GdF3 as a promising phosphopeptide affinity probe

ChemComm COMMUNICATION

Cite this: Chem. Commun., 2014, 50, 11572 Received 28th May 2014, Accepted 13th August 2014

GdF3 as a promising phosphopeptide affinity probe and dephospho-labelling medium: experiments and theoretical explanation† Li-Ping Li,ab Jun-Zi Liu,c Lin-Nan Xu,ab Ze Li,ab Yu Bai,*ab Yun-Long Xiaoc and Hu-Wei Liuab

DOI: 10.1039/c4cc04090b www.rsc.org/chemcomm

Bone-like GdF3 was synthesized and applied for phosphopeptide enrichment for the first time. As a new kind of efficient phosphopeptide affinity probe, GdF3 exhibits high efficiency in the mediation of the dephosphorylation reaction. In addition, DFT calculations were introduced to theoretically explain the unique property of GdF3 compared to GdPO4, which is promising and can be potentially significant in protein phosphorylation research.

Reversible protein phosphorylation occurs at the hydroxyl groups from serine, threonine or tyrosine residues in eukaryotic cells, and plays an essential role in regulating various biological processes.1,2 Therefore, analyses of phosphorylated proteins/peptides are crucial for deciphering their related biological processes. Despite the rapid development of MS-based techniques for proteomic analysis in the past two decades, it remains a major challenge to detect phosphorylated peptides from complex protein digests due to their low abundance and high dynamic range. Recently researchers have made great efforts in developing phosphopeptide enrichment strategies, among which metal oxide chromatography (MOAC) has attracted increasing interest. Various metal oxides3–13 have been proved to show a strong affinity towards phosphopeptides. Most metal oxides show similar performance in phosphopeptide enrichment, and researchers are still searching for new metal-based affinity probes with novel properties.14 Recently our group proposed bimetal ZnSn(OH)6 (ref. 15) and Zhong’s group proposed trimetal NiZnFe2O4 (ref. 16) as selective affinity probes for multiply phosphorylated peptides.

a

Beijing National Laboratory for Molecular Sciences, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. E-mail: [email protected]; Tel: +86 10 6275 8198 b Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China c Institute of Theoretical and Computational Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China † Electronic supplementary information (ESI) available: Experimental procedures, characterization of materials, detected phosphopeptides and their sequences. See DOI: 10.1039/c4cc04090b

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And rare earth nanomaterials, including CeO217 and REPO4 (RE = Y, Gd, Yb),18 have been proposed for phosphopeptide capturing as well as labelling, because they can catalyze the dephosphorylation reaction resulting in a characteristic mass shift of 80 Da (–HPO3) in the mass spectra. Previously, this kind of dephosphorylation reaction, which is actually the hydrolysis of the phosphate esters, was usually catalyzed by phosphatase.19 Despite the hydrolysis reaction, phosphate moieties can also undergo a beta-elimination reaction under strongly alkaline conditions, resulting in irreversible dephosphorylation processes and dehydroalanyl residues (loss of H3PO4).20 This method has been applied for phosphospecific proteolysis for decades with the disadvantages such as strongly alkaline conditions and high sample consumption. It is suggested that it will be promising and extremely significant if nanomaterial-mediated dephosphorylation can be proposed. So far, the dephosphorylation function of these nanomaterials was not well investigated and searching for ideal affinity probes with higher dephosphorylation activity is still restricted to the unclear theoretical understanding. Herein the lanthanide nanomaterial of GdF3 was synthesized and applied for phosphopeptide enrichment, and its higher activity to mediate the dephosphorylation process over GdPO4 was discovered. For further understanding of its unique property, we performed a density functional theory (DFT) calculation on GdPO4-mediated/GdF3-mediated dephosphorylation reactions. This is the first report of GdF3 in phosphopeptide capturing and labelling, and also the first illuminate exploration of the related dephosphorylation mechanism by computation. GdF3 nanomaterials were synthesized by a one-step ionic liquid (IL)-mediated hydrothermal method. Compared to previous reports,21,22 this method is more user friendly and environmentally friendly to generate large-scale uniform nanomaterials with good biocompatibility for further applications. The as-synthesized GdF3 was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and X-ray diffraction (XRD). The TEM image (Fig. 1a) shows the bone-like shape of the nanomaterial, and the related selected-area electron diffraction

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Fig. 1 TEM image with the SAED pattern (a), SEM image (b) and XRD pattern (c) of GdF3.

(SAED) pattern shows characteristic rings of GdF3. The SEM image in Fig. 1b confirms the large-scale formation of the GdF3 boneshaped nanomaterial, and the X-ray diffraction (XRD) pattern shown in Fig. 1c reveals that all the diffraction peaks can be ascribed to GdF3 (JCPDS No. 41-1445), which is consistent with the SAED results. GdPO4 was synthesized for comparison of its performance with that of GdF3 (see Fig. S1, ESI†). The as-synthesized GdF3 nanomaterial was applied for phosphopeptide capturing from the digests of a standard protein b-casein. Typically the phosphopeptides were bound to GdF3 under acidic conditions, eluted in ammonia solution under alkaline conditions, and then analyzed by MALDI ToF MS (Scheme 1(a)). As shown in Fig. 2(b), after GdF3 enrichment the signals of phosphopeptides (marked as b) and their dephosphorylated species ((b1-HPO3) marked as b#) dominate the mass spectrum. When the protein concentration was decreased to 4 10 10 M, two phosphorylated peptides with their dephosphorylated peptides can still be detected. To further evaluate the efficiency of this new affinity probe, phosphopeptide trapping from complex digests of BSA and b-casein (a molar ratio of 1000 : 1, with a b-casein concentration of 4 1 0 8 M) using GdF3 was performed. Due to the high sample complexity, the phosphopeptide signals can hardly be seen when this mixture was directly analyzed by MS. After GdF3 treatment, only phosphopeptides and their dephosphorylated peptides can be detected. All the above results demonstrate that GdF3 can be a new promising affinity probe for phosphopeptide capturing and labelling. GdPO4 has been reported previously with dephosphorylation effects18 without deep exploration and theoretical explanation. Here we speculated on a more efficient dephosphorylation reaction mediated by GdF3 since the electronegativity of F is stronger. For comparison, the same experimental procedure was performed using GdPO4. It has been observed that weaker peaks of dephosphorylated peptides are generated from GdPO4 compared to those of GdF3. As shown in Scheme 1(b) and (c),


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Scheme 1 Schematic of GdF3 as a new affinity probe for phosphopeptide capturing and labelling; (a) scheme of the experiment protocol; (b), (c) dephosphorylation reactions mediated by GdF3.

Fig. 2 MALDI ToF mass spectra of (a) 4 10 7 M b-casein digests without pretreatment, (b) 4 10 7 M b-casein digests after GdF3 treatment, (c) 4 10 10 M b-casein digests after GdF3 treatment, (d) the digests from a mixture of BSA and b-casein (1000 : 1 n/n) without pretreatment, and (e) the digests from a mixture of BSA and b-casein (1000 : 1 n/n) after GdF3 treatment. (Phosphopeptides marked as b; phosphopeptides after loss of HPO3 marked as b#.)

there are two routes of dephosphorylation, and the products from the second route (b-H3PO4, marked as b*) were observed in the MALDI ToF MS analysis of peptides eluted from GdF3 (Fig. 3(a)). We know that the intensity of dephosphorylated peptide peaks (b-HPO3) in MALDI ToF MS spectra can also be influenced by laser energy. Besides paralleled conditions including well controlled laser energy, we further evaluated the effect by analyzing the eluted peptides after treatment by GdF3 or GdPO4 using LC-MS. Taking the phosphopeptide b1 and its dephosphorylated peptide b1# as examples, we calculated the peak area ratio of (b1-HPO3)/b1 and (b1-H3PO4)/b1 to represent the different efficiencies of GdF3 or GdPO4 in mediating dephosphorylation (see Fig. 3(b)).

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Fig. 4 Reaction routes of GdF3-mediated or GdPO4-mediated dephosphorylation. (H2O: 76.419737 a.u., 1 a.u. = 2625.50 kJ mol 1 = 27.211 eV)

Fig. 3 Comparison of the experimental results of GdF3 and GdPO4. (a) MALDI ToF mass spectra of 4 10 7 M b-casein digests after GdF3 and GdPO4 treatment and the inset is a partial enlargement; (b) chromatographic peak ratio of (b1-HPO3)/b1 and (b1-H3PO4)/b1, n = 3.

Based on the above results, DFT calculations were further carried out to study the mechanism behind the experimental phenomena. We calculated these molecules using unrestricted density functional theory with the hybrid functional B3LYP. The Stuttgart effective core potential was used for gadolinium and the 6-31G(d) basis set was used for the rest of the light element atoms. All calculations were performed using the Gaussian09 package.23 As shown in Fig. 4, we designed simplified model reaction routes for computation, which show the dephosphorylation processes with the cleavage of P–O bonds (generating MS signals of (b-HPO3)) or C–O bonds (generating MS signals of (b-H3PO4)). GdF3 and GdPO4 participate in individual reactions and form phosphate complexes (Fig. S4, ESI†). The reaction energy for two reactions can be utilized to reflect the different mediation effects of GdF3 and GdPO4. The total electronic energy of each molecule was calculated with its optimized geometry, and the energy changes of reactions can be obtained as follows: DE1(a) =

0.258114 a.u., DE1(b) =

0.251056 a.u.

DE2(a) =

0.245636 a.u., DE2(b) =

0.238578 a.u.

From the inequalities |DE1(a)|4 |DE1(b)| and |DE2(a)|4 |DE2(b)|, we can conclude that for both route 1 and route 2, dephosphorylation reactions mediated by GdF3 have a higher energy release, which indicates that GdF3 has a higher activity in mediating dephosphorylation reactions than GdPO4. This conclusion matched what we speculated. On the other hand,

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from the inequalities |DE1(a)|4 |DE2(a)| and |DE1(b)|4 |DE2(b)|, we can conclude that for either GdF3 or GdPO4, reaction route 1 is the energy-favorable reaction route, as we also observed a higher ratio of (b1-HPO3)/b1 than (b1-H3PO4)/ b1. Actually due to the low ratio of (b1-H3PO4)/b1, the signals of (b1-H3PO4) were rarely detected in MALDI ToF MS in previous reports. Our experimental observations can be reasonably explained by the computational results. Theoretical calculations play an illuminated role in understanding the nanomaterial–phosphopeptide interaction. In conclusion, GdF3 was proposed as a new affinity probe for phosphopeptide capturing with high efficiency for the mediation of dephosphorylation, and DFT calculations were introduced as a powerful tool to decipher the different properties of GdF3 over GdPO4. We believe that GdF3 can be an important complement for the existing affinity probes. On the other hand, though increasing kinds of metal nanomaterials have been applied for phosphopeptide enrichment and dephosphorylation labelling, the chemical mechanism remains unclear, resulting in the lack of effective guidance in the development of novel affinity probes. Here simplified theoretical calculations provided a possible mechanism for dephosphorylation processes. The mediation of the dephosphorylation reaction of GaF3 is promising and can be potentially significant in protein phosphorylation research. This work was supported by the National Natural Science Foundation of China (21322505, 21175008), the Ministry of Science and Technology of China (2012IM030900), and The Project Sponsored by the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. We thank Dr Xiaoyun Liu and Ms Yanhua Liu for their help in LC-MS analysis.

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