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ICPMS-based Specific Quantification of Phosphotyrosine: A Gallium-tagging and Tyrosine-phosphatase Mediated Strategy Nannan Tang, Zhaoxin Li, Limin Yang, and Qiuquan Wang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02979 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 6, 2016

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Analytical Chemistry

ICPMS-based Specific Quantification of Phosphotyrosine: A Gallium-tagging and Tyrosine-phosphatase Mediated Strategy Nannan Tang,† Zhaoxin Li,† Limin Yang,† and Qiuquan Wang*,†,‡ †

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: [email protected]; phone: +86 (0)592 2181796 ‡ State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China

ABSTRACT: Low-abundance tyrosine phosphorylation is crucial to not only normal but also aberrant life processes. We designed and synthesized a photocleavable magnetic nanoparticle-based gallium tag for tagging and enrichment as well as UVrelease of the phosphate-bearing molecules/ions in cells. HPLC/71Ga speciesunspecific isotope dilution (71Ga-SUID) ICP-MS was subsequently developed for specific and absolute quantification of phosphotyrosine (pY) under the assistance of a protein tyrosine phosphatase-1B (PTP-1B). pY quantification was thus achieved via determination of Ga in the Ga-phosphate complexes that come exclusively from the Ga-tagged pY. In this way, the method detection limit of pY reached down to 30 amol with the RSD lower than 5.70 % (n = 5 at pmol level). Feasibility of this proposed method was validated using VNQIGTLSEpYIK, VNQIGTLpSEpYIK and extracellular regulated protein kinase 1 peptide (pTEpY-) standards with the recovery more than 96 % (n = 5). It was applied to the absolute quantification of pY in human breast cancer MCF-7 cells, indicating that pY increased by 1.60 nmol (61.1 %) in 3.0 × 106 MCF-7 cells after 100 nM insulin stimulation. We believe that, not limited to pY quantification, this element-tagging and proteasespecific reaction mediated ICP-MS methodology will pave a simple path for ever more applications of ICP-MS to the studies of quantitative protein post-translational modifications (PTMs) when suitable element-tags are designed and specific proteases are available towards targeted PTMs. Protein phosphorylation is one of the most important posttranslational modifications (PTMs) involved critically in many significant cellular processes.1 It predominantly takes place at serine, threonine and tyrosine residues of the proteins in eukaryotes at any given time, and their relative abundance of phosphoserine (pS), phosphothreonine (pT) and phosphotyrosine (pY) have been estimated to be 86.4 %, 11.8 % and 1.8 %.2 Despite lower abundance compared with pS and pT, pY signaling is more tightly regulated and plays significant roles in not only normal life processes but also in the mediation of disease pathogenesis and oncogenic transformation.3,4 In order to determine pY for understanding the mechanism of tyrosine phosphorylation and its biological roles, SILAC and iTRAQ as well as stable isotope dimethyl labeling have been recently applied to reflect the relative ratio of the tyrosine-phosphorylated peptides/proteins using soft ionsource electrospray ionization tandem mass spectrometry (ESIMS/MS) together with necessary enrichment based on the immunoprecipitation with anti-phosphotyrosine antibodies and/or immobilized metal affinity chromatography (IMAC).5-9 Selected reaction monitoring (SRM) and/or multiple reaction monitoring (MRM) with synthesized individual isotopic signature peptide standards could achieve absolute quantification of multisite phosphorylation.10,11 Actually, one has to bear the cost of the assay in addition to the risk arising from possible imprecise and incomplete proteolysis; and sensitivity of these ESI-MS/MS based approaches still needs to be improved. On the other hand, highly sensitive inductively coupled plasma mass spectrometry (ICPMS) has a unique feature that signal intensity of an element/isotope is almost independent of its chemical forms because all molecules in the sample are eventually converted into atoms and elemental ions in the hard ICP ion-source. Therefore, accurate isotope dilution quantification can be performed using only one

species-unspecific isotope standard.12 More importantly, a novel concept of chemical hub proposed for integrating structureselective ESI-MS and element/isotope-specific ICP-MS allows one to know what have been quantified by ICP-MS.13 State-ofthe-art chemoselective and biospecific element-tagging strategies developed in the last two decades enable ICP-MS to be a simple and effective tool for the detection and quantification of proteins, nucleic acids and even cells.14-27 Evaluation of the global peptide/protein phosphorylation (pS + pT + pY) degree was also performed by determining the stoichiometric phosphorus to sulfur (31P+ to 32S+) ratio, and naturally occurring phosphorus with bis(4nitrophenyl) phosphate as an internal standard; the inevitable polyatomic interferences such as 14N16O1H+ to the monoisotopic 31 + P could be overcome using dynamic reaction cell and tandem ICP-MS via monitoring 31P16O+.28-31 However, the relative low ionization efficiency (33 %) of phosphorus in Ar-based ICP due to its relative high first ionization potential (10.49 eV) still restrict further improvement of detection limit (DL).32 More seriously, specific quantification of pY out of the global protein phosphorylation using ICP-MS has not been achieved to date when considering the coexistence of not only pS and pT but also other phosphate-containing molecules/ions (pX) in a complicated biological sample. As we know, Ga-associated IMAC demonstrated a highly efficient enrichment of pS, pT and pY as well as pX such as phospholipids prior to MS, and stability of the peptide phosphate ester bond during collision-activated dissociation ESI-MS/MS could be improved by the protection of a dinuclear Ga-complex, suggesting a highly specific interaction between Ga and phosphate ester group with an apparent association constant of (3.08 ± 0.31)×106 M-1.33-35 Moreover, Ga has no biological background; and its low-

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er first ionization potential (5.99 eV) compared with those of P and Ar (15.76 eV) results in almost 100 % ionization efficiency in Ar-based ICP-MS with two stable isotopic mass signals (69Ga+ and 71Ga+). Therefore, Ga might be an exogenous element-tag of choice for further improving the DLs of pS, pT and pY as well as pX using ICP-MS. Different from our previous work for quantifying targeted biomolecules and cells via directly chemoselective and activity-based Eu- and Hg-tagging strategies,36-42 our hypothesis in this study was to construct a photocleavable magnetic nanoparticle-based Ga-tag (MNP-based Ga-tag) for tagging and enrichment of pS, pT, pY and pX, absolute and specific pY quantification could be subsequently achieved using HPLC/71Gaspecies unspecific isotope-dilution (71Ga-SUID) ICP-MS when applying a specific tyrosine phosphatase (Scheme 1). The photocleavable MNP-based Ga-tag contains a dimetallic Ga-complex moiety for targeting the phosphate ester motif and ICP-MS readout; it links to Fe3O4@SiO2 MNPs through a photocleavable o-nitrobenzyl ether for handy magnetic separation and subsquent UV-induced release of the Ga-tagged and enriched molecules/ions. We expected that this Ga-tagging and tyrosinephosphatase mediated strategy affords a simple way to sensitive, specific and absolute quantification of pY using HPLC/71GaSUID ICP-MS regardless of the coexistence of pS, pT and pX. Scheme 1. A photocleavable MNP-based Ga-tag for tagging, enrichment and UV-release of phosphorylated and phosphate-bearing molecules/ions; and specific pY quantification using HPLC/71Ga-SUID ICP-MS under the assistance of a protein tyrosine-specific phosphatase PTP-1B.

4-(2-aminoethyl)-2,6-bis((bis(pyridin-2-lmethyl)amino)methyl) phenol (1), photocleavable Fmoc-nitrobenzyl-NHS linker (2), photocleavable ligand (3), photocleavable Ga-tag (4) and photocleavable MNP-based Ga-tag (5) were fabricated via classical organic reactions (Figure S1 and Supporting Information). All the synthesized intermediates and final products were purified with semi-preparation HPLC and characterized using 1HNMR and ESI-MS (Figures S2-9). Synthesis and characterization of Fe3O4@SiO2-COOH MNPs are described in Supporting Information (Figures S10-11). TEM images told us that the synthesized Fe3O4@SiO2-COOH MNPs were uniform with an average diameter of 347 ± 5 nm containing a silica layer of 27 ± 2 nm, and its hydrodynamic diameter (Dh) was 398 ± 3 nm determined using the dynamic light scattering technique. The ξ potentials of Fe3O4@SiO2, Fe3O4@SiO2-NH2 and Fe3O4@SiO2-COOH MNPs are -25.13 ± 1.34 mV, 33.03 ± 0.58 mV and -35.37 ± 1.62 mV,

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suggesting successful modifications of the -NH2 and then -COOH groups. In order to construct the MNP-based photocleavable Gatag 5, 4 was conjugated onto the MNP-surface through the typical amide condensation reaction. Change in the ξ value from -35.37 ± 1.62 mV of Fe3O4@SiO2-COOH MNPs to 29.4 ± 0.78 mV the photocleavable MNP-based Ga-tag 5 and corresponding increase in Dh to 407 ± 3 nm confirmed a successful conjugation. In such a way, 9.16 × 104 Ga-tags were anchored on each MNP as determined using ICP-MS (see Supporting Information). Subsequently, pH-dependent stability of Ga3+ in the photocleavable Ga-tag 4 was investigated using HPLC. More hydrophobic ligand 3 resulted from the dissociation of Ga3+ out of 4 was eluted at 27.3 min when pH was lower than 2.5 and/or greater than 7.5; while the intact 4 at 23.1 min suggested that Ga3+ in 4 was stable in the pH range 2.5 to 7.5 (Figure S12). UV-cleavable efficiency of 4 was then investigated with and without UVirradiation (λ = 365 nm). As expected, the intact 4 was eluted at 23.1 min on a C18 column (Figure 1a) with the deconvolution molecular weight (DM) of 1126 Da (Figure 1c, m/z at [563]2+ and [375]3+). After 45 min UV irradiation, the intact 4 disappeared while a new peak was observed at 11.2 min (Figure 1b). This new UV-released product was assigned to [1-Ga2(OH)2]2+ (Figure 1d, m/z at [366]2+ and [244]3+, DM = 731 Da), being consistent with the photocleavage mechanism of o-nitrobenzyl ether compound,43 implying that the UV-cleavage completes after a short time UV irradiation. Next, tagging properties of the Ga-tag 4 and Ga-tag 5 towards phosphorylated peptide/protein standards VNQIGTLSEpSIK, VNQIGTLSEpTIK, VNQIGTLSEpYIK, VNQIGTLpSEpSIK, VNQIGTLpSEpYIK and ERK1 (extracellular regulated protein kinase 1) peptide with -pTEpY- in the TEY motif as well as βcasein with multiple pS (IVEpSLpSpSpSEESITRINKKIEKFQpSEEQ-) (Table S1) were studied. For the mono-phosphorylated VNQIGTLSEpSIK, VNQIGTLSEpTIK and VNQIGTLSEpYIK (Figures S13a-c), DMs of the 4-Ga-tagged VNQIGTLSEpSIK, VNQIGTLSEpTIK and VNQIGTLSEpYIK at m/z [615]4+ (Figure S13h), [618]4+ (Figure S13i) and [634]4+ (Figure S13j) are consistent with their theoretical molecular weights (TMs). Similar results were obtained in the case of 5. After UV-irradiation, the observed UVreleased 1-Ga-VNQIGTLSEpSIK, 1-Ga-VNQIGTLSEpTIK and 1-Ga-VNQIGTLSEpYIK at m/z [688]3+ (Figure S13o), [692]3+ (Figure S13p) and [713]3+ (Figure S13q) are well in agreement with their corresponding TMs. These results obtained from ESIMS indicated that all the mono-phosphorylated peptide standards were tagged quantitatively by one Ga-tag with 2:1 stoichiometry between Ga and the phosphate group. However, only one Ga-tag was found to label VNQIGTLpSEpSIK (Figure S13d). The 4-Gatagged VNQIGTLpSEpSIK was observed at m/z [635]4+ (Figure S13k) and the UV-released 1-Ga-VNQIGTLpSEpSIK at m/z [714]3+ (Figure S13r). Three Ga-tags were labeled on one βcasein molecule (Figure S13e), the 4-Ga-tagged β-casein at m/z [909]30+ (Figure S13l) and the UV-released 1-Ga-β-casein m/z [870]30+ (Figure S13s). In the cases of VNQIGTLpSEpYIK (Figure S13f) and ERK1 peptide (Figure S13g) that contain adjacent pY and pS or pT linked via E, the determined DMs also suggested that only one Ga-tag tagged on VNQIGTLpSEpYIK and ERK1 peptide as the 4-Ga-tagged VNQIGTLpSEpYIK at m/z [654]4+ (Figure S13m), the UV-released 1-GaVNQIGTLpSEpYIK at m/z [740]3+ (Figure S13t) and the 4-Gatagged ERK1 peptide at m/z [1650]4+ (Figure S13n), the UVreleased 1-Ga-ERK1 peptide at m/z [1551]4+ (Figure S13u) were determined. Although these ESI-MS results could not confidently discriminate which phosphorylated site was labeled by Ga-tag at this moment, we speculated that pY pre-empts to be tagged considering the length and inductive effect of phenyl ring in Y compared to the zero-length in S and strong steric hindrance from the side -CH3 in T linked to the phosphate group, in addition to the


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greater affinity of Ga to phosphate than carboxyl (in E) at lower pH condition.44

Figure 1. HPLC/71Ga-SUID ICP-MS chromatograms of the photocleavable Ga-tag 4 before (a) and after (b) UV irradiation (λ = 365 nm); and ESI-MS spectra of 4 (c) and UV-released [1-Ga2(OH)2]2+ complex (d).

When a simple mixture of VNQIGTLSEpSIK (1.48 nmol), VNQIGTLSEpTIK (1.59 nmol) and VNQIGTLSEpYIK (4.50 nmol) was analyzed by HPLC/71Ga-SUID ICP-MS after tagging and enrichment with the Ga-tag 5 thru magnetic separation and UV-irradiation, the UV-released 1-Ga-VNQIGTLSEpYIK could be separated fortunately from the Ga-tagged pS and pT peptides and the free Ga-tags as well (Figures 2a, a’ and 2b, b’). VNQIGTLSEpYIK was thus quantified as 4.38 ± 0.12 nmol with the recovery of 97.3 % (n = 5) according to the Ga-mass flow equation in Supporting Information. Although the pS and pT peptides partly overlapped under the chromatographic conditions, the global phosphorylation level of this artificial sample could be quantified being 7.29 ± 0.27 nmol with the recovery of 96.3 % (n = 5) via integrating the Ga-peaks areas except that of the free Gatags in the Ga-mass flow chromatogram (Figure 2a’). It is worthy of mentioning that peak overlapping is a frequently encountered situation when HPLC separates proteins and their proteolytic digests of a real biological sample before detection. When the peptides mixture underwent tyrosine-specific dephosphorylation with PTP-1B (10 min incubation),45 the Ga-tagged VNQIGTLSEpYIK disappeared and a new peak appeared in the meanwhile at a shorter retention time of 6.8 min; while the Gatagged pS and pT peptides were observed still at their right position without change in their peak areas (Figures 2b and b’). The more hydrophilic new peak at 6.8 min is 1-Ga-phosphate complex ([1-Ga2(HPO4)]2+/[1-Ga2(HPO4)(OH2)]2+) as identified using ESIMS (Figure 2d inserted mass spectrum, m/z at [397]2+ and [406]2+), suggesting that pY dephosphorylation by PTP-1B is specific and efficient. Importantly, quantification of pY could be achieved via determination of the 1-Ga-phosphate complex, which came solely from pY. In this way, VNQIGTLSEpYIK was quantified to be 4.33 ± 0.11 nmol with the recovery of 96.2 % (n = 5), being in agreement with that determined through the intact Ga-tagged peptide. Moreover, as a typical signaling protein, ERK1 has moderate complexity that is phosphorylated on T and Y residues within its activation loop (-202pTE204pY-).46 The human ERK1 peptide (1.67 nmol) was then employed to validate our proposed method for specific quantification of pY in one peptide. Results obtained without and with PTP-1B dephosphorylation are shown in Figures 2c, c’, 2d and d’. The Ga-tagged ERK1 peptide disappeared at its retention time after PTP-1B dephosphorylation while the 1-Ga-phosphate complex appeared at 6.8 min as expected. These results indicated the selectivity of Gatagging towards pY when adjacent free -COOH-bearing E and pT exist in the same peptide, confirming the speculation that pY preempts to be tagged from ESI-MS studies. The content of ERK1 peptide was quantified via the 1-Ga-phosphate complex (1.61 ±

0.14 nmol with the recovery of 96.4 % (n = 5)) after PTP-1B dephosphorylation. The PTP-1B generated 1-Ga-phosphate complex is particularly significant for an accurate and specific pY quantification, especially in the case when ICP-MS was employed as a detector, because it can be separated much easier and far from the UV-released Ga-tagged pT and pS peptides/proteins as well as possible pX on a conventional HPLC, avoiding the inevitable chromatographic overlapping of them in a real biological sample. It should be pointed out that the pY quantified via 1-Gaphosphate complex is the global pY level of all the tyrosinephosphorylated peptides/proteins on a proteomic level. In the case of more than one pY abutting in one peptide/protein resulting in possible lost labeling of pY, in situ remedial Ga-tagging will happen to grasp the PTP-1B dephosphorylated phosphates considering that there are up to 9.16 × 104 Ga-tags modified on the surface of the MNPs. The specific quantification of pY was thus accurate in any case. But neither the pS and/or pT nor the global protein phosphorylation (pS + pT + pY) level could be accurately determined due to either possible chromatographic overlapping of themselves and with possible pX or discriminate and incomplete Ga-tagging towards the multiple phosphorylated-sites.

Figure 2. HPLC/71Ga-SUID ICP-MS chromatograms of a Ga-tagged phosphorylated peptides mixture without (a) and with (b) PTP-1B dephosphorylation and their corresponding Ga mass flow chromatograms (a’) and (b’); and those of Ga-tagged ERK1 peptide without (c) and with (d) PTP-1B dephosphorylation and their corresponding Ga mass flow chromatograms (c’) and (d’).

Based on the instrument DL (3σ) of Ga, 4.8 fmol, with RSD lower than 5.70 % (n = 5 at pmol level) in ICP-MS, the corresponding method detection limit (MDL) of pY was calculated as low as 30 amol considering that one Ga-tag contains two Ga3+ and the enrichment factor is 80 according to our practical experiment procedures (Supporting Information). This MDL is about 220fold lower than the reported 6.6 fmol with P using ICP-QQQMS,30 more than 16-fold lower than 500 amol (endogenous pY peptide of tryptic peptide mixture derived from Jurkat T cellular extract)10 using SRM mass spectrometry, and much lower than 1.0 pmol (tyrosine phosphorylated peptide (Tb)2-cggA)47 using timeresolved luminescence technique as well. It is generally accepted that tyrosine phosphorylation is coordinately regulated by the balanced action of protein tyrosine kinases and protein tyrosine phosphatases in cells.48,49 This dynamic equilibrium may be broken by stimulating the intracellular signaling pathway with insulin to prompt phosphorylation of its own receptor.50 After incubation without (control group) and with (stimula-



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tion group) 100 nM human insulin for 10 min, the lysates of 3 × 106 MCF-7 cells were subjected to HPLC/71Ga-SUID ICP-MS together with the Ga-tag 5 to evaluate pY level (Supporting Information). The Ga-tagged and enriched pS, pT, pY and pX were dephosphorylated with addition of 0 µg, 3 µg, 5 µg and 7 µg PTP1B. After UV-release, pY level was quantified via the determination of Ga in the 1-Ga-phosphate complex (Figure S14 and Table S2). It should be noted that there are free phosphoric acid and inorganic phosphates in the MCF-7 cells lysates, which would be tagged to produce the 1-Ga-phosphate complex and corresponding analogs of similar chromatographic behivior. Although these small phosphate-bearing compounds can be easily trapped down from the cell lysate using a size-exclusion column before Gatagging and enrichment procedures, we still determined the possible triggered background value when PTP-1B was not applied. In the case that the cell lysates were not treated by PTP-1B (0 µg), the background values of the control and insulin stimulation groups were 1.74 ± 0.72 nmol (n = 5) and 2.32 ± 0.76 nmol (n = 5) (Table S2). Although there is no statistical difference between the background levels, there is an apparent difference in the mean value after insulin stimulation, which could be interpreted by the fact that phosphorylation was accompanied with the energytransducing processes mediated by adenosine phosphates, and the resulting adenosine phosphate species might be more effectively labeled by the Ga-tag at the moment, causing the difference in the mean between the control and stimulation group. The quantified pY level of both the control and stimulation group, after subtracting the corresponding background values, increased and leveled off afterwards along with the increase of PTP-1B applied from 3 to 7 µg. Thus, we thought that 7 µg was enough to dephosphorylate pY in the cell lysates. pY were quantified being 4.22 ± 0.72 and 2.62 ± 0.73 nmol in the MCF-7 cells with and without insulin stimulation, implying that the level of pY increased by 1.60 nmol (61.1 %) after insulin stimulation. These results were in accordance with those reported previously that incubation of cells with insulin leads to a transient rise in tyrosine-phosphorylation.51 In conclusion, we present a simple and effective way for specific and absolute quantification of pY using HPLC/71Ga-SUID ICP-MS together with a novel photocleavable MNP-based Ga-tag under assistance of the phosphotyrosine-specific PTP-1B. The MDL of pY was greatly improved down to 30 amol; and specific pY quantification was achieved via determination of Ga in the Ga-phosphate complex that exclusively generated from pY under PTP-1B dephosphorylation. It is the first time to demonstrate a highly sensitive, specific and absolute pY quantification using ICP-MS, pushing ICP-MS into the studies of quantitative protein PTMs. Moreover, this Ga-tagging strategy “freezes” phosphorylation status considering the dynamic feature of protein phosphorylation, offering more realistic results. This study aims to specific quantification of the global pY with the designed and synthesized photocleavable MNP-based Ga-tag. We believe that, not limited to pY quantification, this element-tagging and protease-specific reaction mediated ICP-MS methodology will pave a simple path for ever more applications of ICP-MS to the studies of quantitative PTMs when suitable element-tags are designed and specific proteases are available towards targeted PTMs.

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional details about the reagents, instrumentation, synthetic routes, ESI-MS study of Ga-tagging properties, cell culturing and Ga-tagging methods (PDF).

AUTHOR INFORMATION

Corresponding Author

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*E-mail: [email protected]; Fax: +86 (0)592 2187400.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

This study was financially supported by the National Natural Science Foundation of China (21535007, 21475108 and 21275120) and the National Basic Research 973 Project (2014CB932004) as well as the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (21521004) and Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, IRT13036). We thank Dr. Xin Jiang and Prof. Haifeng Chen of School of Pharmaceutical Science, Xiamen University, for helping with cell culture and preparation of the cell lysates.

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Analytical Chemistry Figure Captions

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Scheme 1. A photocleavable MNP-based Ga-tag for tagging, enrichment and UV-release of phosphorylated and phosphatebearing molecules/ions; and specific pY quantification using HPLC/71Ga-SUID ICP-MS under the assistance of a protein tyrosine-specific phosphatase PTP-1B. Figure 1. HPLC/71Ga-SUID ICP-MS chromatograms of the photocleavable Ga-tag 4 before (a) and after (b) UV irradiation (λ = 365 nm); and ESI-MS spectra of 4 (c) and UV-released [1Ga2(OH)2]2+ complex (d). Figure 2. HPLC/71Ga-SUID ICP-MS chromatograms of a Gatagged phosphorylated peptides mixture without (a) and with (b) PTP-1B dephosphorylation and their corresponding Ga mass flow chromatograms (a’) and (b’); and those of Ga-tagged ERK1 peptide without (c) and with (d) PTP-1B dephosphorylation and their corresponding Ga mass flow chromatograms (c’) and (d’).


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96x82mm (300 x 300 DPI)


ICPMS-Based Specific Quantification of ... - ACS Publications

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