Fe O NPs. GAPT-Zn chelate groups have strong binding to
3
4
phosphate groups. Molecules and NPs are not drawn to scale. (B) TEM
image and (C) PXRD pattern of as-synthesized Fe -OA/OE NPs.
Inset shows the size distributions of the Fe O -OA/OE (red) and
O
4
3
3
4
3
Fe O -GAPT-Zn (blue) (D) FTIR of Fe O -OA/OE (blue), Fe O -
teins and the low stoichiometry of phosphorylation. Mass
3 4 3 4 3 4
+
NH /PEG (red), and Fe O -GAPT (green) NPs. (E) TEM image of
3
3
4
spectrometry (MS) has become the method of choice for the
3
,4
the functionalized Fe O -GAPT-Zn NPs. (F) TGA analysis of Fe O -
3
4
3
4
analysis of protein phosphorylation, but it is nearly impossible
to analyze phosphoproteins directly from the complex proteome
by MS without a specific enrichment procedure.
OA/OE(blue), APTMS and Si-PEG (red), and GAPT coupled to
APTMS (green). (G) Functionalized Fe O -GAPT-Zn NPs well-
3
4
dispersed (left) and collected by a magnet (right) in aqueous solutions.
In the past decade, a plethora of methods have been developed
for the enrichment and MS analysis of phosphopeptides from
7
a,c
3
−5
affinity chromatography (IMAC)
can enrich proteins with
protein digests. The digestion of each protein into hundreds
of peptides significantly increases the overall complexity. In
contrast, enriched intact phosphoproteins can be directly
analyzed by top-down MS for comprehensive characterization
phosphorylated Ser, Thr, and Tyr residues without bias;
however, they have low specificity, efficiency, and poor
reproducibility. Thus, the effective capture and universal
enrichment of phosphoproteins from complex mixtures remains
a significant challenge. Herein, we have designed and synthesized
functionalized multivalent superparamagnetic nanoparticles
NPs) to universally and effectively enrich phosphoproteins
from complex mixtures. The effectiveness of NP-enabled
8
2b,6
7
of phosphorylation. However, very few methods are available
for the enrichment of phosphoproteins and each has different
major drawbacks. Although phospho-specific anti-phosphotyr-
7
a
(
osine antibodies have high affinity, the enrichment of
phosphoserine/threonine containing proteins (which represent
3
,4
>
99% of all phosphoproteins) remains a challenge due to the
4a,7a
low affinity and specificity of phospho-Ser/Thr antibodies.
Received: November 18, 2014
The enrichment strategies employing immobilized metal ion
©
XXXX American Chemical Society
A
DOI: 10.1021/ja511833y
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
phosphoprotein enrichment from complex cell and tissue lysates
was demonstrated by SDS-PAGE analysis with a phosphopro-
tein-specific stain, and furthermore by top-down MS analyses of
the enriched phosphoproteins (Figure S1).
In our approach, we utilized superparamagnetic iron oxide
(
(
magnetite, Fe O ) NPs with a small diameter ranging 4−6 nm
3
4
Figure 1). The small size of the NPs gives them a high surface-
to-volume ratio, which allows for easy modification of the NPs’
surface with multivalent ligand molecules properly designed to
9
have a specific interaction with target proteins. Furthermore, the
size of NPs is comparable to the size of proteins, therefore
enabling the NPs to (i) penetrate better in protein mixtures,
leading to a higher binding rate; (ii) reduce the probability that
9b,c,10
proteins are denatured; and (iii) have good solubility.
Furthermore, Fe O NPs of this small size (below 20 nm) are
3
4
1
1
Figure 2. (A) Workflow of the capture and enrichment strategy using
the functionalized NPs. (B) SDS-PAGE analysis with Sypro Ruby (top)
and Pro-Q Diamond (bottom) staining confirmed the highly specific
enrichment of phosphoproteins (β-casein and pepsin) from standard
protein mixtures containing non-phosphoprotein (BSA). M, molecular
markers; LM, loading mixture before enrichment; FT, flow-through;
and E, elution after enrichment. (C,D) Global enrichment of
phosphoproteins from (C) HEK 293 cell lysate (equal amount loading,
superparamagnetic; therefore, unlike bulky micron-sized
magnetic beads, the NPs are not spontaneously magnetic
without external magnetic field. This property prevents the
NPs from aggregating by themselves due to mutual magnetic
attraction, but enables them to aggregate and precipitate readily
under applied magnetic fields. Crucial to the specific capture,
enrichment, and release of the phosphoproteins is the use of a
dinuclear Zn (II)-dipicolylamine (Zn-DPA) complex as an
affinity ligand to specifically bind to the phosphate groups
1
5 μg) and (D) swine heart tissue extract (equal amount loading, 10 μg)
as demonstrated by SDS-PAGE analysis with Sypro Ruby (left side) and
Pro-Q Diamond staining (right side).
(
Figure 1A). The Zn-DPA coordination complex has a vacancy
2
−
on two Zn metal ions that the phosphate dianion (ROPO ) can
access to form the complex ROPO3 -(2Zn-DPA) , and
3
2
−
3+
14a
the existence of Si-O-R and C-O-C, EO CH , and the sharp
2
−
1
therefore is known to exhibit a high affinity toward anionic
peak around 1630 cm of Fe O -GAPT NPs proved the
3
4
2
−
−
phosphorylated chemical species (against SO , CH COO ,
4
3
existence of the amide bond (Figure 1D). In addition,
thermogravimetric analysis (TGA) of the as-synthesized Fe O ,
−
12
and Cl ) in aqueous solution at a neutral pH. Therefore,
3
4
2
+
+
acrylamides containing copolymerized M -DPA complexes (so-
called “phos-tag”) have been used for phosphate-affinity gel
electrophoresis and visualization of proteins with phosphoryl-
ated Ser, Thr, and Tyr residues without bias.
We synthesized Fe O NPs functionalized with Zn-DPA
Fe O -NH /PEG, and Fe O -GAPT NPs showed a different
3
4
3
3
4
weight loss (%) for each of the surface-modified NPs that was
commensurate with the corresponding ligand molecules (Figure
1F). After activating Fe O -GAPT NPs with Zn ions, we used X-
12
3
4
3
4
ray photon spectroscopy (XPS) to observe the characteristic Zn
2p peak at 1020 eV (Figure S2) and confirm the presence of Zn
on the NP surface. The resulting NPs can be dispersed in
aqueous solution and gathered using a magnet (Figure 1G).
Next, we performed phosphoprotein enrichment experiments
using the functionalized Fe O -GAPT-Zn NPs (Figure 2A) as
ligands that are linked via glutaric acid (hereafter referred to as
GAPT), as illustrated in Figure 1A. First, oleic acid (OA) and
oleylamine (OE)-capped Fe O NPs were synthesized as
3
4
13
reported previously (see SI for detailed procedures). Trans-
mission electron microscopy (TEM) imaging (Figure 1B)
revealed that the size of as-synthesized Fe O -OA/OE NPs
3
4
3
4
follows: (1) mixing and agitating the protein mixture with the
NPs, (2) removing the unbound non-phosphoproteins as flow-
through, and (3) elution of the phosphoproteins. We tested the
phosphoprotein enrichment from standard protein mixtures
containing either β-casein or pepsin (both of which are
phosphoproteins) and bovine serum albumin (BSA, a non-
phosphoprotein) in the same mass-to-volume ratio. The best
enrichment results were obtained using a buffer solution
containing 50 mM HEPES and 150 mM NaCl (pH 7.7) for
binding and subsequent washing, and a buffer containing 100
mM Na HPO and 200 mM NaCl (pH adjusted to 7.3 with HCl)
was 4.6 ± 0.7 nm (red size distribution histogram in Figure 1C
inset). Powder X-ray diffraction (PXRD) confirmed the Fe O4
3
NPs to be magnetite (Figure 1C). Next, the hydrophobic OA and
OE ligands on these intermediate Fe O NPs were exchanged
3
4
with 3-aminopropyl trimethoxysilane (APTMS) and 2-methoxy
polyethyleneoxy) propyl trimethoxysilane (hereafter referred to
(
+
14
as Si-PEG), to produce Fe O -NH /PEG (Figure 1A, P1).
3
4
3
These PEG groups, which contain a polyethyleneoxy moiety,
decrease nonspecific interactions between proteins and the NPs
15
and improve the solubility of the NPs in aqueous buffers.
Subsequently, the Zn-chelating ligand molecules, GAPT, which
2
4
for elution. The large excess of phosphate ions in the elution
buffer out-competes the phosphate groups on the captured
phosphoproteins for binding to the NPs, thereby releasing the
phosphoproteins.
16
were synthesized following a previous report, were coupled to
the free amino groups of the APTMS molecules on the NPs.
Finally, the Fe O -GAPT NPs (Figure 1A, P2) were activated
3
4
with 10 mM ZnCl (aq) to generate Fe O -GAPT-Zn NPs
We used SDS-PAGE to demonstrate the selective enrichment
of phosphoproteins by the functionalized NPs (Figure 2B). The
gel was first stained by Pro-Q Diamond fluorescent dye to
visualize phosphoproteins and then by Sypro Ruby dye to
observe the profile of total proteins (including both non-
phosphoproteins and phosphoproteins) in the gel. After
enrichment, in the elution solutions the bands corresponding
to β-casein and pepsin were far more prominent, whereas the
2
3
4
(
Figure 1A, P3). TEM showed that the average size of Fe O -
3 4
GAPT-Zn NPs increased to 5.4 ± 0.6 nm (blue histogram in
Figure 1C inset) except that its aggregation was slightly increased
(Figure 1E). Fourier transform infrared spectroscopy (FTIR)
was used to confirm the proper functionalization of NPs with the
−
1
ligand molecules. The broad and strong band around 1022 cm
−1
and peaks around 1200 cm of Fe O -NH /PEG NPs indicated
3
4
3
B
DOI: 10.1021/ja511833y
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
non-phosphoprotein, BSA, was nearly absent (Figure 2B, top). In
contrast, the flow-through solution contained significantly higher
amount of BSA. Moreover, Pro-Q Diamond stained only β-
casein or pepsin, not BSA (Figure 2B, bottom), confirming that
the eluted β-casein and pepsin are indeed phosphoproteins.
These results show that the Fe O -GAPT-Zn NPs can selectively
3
4
bind to phosphoproteins in mixtures containing non-phospho-
proteins. To confirm the importance of the metal-ion chelating
group GAPT-Zn for the specific binding to phosphate groups, we
compared protein binding using three different control NPs: (1)
Figure 3. Representative intact protein MS spectra, before and after
enrichment, confirming the highly specific enrichment of phosphopro-
tein from a swine heart tissue extract. (A) Low abundance
phosphoprotein that is not detectable before enrichment (top) is
detected after enrichment (bottom). (B) Phosphoprotein with very low
phosphorylation occupancy that the unphosphorylated form (Un-P) is
predominant before the enrichment but the phosphorylated form (+P)
2
+
Fe O -GAPT NPs that were not activated by Zn ; (2) Fe O -
3
4
3
4
+
NH /PEG NPs that were functionalized with positively charged
3
ligands, but without GAPT ligands; and (3) Fe O -PEG NPs that
3
4
were functionalized only with PEG groups. All of the control NPs
showed poor affinity or reversible binding toward phosphopro-
teins in the same enrichment experiments with the standard
protein mixtures (Figure S3). These control experiments
unequivocally confirmed that the specific binding of phospho-
proteins occurs via interactions between the phosphate group
becomes dominant after the enrichment. M = most abundant molecular
r
weight. +HPO , the covalent addition of a phosphate group (+80 Da).
3
and the GAPT-Zn ligand complex on the surface of the Fe O -
GAPT-Zn NPs.
3
4
enrichment by LC-MS. The LM and E solutions from swine
heart tissue extract without digestion were desalted, concen-
trated, and separated by reverse phase chromatography.
Subsequent MS analysis of the LM revealed that most of the
detected proteins are highly abundant blood proteins, such as
hemoglobin subunit α (see Figure S6) or β, and myoglobin.
However, these blood proteins were either not detected or
dramatically decreased by MS in the E solution (Figure S7A).
This suggests that these highly abundant non-phosphoproteins
were not captured by the NPs and, consequently, were removed
during the washing step.
To further evaluate the specificity of the phosphoprotein
enrichment, we systematically increased the mass ratio of BSA:β-
casein from 9:1 to 99:1 while holding the amount of β-casein
constant at 200 μg. The β-casein was clearly enriched even in the
mixture containing an overwhelming amount of BSA (BSA:β-
casein = 99:1) (see Figure S4A,B). We determined the
percentage of protein recovery and the enrichment factor
(defined as the gain in the relative ratio of the phosphoprotein to
non-phosphoprotein) to assess the performance of phospho-
protein enrichment (Figure S4C,D). For a mixture of 99:1 BSA
to β-casein, the enrichment factor was over 140-fold (Figure
S4D), which could almost be considered “purification” of
phosphoproteins. The enrichment performance of Fe O -
Importantly, the top-down MS data clearly showed that
phosphoproteins in swine heart tissue extracts were enriched by
the Fe O -GAPT-Zn NPs, even in the presence of highly
3
4
3
4
abundant blood proteins (vide supra). Many of the detected
phosphoproteins have very low abundance in comparison to
non-phosphoproteins and/or low stoichiometry (low phosphor-
ylation occupancy) in the pre-enrichment samples; however,
these phosphoproteins were significantly enriched in the post-
enrichment samples, some with more than one phosphorylation
detected for the same protein (mass increases of multiples of 80
Da) (Figures 3 and S7B). As a representative example, after
enrichment, a protein with M 11 657.52 (M , most abundant
GAPT-Zn NPs was also compared with an IMAC-based
phosphoprotein enrichment kit (Thermo). For the same
enrichment experiment of a mixture of 99:1 BSA to β-casein,
our NPs showed significantly reduced nonspecific binding and
greatly outperformed this IMAC-based material (Figure S5).
We further assessed the enrichment of phosphoproteins from
human embryonic kidney (HEK) 293 cell lysate (Figure 2C) and
swine heart tissue extract (Figure 2D), two highly complex
mixtures, using the Fe O -GAPT-Zn NPs. We loaded equal
r
r
3
4
molecular weight) was detected by top-down MS together with
two additional peaks with 80 Da mass increases (labeled +HPO3)
(Figure 3A, bottom), which correspond to multiple phosphory-
lated forms of the protein. However, none of these peaks were
detected in the MS before enrichment (Figure 3A, top) implying
they are all phosphorylated protein forms that have low
abundance. Another representative MS of the original protein
amounts of proteins before and after enrichment on the SDS-
PAGE gel, stained with Pro-Q Diamond first, destained, and
restained with Sypro Ruby (for total protein detection). Despite
the equal amount loading as confirmed by the similar total
intensities of the loading mixture before enrichment (LM), flow-
through (FT), and the elution after enrichment (E) lanes stained
by Sypro Ruby, the Pro-Q Diamond-stained LM and FT lanes
showed significantly lower intensity than the E lane, suggesting
that most of the proteins in the LM and FT are non-
phosphoproteins. Furthermore, the banding patterns of the E
lane stained with both Pro-Q Diamond and Sypro Ruby are
highly similar, in contrast to the LM and FT lanes which are very
different between the Pro-Q Diamond and Sypro Ruby stains,
indicating the enriched proteins are predominantly phospho-
proteins. These results clearly indicate that the Fe O -GAPT-Zn
mixture before enrichment displayed a protein with M 8808.18
r
(Figure 3B, top, labeled Un-P). After enrichment, a protein with
M 8888.19 with a mass increase of 80 Da was detected in the MS
r
instead (Figure 3B, bottom, labeled +P). This clearly shows that
the relative abundance of the phosphorylated species (Mr
8888.19) is significantly increased compared to the non-
phosphorylated species (M 8808.18) after enrichment. It should
r
2+
also be noted that a few Zn -binding proteins, such as a
17
parathymosin-like protein (Figure S8), were also detected in
the E fractions due to its high affinity to GAPT-Zn ligands on the
NPs. Thus, top-down MS analysis confirmed that the number
and amount of phosphoproteins in the E fraction were
significantly increased compared to the LM, which was
dominated by overwhelmingly abundant blood proteins. The
3
4
NPs can specifically and effectively enrich phosphoproteins from
complex biological samples with high affinity and efficiency.
To demonstrate our NP enrichment strategy is compatible
with top-down MS, we have examined the intact proteins present
in the complex swine heart tissue extracts before and after
C
DOI: 10.1021/ja511833y
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
fact that no high-mass phosphoproteins were identified in this
top-down MS study is mostly likely due to the exponential decay
in the signal-to-noise ratio that occurs with increasing mass in
ACKNOWLEDGMENTS
■
This work was supported by the NIH R21 EB013847 (to both
S.J. and Y. G.). Y. G. also thanks the support by NIH R01
HL096971 and R01 HL109810. We would like to thank Ying-
Hua Chang for critical reading of this manuscript and Yi Zhang
for assistance in NPs synthesis.
1
8a
high-resolution MS
and the use of Q-Exactive mass
spectrometer, which is not optimized for high-mass protein
1
8b
detection. However, high-mass proteins (20−150 kDa) were
clearly detected by SDS-gel analyses of the E fractions after
phosphoenrichment from HEK293 cell lysates (Figure 2C) and
swine heart tissue extracts (Figure 2D). This confirms that high-
mass phosphoproteins were indeed enriched by the NPs.
To recapitulate, we have developed superparamagnetic NPs
that are functionalized with multivalent ligands to capture and
enrich intact phosphoproteins with high specificity and efficacy.
Such functionalized NPs can have many advantages for
phosphoprotein enrichment: (a) They have a high surface area
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(
(
4
9
,10
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(
4
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1
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*
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*
*
Corresponding Authors
jin@chem.wisc.edu
ge2@wisc.edu
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Notes
The authors declare no competing financial interest.
D
DOI: 10.1021/ja511833y
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX