1. Introduction
Protein phosphorylation plays an important role in cell signal transduction, cell cycle modulation, cytoskeletal regulation, and cell apoptosis [[1], [2], [3]]. Sensitive and high-throughput detection of phosphoproteins offers a straightforward strategy to identify the disease biomarkers and potential drug targets, which are fundamental for diagnostics, prognostics, and disease treatments [4]. Mass spectrometry (MS) is a powerful analytical tool with wide applications in the study of protein phosphorylation, proteomics and metabolomics and small biomolecular [[5], [6], [7], [8]]. Important advances have been made in the development of different MS techniques, including matrix-assisted laser disorption/ionization-time of flight (MALDI-TOF) MS and liquid chromatography-MS (LC-MS) [[9], [10], [11]]. However, there usually exists a very wide concentration range of protein molecules in the body, for instance, in human blood the dynamic concentrations of proteins ranging from mg/mL to pg/mL or more [12]. The signal intensities of the proteins in a low abundance are frequently suppressed by the highly abundant proteins in the mass spectral data, thus becoming difficult for identification. The diverse mechanisms of protein phosphorylation and dephosphorylation in biology also affect their dynamic stoichiometry, which make it challenging to identify and characterize the unknown phosphoproteins [13]. There is a great need to develop selective methods to enrich phosphoproteins for biomedical research.
In the recent progress for phosphorylated proteins/peptides enrichment, there are mainly four categories of techniques with affinity-based materials, including strong cation exchange chromatography (SCX) [14], anti-pTyr antibody, immobilized metal ion (usually Fe3+, Ga3+, Ni2+ and Zr4+) affinity chromatography adsorbents (IMAC) [15], and metal oxide/hydroxide affinity chromatography (MOAC) [16]. Among them, MOAC is the most widely used method for phosphopeptide enrichment. In this technique, phosphopeptides can be specifically adsorbed through bidentate bindings between the phosphate anions of the peptides and the surface of a metal oxide such as TiO2 [17], ZrO2 [18,19], Fe2O3 [20], and Al2O3 [21]. For example, Cai and co-workers developed TiO2-based nanocomposites by co-doping Bi and Fe elements for highly selectively enrichment of phosphopeptides [22]. The metal oxide of ZrO2 is also attractive due to its superior amphoteric surface properties, which facilitate the reversible adsorption and release of the phosphate groups by the pH change.
Here we report the development of core-shell structured Fe3O4@mZrO2 microspheres for highly efficient and selective enrichment of phosphopeptides (Fig. 1). The as-prepared microspheres integrate superparamagnetic particles (Fe3O4 core) and MOAC materials (ZrO2 shell with mesoporous structures) by a high-yield synthetic route. Mesoporous nanomaterials are featured with ultra-high surface areas, tunable pore sizes, and enhanced active sites accessible to the external target molecules [[23], [24], [25]]. For the first time, we have demonstrated that Fe3O4@mZrO2 microspheres enable highly efficient enrichment of mono- and multi-phosphopeptides, and allow for a unique and rapid protocol of in-situ elution for sensitive MS detection. These features promote the limit of detection to the femtomole range for phosphopeptides using our approach. The selective enrichment of the target phosphopeptides by Fe3O4@mZrO2 microspheres surpasses the interference from the mixed proteins and cell lysates. Our approach will facilitate the biomedical research on protein phosphorylation by allowing for sensitive detection of the crucial signaling regulators in a low abundance.
Fig. 1.
Fig. 1.
Schematic illustration of synthesis of Fe3O4@mZrO2 microspheres and selective enrichment of phosphorylated peptides for MALDI-TOF analysis. The scheme highlights the difference between the in-situ elution by our approach and the conventional elution method.
2. Experimental
2.1. Materials and reagents
Iron (III) chloride hexahydrate (FeCl3), ammonium acetate (NH4OAc), ethylene glycol, ammonium hydroxide solution (25∼28 wt%), anhydrous ethanol, and phosphoric acid (8 wt5%) were purchased from Shanghai Chemical Industrial Co. (Shanghai, China). Risodium citrate dehydratet, β-casein, bovine serum albumin (BSA), 1,1-(tosylamido)-2-phenylethyl chloromethyl ketone (TPCK)-treated trypsin (E.G2.4.21.4), urea, dithiothreitol (DTT), iodoacetamide (IAA), zirconium (IV) butoxide solution (80 wt% in 1-butanol), and α-Cyano-4-hydroxycinnamic acid (CHCA, ≥99%, HPLC) were purchased were from Sigma (St. Louis, MO, USA). RPMI 1640 medium was obtained from Hyclone (HyClone Europe, Cramlington, UK). Fetal bovine serum (FBS) was purchased from Gibco (GIBCO Inc., NY, USA). Acetonitrile (ACN, ≥99.9%), trifluoroacetic acid (TFA, ≥99.8%), and DL-Lactic acid (≥90%) were obtained from Aladdin. Deionized water (18.2 MΩ cm) in all the experiments was obtained from a Milli-Q system (Millipore, Bedford, MA, USA). All chemicals used in our experiment were analytical grade without further purification.
2.2. Synthesis of Fe3O4 particles
The Fe3O4 particles were prepared through a modified solvothermal reaction. In brief, 0.65 g FeCl3 and 0.47 g sodium citrate was mixed in 40 mL of ethylene glycol. The mixture was stirred vigorously for 15 min. Then, 2.4 g sodium acetate was added into the mixture and stirred vigorously for 30 min at room temperature. It was transferred into a Teflon-lined stainless-steel autoclave (50 mL), heated to 200 °C for 10 h. After it was cooled to room temperature, the product of Fe3O4 particles in the black was washed with ethanol and double distilled water three times. It was collected with the help of a magnet, and re-dispersed in ethanol for the subsequent experiments.
2.3. Preparation of Fe3O4@ZrO2 core/shell microspheres
The Fe3O4@ZrO2 core/shell microspheres were synthesized by directly coating a ZrO2 layer on the surface of Fe3O4. Briefly, 50 mg Fe3O4 microspheres were dispersed in mixed solvent of ethanol (90 mL) and acetonitrile (30 mL) with the aid of ultrasound, followed by mixing with 3 mL ammonium hydroxide. With the help of mechanical stirrer, 600 μL zirconium butoxide solution (80%) was added into the mixture within 5 min. The reaction of ZrO2 layer coating was performed for 1.5 h at room temperature. The product of Fe3O4@ZrO2 core/shell microspheres was washed with ethanol and double distilled water before collection with a magnet.
2.4. Preparation of Fe3O4@mZrO2 core/shell microspheres with mesoporous structure
The Fe3O4@ZrO2 microspheres (50 mg) dispersed in 60 mL of mixed solvent of ethanol and deionized water (2:1, v/v), and then mixed with 3 mL ammonium hydroxide. The mixture was transferred into a Teflon-lined stainless-steel autoclave (50 mL) and heated at 160 °C for 20 h. After being cooled to room temperature, the product of Fe3O4@mZrO2 core/shell microspheres was washed with ethanol before collection with a magnet.
2.5. Preparation of standard peptide mixtures
β-Casein and BSA were separately dissolved in 25 mM ammonium bicarbonate buffer at pH 8.0 with a final concentration of 1 mg/mL. They were denatured for 10 min at 100 °C. The protein solutions were treated with trypsin (1:25, w/w) for 12 h at 37 °C in the presence of 5% acetonitrile. The final standard peptide mixtures were stored at -20 °C before use.
2.6. Cells culture and preparation of A549 cell lysates
The human lung adenocarcinoma A549 cells were provided by ATCC. It was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotic (100 U/ mL penicillin and 100 μg/mL streptomycin) at 37 °C in a humidified incubator under 5% CO2. The A549 cells were used for subsequent experiments in confluency of 95%.
After centrifuge, A549 cells were washed with ice-cold PBS (pH 7.4) and counted using a hemocytometer. The cells were resuspended in 1 mL lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 8 M urea, protease inhibitors (cocktail), phosphatase inhibitors (1 mM Na3VO4), 100 mM dithiothreitol (DTT) at 4 °C. After lysis for 20 min on ice, the mixture were sonicated (450 W) with several bursts of 5 s using the Vibra-cell TM sonicator (VCX750, Sonics & Materials Inc., Newtown, CT, USA) in an ice bath. The cell debris was eliminated by centrifugation at 14800 rpm at 4 °C for 10 min. The supernatant was collected for protein concentration measurement by Bradford assay. Cell lysate (0.5 mL) was adjusted to pH 8.0 using Tris-HCl and mixed with 5 μL DTT (50 mM) for 30 min at room temperature. Then the mixture was alkylated by 20 μL iodoacetamide (IAA, 50 mM) for 30 min in the dark. After reaction, 5 μL DTT (50 mM) was added to neutralize the excess IAA. Then the solution was diluted 10 times by ammonium bicarbonate (50 mM). The samples were digested by trypsin (enzyme to sample ratio, 1:40 w/w) at 37°C for 12 h. The tryptic digestion was stopped using trifluoroacetic acid (TFA). The products were lyophilized and kept at -20 °C.
2.7. Enrichment of phosphopeptides by Fe3O4@mZrO2 core/shell microspheres
Fe3O4@mZrO2 microspheres were sequentially washed with buffer A (0.4% TFA and 80% ACN) and buffer B (25% lactic acid and 75% buffer A) for twice, then re-dispersed in buffer B with a final concentration of 10 mg/mL. The mixture of β-casein (20 μL, 1 mg/mL) and BSA (20 μL, 1 mg/mL) was diluted by adding 80 μL buffer B and mixed with the Fe3O4@mZrO2 suspension (25 μL) for 30 min at 37 °C. The microspheres were separated from the solution using a magnet and washed with buffer B and buffer A for twice, respectively.
2.8. Analysis of phosphopeptides by MALDI-TOF MS
The microspheres were re-dispersed in 6 μL matrix solution CHCA (20 mg/mL in ACN/water/TFA, 50/49.9/0.1 v %). The matrix was deposited on the MALDI plate. The solution of ammonium hydroxide (1.5 μL 5 v %) was added onto the plate for in-situ elution of peptides from the mesoporous microspheres. The solution of phosphoric acid (1.5 μL, 1 v %) was added on the sample spot. After the spots were dried, the matrix solution of CHCA (1.5 μL) was applied onto them once again, before the MALDI-TOF MS measurements.
3. Characterization methods and instrumentation
The transmission electron microscopy (TEM) and high-resolution TEM (HR-TEM) images were obtained using a Philips CM300 transmission electron microscope operating at an acceleration voltage of 200 kV. The scanning electron microscopy (SEM) images of these nanomaterials were acquired with a FEI Quanta 200 F scanning electron microscope. The XRD experiments were performed on a Shimadzu XRD-6000 X-ray diffractometer equipped with Cuka radiation (λ = 0.15406 nm). The size distribution and zeta potential of nanoparticles were measured by the dynamic light scattering equipment (Zetasizer Nano Zen3690, Malvern Instruments, UK). MALDI-TOF MS analysis was performed on a Bruker BIFLEX III mass spectrometer (Bruker Daltonics, Germany) equipped with reflection mode for the positive-ion with the Nd:YAG laser at 355 nm.
4. Results and discussion
4.1. Synthesis of Fe3O4@mZrO2 core/shell microspheres
The synthesis of the Fe3O4@mZrO2 core/shell microspheres with a subsequent application in selective enrichment of phosphopeptides was illustrated by Fig. 1. Superparamagnetic Fe3O4 particles were synthesized by a modified solvothermal reaction reported previously [26]. Citrate groups were introduced onto the surface of Fe3O4 particles as ligands. The as-prepared nanoparticles were spherical in the diameter of 250 nm on the average (Fig. 2). The TEM and SEM images consistently suggested that the size of the superparamagnetic Fe3O4 core particles were uniform (Fig. 2a, d). The particles were not aggregated with each other, indicating an excellent dispersibility in solvents. The measurements of DLS verified the diameter of Fe3O4 particles (250 nm approximately) in a relatively narrow particle size distribution. The surface of the nanoparticles was negatively charged with a zeta potential of -24.8 mV (Supporting Information, Fig. S1).
Fig. 2.
Fig. 2.
SEM images of (a) Fe3O4 particles, (b) Fe3O4@ZrO2 microspheres, and (c) Fe3O4@mZrO2 microspheres. TEM images of (d) Fe3O4 particles, (e) Fe3O4@ZrO2 microspheres, and (f) Fe3O4@mZrO2 microspheres with an inset for the zoomed-in view. Scale bar: 50 nm. DLS data for (g) Fe3O4 particles, (h) Fe3O4@ZrO2 microspheres, and (i) Fe3O4@mZrO2 microspheres.
The Fe3O4 particles were subsequently subjected to the hydrothermal treatments in two steps, in order to synthesize the core-shell Fe3O4@mZrO2 microspheres with a mesoporous structure [27,28]. During this procedure, the ammonia played a pivotal role in catalyzing the formation of the shell layer of ZrO2 on the particle surface through electrostatic charge mediation. The particle diameter was increased to nearly 500 nm with the ZrO2 layer (Fig. 2b, e). The energy-dispersive X-ray analysis (EDXA) spectrum verified the elemental compositions, including Zr, Fe, and O elements in the as-prepared particles (Fig. S2). Highly ordered mesoporous structures were presented on the ZrO2 layer (Fig. 2c, f). The specific surface area of Fe3O4@mZrO2 microspheres was evaluated by Brunauer-Emmett-Teller (BET) analysis. There was a typical type IV curve in the isotherm of nitrogen adsorption-desorption experiments, which validated the mesoporous structures (Fig. 3a). The specific surface area was calculated to be 338.3 m3/g for Fe3O4@mZrO2 microspheres, a value higher than many other mesoporous materials reported in the literature [29,30]. The calculation by using the Barrett-Joyner-Halenda (BJH) method (Fig. 3b) revealed a pore size of 5.5 nm on the average, indicating the possibility of allowing proteins and peptides to adsorb on or desorb from Fe3O4@mZrO2 microspheres through the pores.
Fig. 3.
Fig. 3.
Characterization of different particles. (a) Nitrogen adsorption - desorption isotherms. (b)BJH pore-size distribution curves for Fe3O4@mZrO2 microspheres. (c) Wide-angle X-ray diffraction (XRD) patterns of Fe3O4 particles, Fe3O4 microspheres, and Fe3O4@ZrO2 microspheres. (d)Magnetic hysteresis curves of Fe3O4 particles, Fe3O4@ZrO2 microspheres, and Fe3O4@mZrO2 microspheres.
4.2. Characterization of Fe3O4@mZrO2 core/shell microspheres
In the X-ray diffraction (XRD) spectra, the powder diffraction data of Fe3O4 particles included the characteristic peaks at 30°, 35°, 43°, 53°, 57°, and 63° (the black curve in Fig. 3c). They were respectively attributed to (220), (311), (400), (422), (511), and (440) planes for the sample of Fe3O4 [31]. Good crystallization of Fe3O4 was revealed by these intensive, well-shaped characteristic peaks. After introducing the ZrO2 layer, the characteristic XRD peaks of Fe3O4 were attenuated for the sample of Fe3O4@ZrO2 (the cyan curve in Fig. 3c). The hydrothermal treatment for the mesoporous structures might change the crystallinity of ZrO2, leading to the emerge of two new XRD peaks at 50° and 60° contributed by ZrO2 in tetragonal phase (the red curve in Fig. 3c). The peaks at 30° and 35° of ZrO2 and Fe3O4 overlapped, while compared with the pattern of Fe3O4, the intensity ratio of peak at 30° to 35° was increased, which meant ZrO2 contributed to these two peaks. And compared with Fe3O4@ZrO2, the appearance of the characteristic peaks of ZrO2 manifested that the zirconia layers had undergone changes from amorphous to crystalline.
The magnetic properties of the as-prepared particles were investigated with a vibrating-sample magnetometer at room temperature (25 °C). As shown in Fig. 3d, the saturation magnetization (Ms) value of the Fe3O4 particles was determined to be 67.7 emu/g by this method. In addition, the Ms values of Fe3O4@ZrO2 and Fe3O4@mZrO2 were measured, i.e. 20.5 emu/g and 19.9 emu/g, respectively. In the absence of an external magnetic field, the hysteresis analysis suggested that all these particles exhibited strong compulsion, corresponding to zero magnetic remanence and coercivity. We further demonstrated that Fe3O4 particles and Fe3O4@mZrO2 microspheres maintained an excellent superparamagnetic property by dispersing themselves in the aqueous solutions very well and responding to the external magnet rapidly (Fig. S1 a, b). The zeta potential of Fe3O4 particles was measured as -25 mV, due to contribution of the carboxyl groups on the particle surface. The zeta potentials of Fe3O4@ZrO2 or Fe3O4@mZrO2 microspheres were less negative because of the surface coating change by the ZrO2 layer. Collectively, the experiments indicated that the as-prepared superparamagnetic mesoporous Fe3O4@mZrO2 was promising for efficient enrichment of specific biomolecules.
4.3. Efficient enrichment of phosphopeptides by Fe3O4@mZrO2 core/shell microspheres
The tryptic digests of bovine β-casein were tested in order to evaluate the performance of Fe3O4@mZrO2 in enriching phosphopeptides [32]. The conventional enrichment techniques require several steps including adsorption, desorption, and liquid transfer, thus suffering from the shortcoming of losing phosphopeptides, especially multiphosphopeptides. We designed a simplified sample-handling protocol featuring analyte elution in-situ with Fe3O4@mZrO2 core/shell microspheres (Fig. 1). We demonstrated that our method could save the valuable samples by minimizing the undesired loss of multi-phosphorylated peptides. The residual phosphoric acid by this in-situ elution protocol can facilitate ionization of phosphopeptides and enhance the sensitivity of MS detection [33]. As shown in Fig. 4, tryptic β-casein digests titrated at different concentrations (140 nM to 140 pM) were enriched by Fe3O4@mZrO2 microspheres (0.1 mg), followed by in-situ elution for MALDI-TOF MS analysis. For the purpose of comparison, we also performed MS analyses on the control samples without enrichment, where the peaks of non-phosphorylated peptides were predominant in the mass spectrum (Fig. S3). Due to the effect of suppression by these peaks, it was difficult to identify any phosphopeptides in the samples (Fig. S3). In contrast, enrichment with Fe3O4@mZrO2 microspheres resulted in appearance of the significantly enhanced peaks at m/z 2061.83, 2556.09, and 3122.27, which were corresponding to three characteristic phosphopeptides in the tryptic β-casein digests (Fig. 4a-d). The signal-to-noise ratios of these characteristic peaks were high, therefore easy for identification. Even when the concentration of the tryptic β-casein digests was diluted to 140 pM, the signal-to-noise ratios of all these three characteristic peaks were higher than 10. The limit of detection (LOD) was determined to be 76 pM (i.e. 9.0 fmol) for m/z 3122.27, when the S/N ratio was approaching 3 based on the mass spectral data of the dilution series (Fig. S4), suggesting a femtomole detection limit for the tryptic digests (Fig. 4d). Interestingly, our approach allowed for robust identification of multiphosphorylated peptides from a series of the digest mixture with different dilution. For instance, as highlighted in the inset of Fig. 4a, the characteristic peak at m/z 3122.27 was attributed to a tetra-phosphopeptides containing 4 phosphate groups at four different sites. Our approach not only detected this tetra-phosphopeptides at m/z 3122.27, but also its homologous peptides at m/z 3042.829, 3025.492, 2966.361 and 2927.465 (marked by “#” in Fig. 4a). The molecular weight difference between these peaks was constant (98 Da or 80 Da), corresponding to a phosphate group (H3PO4) or metastable (HPO3) [34]. Therefore, both the mono-phosphopeptides and multi-phosphopeptides can be successfully identified in our MS data. The performance in the detection of the phosphopeptide homologs was reproducible even in the most diluted samples (Fig. 4d). Using the conventional procedure of elution and liquid transfer, the five characteristic peaks of multiphosphorylated peptides/homologs around m/z 3122.27 were lost (Fig S5). These experiments validated that Fe3O4@mZrO2 microspheres with in-situ elution can serve as a reliable tool to detect both mono- and multi-phosphorylated peptides, better than the traditional enrichment techniques (Table 1) [22,[35], [36], [37]].
Fig. 4.
Fig. 4.
Evaluation of the enrichment performance of Fe3O4@mZrO2 microspheres. MALDI-TOF mass spectra for the different concentrations of tryptic digests of β-casein after enrichment using Fe3O4@mZrO2 microspheres. Concentrations of β-casein digests. (a) 140 nM, (b) 14.0 nM (c) 1.40 nM, (d) 140 pM. “*” and “#” indicate phosphorylated peptides and their dephosphorylated homologs, respectively.
Table 1 Performance comparison between Fe3O4@mZrO2 microspheres against the other enrichment materials in the literature reports.
4.4. Selective enrichment of phosphopeptides by Fe3O4@mZrO2 core/shell microspheres
We further examined the selectivity of the Fe3O4@mZrO2 core/shell microspheres for phosphopeptide enrichment from complicated biological samples. The tryptic digests of β-casein and BSA were mixed with a series of mass ratios (1:1, 1:10, 1:100, 1:1000), followed by the enrichment using Fe3O4@mZrO2 microspheres and in-situ elution for MS analysis. Our method can detect the characteristic peaks of mono- and multi-phosphorylated peptides from β-casein digests, exhibiting high tolerance to the background interference from BSA digests (Fig. 5a-d). At the extremely low mixing ratio (β-casein: BSA = 1:1000, w/w), our method still detected the signals from phosphopeptides with decent signal-to-noise ratios, (e.g. S/N 54 at m/z 3122.27). In the control experiments, Fe3O4@ZrO2 microspheres without the mesoporous structures (Fig. S6) and the commercial tips containing TiO2 nanoparticles (Fig. S7) were tested using the mixed digests of β-casein and BSA. As shown in Fig. S6, the phosphopeptide signal intensities at the above characteristic peaks were reduced using Fe3O4@ZrO2 microspheres. Depending on the mixing ratios of the samples, their S/N ratios were attenuated by several folds up to dozens of folds (Fig. S6) in comparison to those using Fe3O4@mZrO2 microspheres in the identical conditions (Fig. 5). The comparison verified that the mesoporous structures in the ZrO2 layer were indispensable for the high performance of Fe3O4@mZrO2 microspheres in phosphopeptide enrichment. The tests using the commercial tips containing TiO2 nanoparticles detected the phosphopeptides at m/z 2061.83, 2556.09, and 3122.27, with relatively lower S/N ratios (Fig. S7). The capability of the commercial tips for enrichment of multiphosphorylated peptides was limited (Fig. S7), compared to Fe3O4@mZrO2 microspheres.
Fig. 5.
Fig. 5.
Evaluation of enrichment specificity of Fe3O4@mZrO2 microspheres. MALDI-TOF mass spectra of Fe3O4@mZrO2 enriched peptides for the different mixing ratios of tryptic digests of β-casein and BSA, (a) 1:1, (b)1:10, (c)1:100, (d)1:1000. The original concentration of β-casein: 70 μM. “*” and “#” indicate phosphorylated peptides and their dephosphorylated homologs, respectively.
A549 cell lysates were mixed with β-casein digests to increase the background complexity of the samples, in order to further evaluate the enrichment performance of Fe3O4@mZrO2. A549 cell line is an adenocarcinomic cell line categorized as a non-small-cell lung carcinoma (NSCLC), which has been reported to be common and accounts for more than 85% of all cases of lung cancer [38]. Detection of the upregulated phosphoproteins may provide important information on the signaling pathways of cancer cell adhesion, proliferation, and migration in NSCLC [39]. As shown in Fig. 6, the spiked phosphopeptides including the tetraphosphorylated peptide at m/z 3122.27 were selectively enriched with relatively high signal-to-noise ratio by Fe3O4@mZrO2 microspheres, when the target phosphopeptides from β-casein digests in gradient dilutions (from 14.0 μM to 140 nM) were mixed with the A549 cell lysates. Consistently, our approach was able to detect the mono- and multi-phosphorylated peptides including their dephosphorized homologs in the diluted concentration of 140 nM. Below this concentration, the signal intensities of the background proteins from A549 cell lysates became predominant, thus suppressing these three characteristic peaks from the spiked β-casein digests.
Fig. 6.
Fig. 6.
MALDI-TOF mass spectra of the tryptic digest mixture of β-casein and cell extracts (A549) with different concentrations of β-casein. (a) 14.0 μM; (b) 1.40 μM; (c) 140 nM; (d) 14.0 nM. “*” and “#” indicate phosphorylated peptides and their dephosphorylated homologs, respectively.
We attempted to analyze the mass spectrometric data of A549 cell lysate after enrichment with Fe3O4@mZrO2 microspheres, and compared the sequences of the enriched phosphopeptides against the references in the UniProt protein database. We identified a panel of the endogenous phosphorylated peptides relevant to the abnormally up-regulated protein expression in lung cancers including NSCLC. Some of them have been proposed to have diagnostic values as the potential cancer biomarkers in the literature [[38], [39], [40]], including thioredoxin (TXN), ribosomal protein L39 and S30, histone H2A.2, p53, and HSP90 etc. (Table. S1). Further investigation is necessary in order to apply this enrichment technique onto the clinical samples. It shall be cautious to reuse the material of Fe3O4@mZrO2 microspheres. This manuscript presents two different protocols of using Fe3O4@mZrO2 microspheres for phosphopeptide enrichment. As for the scenario I (in situ elution), it might not be convenient to recover the microspheres from the MALDI plate after the use. There could also be a concern of contamination between different samples after recycling the materials. As for the scenario II (the conventional elution), after separation of the microspheres, a thoroughly-cleaning procedure would be required in order to minimize the sample contamination issue for the purpose of reuse.
5. Conclusion
In summary, we have developed Fe3O4@mZrO2 core-shell microspheres and demonstrated their applications in the enrichment of phosphopeptides. Due to the unique properties of the metal oxide of ZrO2 in the mesoporous structure, this technology enables highly efficient enrichment of mono- and multi-phosphorylated peptides simultaneously and in-situ elution for sensitive detection. The limit of detection for the target phosphopeptides can achieve femtomole range by our approach. Our experiments suggest a reliable performance for this technique to selectively enrich phosphopeptides from various types of complex samples. Therefore, it promises important applications in phosphoproteomics through the enrichment of the critical cell signaling regulators which might be missed otherwise.
Acknowledgements
This work is supported by the National Key Research and Development Program of China (2017YFE0131700), and the National Natural Science Foundation of China (21874096); a project supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the 111 Project, Joint International Research Laboratory of Carbon-Based Functional Materials and Devices, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.jmst.2020.02.091.
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Cell signaling mechanisms often transmit information via posttranslational protein modifications, most importantly reversible protein phosphorylation. Here we develop and apply a general mass spectrometric technology for identification and quantitation of phosphorylation sites as a function of stimulus, time, and subcellular location. We have detected 6,600 phosphorylation sites on 2,244 proteins and have determined their temporal dynamics after stimulating HeLa cells with epidermal growth factor (EGF) and recorded them in the Phosida database. Fourteen percent of phosphorylation sites are modulated at least 2-fold by EGF, and these were classified by their temporal profiles. Surprisingly, a majority of proteins contain multiple phosphorylation sites showing different kinetics, suggesting that they serve as platforms for integrating signals. In addition to protein kinase cascades, the targets of reversible phosphorylation include ubiquitin ligases, guanine nucleotide exchange factors, and at least 46 different transcriptional regulators. The dynamic phosphoproteome provides a missing link in a global, integrative view of cellular regulation.
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Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis, but aberrant protein phosphorylation regulation leads to various diseases. The study of signaling regulation by protein phosphorylation is complicated in part by the sheer scope of the kinome and phosphoproteome, dependence of signaling protein functionality on cellular localization, and the complex multivariate relationships that exist between protein phosphorylation dynamics and the cellular phenotypes they control. Additional complexities arise from the ability of microenvironmental factors to influence phosphorylation-dependent signaling and from the tendency for some signaling processes to occur heterogeneously among cells. These considerations should be taken into account when measuring cell signaling regulation by protein phosphorylation.
The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) is now recognized as a critical regulator of many physiological and pathophysiological processes, including cancer, atherosclerosis, diabetes and osteoporosis. S1P is produced in cells by two sphingosine kinase isoenzymes, SphK1 and SphK2. Many cells secrete S1P, which can then act in an autocrine or paracrine manner. Most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. More recently, it was shown that S1P also has important intracellular targets involved in inflammation, cancer and Alzheimer's disease. This suggests that S1P actions are much more complex than previously thought, with important ramifications for development of therapeutics. This review highlights recent advances in our understanding of the mechanisms of action of S1P and its roles in disease.
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Enrichment, separation and mass spectrometric analysis of biomolecules carrying a phosphate group plays an important role in current analytical chemistry. Application areas range from the preparative enrichment of phospholipids for biotechnological purposes and the separation and purification of plasmid DNA or mRNA to the specific preconcentration of phosphoproteins and -peptides to facilitate their later identification and characterization by mass spectrometry. Most of the recent improvements in this field were triggered by the need for phosphopeptide enrichment technology for the analysis of cellular protein phosphorylation events with the help of liquid chromatography-mass spectrometry. The high sensitivity of mass spectrometry and the possibility to combine this technique with different separation modes in liquid chromatography have made it the method of choice for proteome analysis. However, in the case of phosphoprotein analysis, the low abundance of the resulting phosphopeptides and their low quality fragment spectra interfere with the identification of phosphorylation events. Recent developments in phosphopeptide enrichment and fragmentation technologies successfully helped to overcome these limitations. In this review, we will focus on sample preparation techniques in the field of phosphoproteomics, but also highlight recent advancements for the analysis of other phosphorylated biomolecules.
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In signal transduction in eukaryotes, protein phosphorylation is a key event. To understand signaling processes, we must first acquire an inventory of phosphoproteins and their phosphorylation sites under different conditions. Because phosphorylation is a dynamic process, elucidation of signaling networks also requires quantitation of these phosphorylation events. In this article, we outline several methods for enrichment of phosphorylated proteins and peptides and discuss various options for their identification and quantitation with special emphasis on mass spectrometry-based techniques.
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Recent advances in single-cell analysis have unraveled substantial heterogeneity among seemingly identical cells at genomic and transcriptomic levels. These discoveries have urged scientists to develop new tools that are capable of investigating single cells from a broader set of
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In this work, we present, to our knowledge, the first demonstration of the utility of iron oxide magnetic microspheres coated with gallium oxide for the highly selective enrichment of phosphopeptide prior to mass spectrometric analysis. These microspheres that we prepared not only have a shell of gallium oxide, giving them a high-trapping capacity for the phosphopeptides, but also their magnetic property enables easy isolation by positioning an external magnetic field. Tryptic digest products of phosphoproteins including beta-casein, ovalbumin, casein, as well as five protein mixtures were used as the samples to exemplify the feasibility of this approach. In very short time (only 0.5 min), phosphopeptides sufficient for characterization by MALDI-TOF-MS were selectively enriched by the Ga(2)O(3)-coated Fe(3)O(4) microspheres. The performance of the Ga(2)O(3)-coated Fe(3)O(4) microspheres were further compared with Fe(3+)-immobilized magnetic silica microspheres, commercial Fe(3+)-IMAC resin, and TiO2 beads for enrichment of peptides originating from tryptic digestion of beta-casein and BSA with a molar ratio of 1:50, and the results proved a stronger selective ability of Ga(2)O(3)-coated Fe(3)O(4) microspheres over the other materials. Finally, the Ga(2)O(3)-coated Fe(3)O(4) microspheres were successfully utilized for enrichment of phosphopeptides from digestion products of rat liver extract. All results show that Ga(2)O(3)-coated Fe(3)O(4) microsphere is an effective material for selective isolation and concentration of phosphopeptides.
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Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the analysis of protein phosphorylation by detection of phosphopeptides in phosphoprotein digest. Enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC) followed with MALDI analysis is the common approach. However, the pH for loading and elution of phosphopeptides is incompatible with protein digestion as well as the preparation of the MALDI matrix solution. Therefore, some pretreatment steps, such as pH adjustment and desalting, are required, which make the approach tedious and insensitive. In this study, a strong anion-exchange (SAX) capillary monolith was prepared to enrich phosphopeptides from protein digest for MALDI-TOF MS analysis. It was found that phosphopeptides could be specifically retained on the SAX column at high pH around 8 and could be eluted by 5% formic acid. Thus, the protein digests without any pretreatment could be loaded onto the SAX column under basic pH condition; after removing nonphosphopeptides by washing, the bound phosphopeptides could be eluted directly onto a MALDI target and analyzed by MALDI-TOF MS. This approach significantly simplified the analytical procedures and reduced the sample loss. Because of the excellent MALDI MS compatible procedure and the microscale SAX column, a detection limit as low as 50 amol for the analysis of phosphopeptides from beta-casein digest was achieved. To circumvent the inconvenience of the sample loading, a new simple sample introducing method based on capillary action was proposed, which further reduced the detection limit to 10 amol.
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A need exists for technologies that permit the direct quantification of differences in protein and posttranslationally modified protein expression levels. Here we present a strategy for the absolute quantification (termed AQUA) of proteins and their modification states. Peptides are synthesized with incorporated stable isotopes as ideal internal standards to mimic native peptides formed by proteolysis. These synthetic peptides can also be prepared with covalent modifications (e.g., phosphorylation, methylation, acetylation, etc.) that are chemically identical to naturally occurring posttranslational modifications. Such AQUA internal standard peptides are then used to precisely and quantitatively measure the absolute levels of proteins and posttranslationally modified proteins after proteolysis by using a selected reaction monitoring analysis in a tandem mass spectrometer. In the present work, the AQUA strategy was used to (i) quantify low abundance yeast proteins involved in gene silencing, (ii) quantitatively determine the cell cycle-dependent phosphorylation of Ser-1126 of human separase protein, and (iii) identify kinases capable of phosphorylating Ser-1501 of separase in an in vitro kinase assay. The methods described here represent focused, alternative approaches for studying the dynamically changing proteome.
Next-generation sequencing allows the analysis of genomes, including those representing disease states. However, the causes of most disorders are multifactorial, and systems-level approaches, including the analysis of proteomes, are required for a more comprehensive understanding. The proteome is extremely multifaceted owing to splicing and protein modifications, and this is further amplified by the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS). MS-based proteomics is starting to mature and to deliver through a combination of developments in instrumentation, sample preparation and computational analysis. Here we describe this emerging next generation of proteomics and highlight recent applications.
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The liver is the largest organ in the body, with many complex, essential functions, such as metabolism, deintoxication, and secretion, often regulated via post-translational modifications, especially phosphorylation. Thus, the detection of phosphoproteins and phosphorylation sites is important to comprehensively explore human liver biological function. The human Chang liver cell line is among the first derived from non-malignant tissue, and its phosphoproteome profile has never been globally analyzed. To develop the complete phosphoproteome and probe the roles of protein phosphorylation in normal human liver, we adopted a shotgun strategy based on strong cation exchange chromatograph, titanium dioxide and LC-MS/MS to isolate and identify phosphorylated proteins. Two types of MS approach, Q-TOF and IT, were used and compared to identify phosphosites from complex protein mixtures of these cells. A total of 1035 phosphorylation sites and 686 phosphorylated peptides were identified from 607 phosphoproteins. A search using the public database of PhosphoSite showed that approximately 344 phosphoproteins and 760 phosphorylation sites appeared to be novel. In addition, N-terminal phosphorylated peptides were a greater fraction of all identified phosphopeptides. With GOfact analysis, we found that most of the identified phosphoproteins are involved in regulating metabolism, consistent with the liver's role as a key metabolic organ.
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In this study, mesoporous TiO(2) microspheres were synthesized by simple hydrothermal reaction, and successfully developed for phosphopeptides enrichment from both standard protein digestion and real biological sample such as rat brain tissue extract. The mesoporous TiO(2) microspheres (the diameter size of about 1.0 microm) obtained by simple hydrothermal method were found to have a specific surface area of 84.98 m(2)/g, which is much larger than smooth TiO(2) microspheres with same size. The surface area of mesoporous TiO(2) microspheres is almost two times of commercial TiO(2) nanoparticle (a diameter of 90 nm). Using standard proteins digestion and real biological samples, the superior selectivity and capacity of mesoporous TiO(2) microspheres for the enrichment of phosphorylated peptides than that of commercial TiO(2) nanoparticles and TiO(2) microspheres was also observed. It has been demonstrated that mesoporous TiO(2) microspheres have powerful potential for selective enrichment of phosphorylated peptides. Moreover, the preparation of the mesoporous TiO(2) microspheres obtained by the hydrothermal reaction is easy, simple and low-cost. These mesoporous TiO(2) microspheres with the ability of large scale synthesis can widely be applied for phosphorylated proteomic research.
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Novel Bi0.15Fe0.15TiO2 nanocomposites (B0.15F0.15TNs) were synthesized for the first time by a modified sol-gel technology and successfully applied to selective extraction and enrichment of phosphopeptides from digested protein mixture solutions and real samples (tissue protein extract from human liver). The codoping of Bi and Fe into TiO2 results in a significant enhancement in both the enrichment efficiency and selectivity. Compared with the commercial available TiO2 extractant, the proposed B0.15F0.15TNs possess a lower detection limit (2 x 10(-9) M) and higher selectivity at a low weight ratio of 1:1200 (phosphopeptides/nonphosphopeptides). Additionally, a total of 223 phosphorylation sites were identified from the human liver lysate after enrichment by the B0.15F0.15TNs. In addition, the synthesis of B0.15F0.15TNs is quite easy, of high yield, and inexpensive.
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Amino functional mesoporous silica SBA-15 materials have been prepared to develop efficient adsorbents of heavy metals in wastewater. Functionalization with amino groups has been carried out by using two independent methods, grafting and co-condensation. Three organic moieties have been selected to incorporate the active amino sites: aminopropyl (H(2)N-(CH(2))(3)-), [2-aminoethylamino]-propyl (H(2)N-(CH(2))(2)-NH-(CH(2))(3)-) and [(2-aminoethylamino)-ethylamino]-propyl (H(2)N-(CH(2))(2)-NH-(CH(2))(2)-NH-(CH(2))(3)-). Materials have been characterized by XRD, nitrogen sorption measurements and chemical analysis. We have found that all materials preserve the mesoscopic order and exhibit suitable textural properties and nitrogen contents to act as potential adsorbents. Metal removal from aqueous solution has been examined for Cu(II), Ni(II), Pb(II), Cd(II), and Zn(II); adsorption performances of materials prepared by the two functionalization methods have been compared. In addition, copper adsorption process has been thoroughly studied from both kinetic and equilibrium points of view for some selected materials. Aqueous Cu(II) adsorption rates show that the overall process is fast and the time evolution can be successfully reproduced with a pseudo-second-order kinetic model. Whole copper adsorption isotherms have been obtained at 25 degrees C. Significant maximum adsorption capacities have been found with excellent behavior at low concentration.
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A mixed-solvent method was developed to coat polystyrene (PS) spheres with smooth, homogeneous shells of amorphous titania by ammonia catalysis. The TEM images showed that, in the presence of ammonia, the thickness of titania shells could be controlled in the range of 8-65 nm by varying the concentration of titanium tetrabutoxide (TBOT) in the ethanol/acetonitrile mixed solvents with an appropriate volume ratio. The diffusion-controlled mechanism of the mixed solvents and the catalysis mechanism of ammonia were investigated. After the calcination of core-shell particles for 2 h at 500 degrees C, spherical hollow titania shells could be obtained, and the surfaces of the particles remained quite smooth and homogeneous. The XRD analysis indicated that calcination promoted the transformation of amorphous titania into an anatase phase.
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Protein phosphorylation plays a key role in most cellular processes. Studying phosphopeptides in complex biological samples has been a great challenge due to their low abundance as well as the coexistence of excessive amounts of salts or surfactants. In this work we demonstrate a general approach for selective separation of phosphopeptides using a class of novel mesoporous nanostructured materials. TiO(2) nanocrystals are first self-assembled into submicrometer clusters containing relatively uniform mesoscale pores and then stabilized by coating with a thin layer of silica. Calcination of the materials at high temperatures connects the neighboring nanocrystals together and enhances the mechanical stability of the clusters and at the same time removes the organic surfactants and makes the TiO(2) surface fully accessible to phosphopeptides. By coating the nanocrystal clusters with a layer of silica before calcination and removing it afterward through chemical etching, we have been able to make the cluster surface hydrophilic and negatively charged, thus enhancing the water dispersibility of the clusters and eventually their accessibility to phosphopeptides. The high selectivity and capacity of these mesoporous TiO(2) clusters have been demonstrated by effectively enriching phosphopeptides from digests of phosphoprotein (alpha- or beta-casein), protein mixtures of beta-casein and bovine serum albumin, milk, and human serum samples. We also demonstrate that the self-assembly process brings the flexibility of incorporation of multiple components, such as superparamagnetic nanocrystals, to further facilitate the peptide separation.
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Precursor ion scans have proven to be extremely useful for the characterization of unseparated peptide mixtures. In conjunction with the nanoelectrospray source, precursor ion scans provide a sensitive tool for the detection of posttranslationally modified peptides and have been used to determine phosphorylation sites of proteins digested in solution. In this report, we extend our previous work to the determination of protein phosphorylation sites of gel-isolated proteins. The in-gel digestion procedure developed in our laboratory for protein microsequencing was found to be suitable for phosphorylation mapping as well. The risk of losing hydrophilic peptides in the desalting step was decreased by using column packing material designed for the purification of oligonucleotides and by adjusting the pH conditions to the needs of phosphopeptide analysis. With this method, the tryptic phosphopeptides of beta-casein were detected after in-gel digestion at a sensitivity of 250 fmol of protein applied to the gel. The phosphorylation sites of two other proteins, Src-delta U and Op18, have similarly been mapped. Subpicomole to low-picomole amounts of protein starting material are needed in general, although we and others have reported attomole sensitivity for the detection of model phosphopeptides using precursor ion scans. This indicates that the success in determining phosphorylation sites depends crucially on the digestion, extraction, and detection efficiency for individual phosphopeptides.
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Qualitative and quantitative characterization of phosphopeptides by means of mass spectrometry (MS) is the main goal of MS-based phosphoproteomics, but suffers from their low abundance in the large haystack of various biological molecules. Herein, we introduce two-dimensional (2D) metal oxides to tackle this biological separation issue. A nanocomposite composed of titanoniobate nanosheets embedded with Fe(3)O(4) nanocrystals (Fe(3)O(4)-TiNbNS) is constructed via a facile cation-exchange approach, and adopted for the capture and isotope labeling of phosphopeptides. In this nanoarchitecture, the 2D titanoniobate nanosheets offer enlarged surface area and a spacious microenvironment for capturing phosphopeptides, while the Fe(3)O(4) nanocrystals not only incorporate a magnetic response into the composite but, more importantly, also disrupt the restacking process between the titanoniobate nanosheets and thus preserve a greater specific surface for binding phosphopeptides. Owing to the extended active surface, abundant Lewis acid sites and excellent magnetic controllability, Fe(3)O(4)-TiNbNS demonstrates superior sensitivity, selectivity and capacity over homogeneous bulk metal oxides, layered oxides, and even restacked nanosheets in phosphopeptide enrichment, and further allows in situ isotope labeling to quantify aberrantly-regulated phosphopeptides from sera of leukemia patients. This composite nanosheet greatly contributes to the MS analysis of phosphopeptides and gives inspiration in the pursuit of 2D structured materials for separation of other biological molecules of interests.
BACKGROUND: Current histopathological classification and TNM staging have limited accuracy in predicting survival and stratifying patients for appropriate treatment. The goal of the study is to determine whether the expression pattern of functionally important regulatory proteins can add additional values for more accurate classification and prognostication of non-small lung cancer (NSCLC). METHODS: The expression of 108 proteins and phosphoproteins in 30 paired NSCLC samples were assessed using Protein Pathway Array (PPA). The differentially expressed proteins were further confirmed using a tissue microarray (TMA) containing 94 NSCLC samples and were correlated with clinical data and survival. RESULTS: Twelve of 108 proteins (p-CREB(Ser133), p-ERK1/2(Thr202/Tyr204), Cyclin B1, p-PDK1(Ser241), CDK4, CDK2, HSP90, CDC2p34, beta-catenin, EGFR, XIAP and PCNA) were selected to build the predictor to classify normal and tumor samples with 97% accuracy. Five proteins (CDC2p34, HSP90, XIAP, CDK4 and CREB) were confirmed to be differentially expressed between NSCLC (n=94) and benign lung tumor (n=19). Over-expression of CDK4 and HSP90 in tumors correlated with a favorable overall survival in all NSCLC patients and the over-expression of p-CREB(Ser133) and CREB in NSCLC correlated with a favorable survival in smokers and those with squamous cell carcinoma, respectively. Finally, the four proteins (CDK4, HSP90, p-CREB and CREB) were used to calculate the risk score of each individual patient with NSCLC to predict survival. CONCLUSION: In summary, our data demonstrated a broad disturbance of functionally important regulatory proteins in NSCLC and some of these can be selected as clinically useful biomarkers for diagnosis, classification and prognosis.
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In the last decade, proteomic analysis has become an integral tool for investigation of tumor biology, complementing the genetic analysis. The idea of proteomics is to characterize proteins by evaluation of their expressions, functions, and interactions. Proteomics may also provide information about post-translational modifications of proteins and evaluate their value as specific disease biomarkers. The major purpose of clinical proteomics studies is to improve diagnostic procedures including the precise evaluation of biological features of tumor cells and to understand the molecular pathogenesis of cancers to invent novel therapeutic strategies and targets. This review briefly describes the latest reports in proteomic studies of NSCLC. It contains a summary of the methods used to detect proteomic markers in different types of biological material and their clinical application as diagnostic, prognostic, and predictive biomarkers compiled on the basis of the most recent literature and our own experience.
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Pemetrexed is an antifolate agent which has been used for treating malignant pleural mesothelioma and non small lung cancer in the clinic as a chemotherapeutic agent. In this study, pemetrexed inhibited cell growth and induced G1 phase arrest in the A549 cell line. To explore the molecular mechanisms of pemetrexed involved in cell growth, we used a two-dimensional polyacrylamide gel electrophoresis (2-DE) proteomics approach to analyze proteins changed in A549 cells treated with pemetrexed. As a result, twenty differentially expressed proteins were identified by ESI-Q-TOF MS/MS analysis in A549 cells incubated with pemetrexed compared with non-treated A549 cells. Three key proteins (GAPDH, HSPB1 and EIF4E) changed in pemetrexed treated A549 cells were validated by Western blotting. Accumulation of GAPDH and decrease of HSPB1 and EIF4E which induce apoptosis through inhibiting phosphorylation of Akt were noted. Expression of p-Akt in A549 cells treated with pemetrexed was reduced. Thus, pemetrexed induced apoptosis in A549 cells through inhibiting the Akt pathway.
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