A chemical "hub" for absolute quantification of a targeted protein: Orthogonal integration of elemental and molecular mass spectrometry

Article abstract of DOI:10.1039/c3cc48460b

A novel element-tagged activity-based photo-cleavable biotinylated chemical "Hub" was designed and synthesized to orthogonally integrate ICP-MS and ESI-MS for absolute targeted-protein quantification. This tetrafunctional chemical "hub" allowed us to quantify a targeted protein using species-unspecific isotope dilution ICP-MS and to know which protein is being quantified using ESI-IT-MS at the same time. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc48460b

Showcasing research from Qiuquan Wang’s
Laboratory/Department of Chemistry, Xiamen University,
Xiamen, China
As featured in:
A chemical “hub” for absolute quantification of a targeted
protein: orthogonal integration of elemental and molecular mass
spectrometry
A novel chemical “hub” was designed and synthesized to
orthogonally integrate ICP-MS and ESI-MS for absolute
targeted-protein quantification. This tetrafunctional chemical
“hub” allowed us to know which protein is being quantified using
ICP-MS and ESI-MS.
See Qiuquan Wang et al.,
Chem. Commun., 2014, 50, 6578.
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A chemical ‘‘hub’’ for absolute quantification of a
targeted protein: orthogonal integration of
elemental and molecular mass spectrometry†
Cite this: Chem. Commun., 2014,
50, 6578
Received 5th November 2013,
Accepted 5th May 2014
Xiaowen Yan,a Zhaoxin Li,a Yong Liang,a Limin Yang,a Bo Zhanga and
Qiuquan Wang*ab
DOI: 10.1039/c3cc48460b
www.rsc.org/chemcomm
A novel element-tagged activity-based photo-cleavable biotinylated Compared with the ESI-/MALDI-MS, elemental mass spectrometry,
chemical ‘‘Hub’’ was designed and synthesized to orthogonally inte- especially the very hard ionization source based inductively coupled
grate ICP-MS and ESI-MS for absolute targeted-protein quantification. plasma mass spectrometry (ICP-MS), has been recently proved to be
This tetrafunctional chemical ‘‘hub’’ allowed us to quantify a targeted an attractive technique for absolute protein quantification using
protein using species-unspecific isotope dilution ICP-MS and to know only one element/isotope standard due to its unique feature that the
which protein is being quantified using ESI-IT-MS at the same time.
signal of an element/isotope naturally in and/or tagged on a protein
is independent of the kind and chemical form of the protein. This
After decades of development, ESI- and/or MALDI-based molecular means that any known species of the element/isotope can be used
mass spectrometry (ESI-/MALDI-MS) with isotope-labeling strate- as a standard for absolute protein quantification using species-
gies is widely used as a relative protein quantification tool in unspecific isotope dilution (SUID) ICP-MS.4 Unfortunately, ICP-MS
addition to its powerful structure-identification ability.1 Together is unable to provide any structural information about the protein to
with the use of stable isotope-labeled proteotypic peptides as be quantified. Because the protein might have and/or be tagged by
internal standards, ESI-MS with its triple-quadrupole mass analyzer an identical element/isotope, not only mutual separation of the
enables the absolute quantification of proteins through multiple targeted one from coexisting proteins but also the stoichiometry
reaction monitoring (MRM) of the proteotypic peptides.2 However, of the element/isotope in/on the protein must be known before
a high-quality MRM quantification calls for the synthesis of the ICP-MS quantification. In this case, the powerful ESI-/MALDI-MS
isotope-labeled proteotypic peptide standards for the targeted should be employed working together with ICP-MS to release the
proteins, whose sequences and properties must be well-known. ‘‘one standard protein quantification’’ advantage of ICP-MS, and
This implies that even known proteins are difficult to be quantified especially to know what protein is being quantified. In this regard,
with this latter technique if the proteotypic peptide standards could we need a chemical ‘‘hub’’ to orthogonally integrate ICP-MS and ESI/
not be accurately synthesized. Moreover, it is also extremely MALDI-MS rather than physically using them in parallel for achieving
difficult and expensive to synthesize the numerous isotope- absolute targeted-protein quantification and identification.
labeled proteotypic peptide standards for the hundreds and
thousands of proteins in a complex proteome.3
In this proof-of-concept study, we report the design and synthesis
of a novel activity-based element-tagged photo-cleavable biotinylated
Absolute protein quantification, as one of the most important chemical ‘‘hub’’ to orthogonally integrate ICP-MS and ESI ion trap
research directions of proteomics, can precisely number the protein MS (ESI-IT-MS) for the first time. In general, the tetrafunctional
copies in a cell or tissue sample, and thus describes the role of the chemical ‘‘hub’’ as shown in Scheme 1 comprises the following four
protein in a complex molecular network of life. It signifies not parts: (1) a sulfonyl fluoride as an example of guiding groups, which
only the exact content of a protein but also comprehensive has specificity towards the hydroxyl group of the serine residue at
molecular information concerning the protein to be quantified. the active site of serine proteases (SPs); (2) a Eu-loaded DOTA to
produce ICP-MS signals for protein quantification; (3) a conjugated
a Department of Chemistry and the MOE Key Laboratory of Spectrochemical
Analysis & Instrumentation, College of Chemistry and Chemical Engineering,
Xiamen University, Xiamen 361005, China. E-mail: qqwang@xmu.edu.cn;
Fax: +86(0)599-2187400
biotin for being captured by streptavidin-coated beads; and (4) a
photo-cleavable o-nitrobenzyl ether linker to release the captured-SP
upon UV irradiation for ESI-IT-MS identification. To build all these
parts into the tetrafunctional chemical ‘‘hub’’, we designed a
synthetic route as illustrated in Fig. 1 (see details in the ESI†).
Briefly, 4-[4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy] butyric acid
(1) was first conjugated with N-Fmoc-1,4-butanediamine (2) to
b State Key Laboratory of Marine Environmental Science, Xiamen University,
Xiamen 361005, China
† Electronic supplementary information (ESI) available: Experimental details and
supporting figures. See DOI: 10.1039/c3cc48460b
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DOTA-NHS (9) to give 10. Next, compound 5 was conjugated with 10
to produce Fmoc-nitrobenzyl-DOTA-azide (11). The Fmoc was sub-
sequently removed from the amine group of compound 11 and
reacted with (+)-Biotin-PEG4-NHS to obtain biotin-nitrobenzyl-
DOTA-azide (12). The Eu3+-loaded 12 was finally conjugated
with compound 13 through the Copper-Catalyzed Azide–Alkyne
Cycloaddition (CuAAC) reaction5 to obtain the tetrafunctional
chemical ‘‘hub’’ 14, biotin-PEG-nitrobenzyl-DOTA-Eu-SF. It was
stable not only during its use but also up to 6 months (longer
time was not tested) if we dissolved it in anhydrous DMSO and
kept in the dark at ꢀ20 1C. The details of the synthetic
processes, purifying method and the related data including
HPLC-UV/MS, 1H-NMR and 13C-NMR are all described in the
ESI† (Fig. S1–S18).
In order to investigate the cleavage efficiency of the ‘‘hub’’ 14,
1 mM 14 in 100 mM Tris-HCl (pH 7.5) was analyzed using HPLC-UV/
ESI-IT-MS without and with the irradiation of UV light (l = 365 nm)
for 20 min. The results obtained indicated that the intact 14 was
eluted at 28 min on a C18 column (1.0 I.D. ꢁ 15.0 mm in length)
(Fig. 2a) under the optimized separation conditions (see the ESI†).
The deconvolution molecular weight (DM) of 14 was determined to
be 1957 Da using ESI-IT-MS (Fig. 2c), identical to its theoretical
molecular weight (TM, 1957 Da); and neither cleavage product nor
forfeiture of Eu3+ was observed, indicating that 14 was stable
during chromatographic separation. From the HPLC chromato-
gram shown in Fig. 2b, on the other hand, we could see that
almost all of 14 disappeared after 20 min of UV irradiation, and
the cleavage efficiency was calculated to be 99% by comparing
the peak areas of 14 before and after UV irradiation. One
cleaved product of 14 was found to be eluted at a retention
time of 23 min (Fig. 2b), and was assigned to be the SF-DOTA-Eu
moiety (TM = 1088 Da) (Fig. 2d), which is consistent with the
photocleavage mechanism of the o-nitrobenzyl ether compound
(Fig. S19 in the ESI†).6 We did not find the other part of UV-cleaved 14.
The reason was speculated to be the low ionization efficiency of this
Scheme 1 Chemical structure of the tetrafunctional chemical ‘‘hub’’ and
procedures for applying it for specifically tagging and capturing as well as
releasing the targeted protease for quantification and identification using
ICP-MS and ESI-MS.
produce compound 3, the hydroxyl group of which was activated
with N,N0-disuccinimidyl carbonate (4) to obtain the photocleavable
linker Fmoc-nitrobenzyl-NHS (5). To synthesize the Eu-chelating
moiety amine-DOTA-azide (10), N-Boc-6-azido-L-norleucine (6) was
first conjugated with 2 to produce compound 7, whose amino group
was deprotected by removing the Boc group in TFA and reacted with
Fig. 2 HPLC-UV (214 nm) chromatograms (a and b) of the tetrafunctional
chemical ‘‘hub’’ 14 and its mass spectra (c and d) before and after UV
irradiation. DM and TM in the mass spectra denote devolution and theoretical
molecular weights.
Fig. 1 Synthetic route of tetrafunctional chemical ‘‘hub’’ 14. Details of the
synthetic procedures are presented in the ESI.†
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biotin-containing part in ESI-IT-MS. In any event, 14 was proven
to undergo swift photolysis under UV irradiation.
The tagging efficacy of 14 towards a typical SP, bovine
chymotrypsin with a DM value of 25 438 Da (Fig. S20a in ESI†)
that was selected as an example in this proof-of-concept study,
was then investigated using ESI-IT-MS. After incubation of
1 mM 14 with freshly prepared chymotrypsin in 100 mM Tris-
HCl (pH 7.5) for 1 hour, the DM value of the tagged chymotrypsin
was found to be 27 376 Da, matching the TM value of 27 375
[25 438 (chymotrypsin) + 1957 (14) – 20 (HF)] Da well (Fig. S20b in
ESI†), suggesting that one chymotrypsin molecule was tagged with
one 14. The difference of 1 Da between the DM and TM values was
due to the relative low-mass resolution of the ESI-IT-MS used in
this study. This result proved that the sulfonyl fluoride group in 14
has high specificity to the hydroxyl group of the serine residue at
the active site of chymotrypsin,7 even though there are numbers of
serine residues and hydroxyl groups in other parts of this protein,
demonstrating an excellent intra-hydroxyl group selectivity.
Subsequently, a sample containing seven non-SP proteins
(RNase A, insulin, Cyt c, lysozyme, BSA, carbonic anhydrase and
ovalbumin) and one SP (chymotrypsin) was chosen to further
evaluate the inter-protein specificity of 14 (Table S1 in the ESI†) via
HPLC coupled with UV detection (214 nm) and 153Eu-SUID ICP-MS.
As shown in Fig. 3a, the eight proteins were separated on a C18
column (see the details in the ESI†), whereas the retention time of
14 was the same as that of Cyt c coincidentally, and BSA partly
overlapped with chymotrypsin under the HPLC conditions
employed in this study. When the effluent from the C18 column
was mixed on-line with the enriched 153Eu standard solution
through a three-way connector, and directly analyzed by ICP-MS
monitoring of the signals of 153Eu and 151Eu isotopes without
protein capture and release steps, only chymotrypsin was deter-
mined (Fig. 3b) due to the element-specific feature of ICP-MS,
suggesting that only chymotrypsin was tagged by 14 and demon-
strating the activity-based inter-protein specificity of 14 towards
chymotrypsin even though there are many serine residues in the
other seven proteins. It should be pointed out that although BSA,
which is a high abundant protein in biological samples, overlapped
with chymotrypsin during the HPLC separation with UV detection,
the interference was blinded when using ICP-MS. This is very
important considering the limited separating ability of current
available separation techniques that frequently encountered the
Fig. 3 HPLC chromatograms of a 14-tagged protein sample determined
using (a) UV (214 nm) and (b) 153Eu species-unspecific isotope dilution ICP-MS,
(c) Eu mass flow chromatogram of the 14-tagged protein transformed from
(b) based on the online isotope dilution equation, (d) the supernatant after the
protein sample was labeled with 14 and captured using streptavidin-coated
magnetic beads, (e) the supernatant determined using UV (214 nm) after the
captured SF-DOTA-Eu tagged chymotrypsin was released after 20 minutes
of UV (365 nm) irradiation, and (f) ESI-IT-MS of peak 6 in (e). 1, RNase A;
2, insulin; 3, Cyt c; 4, lysozyme; 5, BSA; 6, chymotrypsin; 7, carbonic
anhydrase; 8, ovalbumin.
problem to achieve a quantitative separation of the individual isotope dilution equation.8 A limit of detection (LOD, 3s) of
proteins in a complex biological sample. Besides, although the 0.5 fmol chymotrypsin was obtained with RSD lower than 3% (n = 5
signal of 14 might overlap the possible signal of Cyt c in the sample, at pmol level). This LOD is about 40-fold higher than the sensitivity
no signal was observed in the same retention time after 14 was of conventional enzyme-linked immunosorbent assay (ELISA)
UV-cleaved (data not shown here). It is worth mentioning that, (21 fmol), and comparable to the state-of-the-art single-molecule
furthermore, the signal intensity of Eu decreased along with the ELISA (0.4 fmol),9 besides the advantage that this chemical ‘‘hub’’
increase in the acetonitrile content in the HPLC mobile phase as integrated ICP-MS and ESI-IT-MS offers both quantitative and
shown in Fig. 3b, which suggested that the signal response of Eu is structural information. The concentration of chymotrypsin in the
lower at higher retention time (higher acetonitrile content). This mixed protein sample, prepared from the crude chymotrypsin
effect can be effectively calibrated by SUID, since the signal of 151Eu solid powder obtained from Sigma-Aldrich, was determined to be
representing the tagged protein in the sample and that from the (17.1 ꢂ 1.0) mM by integrating the peak area of chymotrypsin as
spiked 153Eu standard solution are affected to the same extent. The shown in Fig. 3c, which corresponded to the chymotrypsin content
chromatograms of 151Eu and 153Eu isotopes were then transformed of (86.9 ꢂ 5.1)% in the crude solid powder with the recovery of
into the Eu mass-flow chromatogram (Fig. 3c) using the online (91.3 ꢂ 5.4)% according to the data provided by the producer.
6580 | Chem. Commun., 2014, 50, 6578--6581
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Finally, in order to evaluate the fishing and releasing ability of chemical ‘‘hub’’ can be easily extended to the quantification and
the chemical ‘‘hub’’ 14 and to perform ESI-IT-MS identification to identification of other targeted proteases such as cysteine, threo-
know what protein is being quantified by ICP-MS, the above protein nine and aspartic proteases.11 We believe that this chemical ‘‘hub’’
sample was used again. The sample was incubated with 14, strategy sets an example, and will open the opportunity not only to
followed by fishing with streptavidin-coated magnetic beads and acquire quantitative information of a targeted known protein but
washing with Tris-HCl buffer (Scheme 1 and details in the ESI†). As also to discover new members in a certain family of proteins, thus
shown in Fig. 3d, the seven proteins except chymotrypsin were providing more and more comprehensive information concerning
observed in the chromatogram, demonstrating a very efficient the proteins for further biological and medical studies in the
fishing of chymotrypsin. After the magnetically separated beads near future.
were resuspended in Tris-HCl buffer and irradiated under UV light
This study was financially supported by the National Basic
for 20 min, the supernatant obtained was analyzed again (Fig. 3e, Research 973 Program (2014CB932004) and the National Natural
peak 6), and the HPLC effluent was subsequently infused into Science Foundation of China (21035006 and 21275120). We
ESI-IT-MS for identification (Fig. 3f). The DM value of the analyte in thank Prof. John Hodgkiss of The University of Hong Kong for
peak 6 was found to be 26 507 Da, which is consistent with the helping with the English in this article.
TM value of 26 506 Da of DOTA-Eu-SF-tagged-chymotrypsin, con-
firming that the protein in peak 6 was chymotrypsin. All these
results demonstrated that 14 was an effective tetrafunctional
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´
´
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