Enrichment of Phosphorylated Peptides with Metal-Organic

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Enrichment of Phosphorylated Peptides with Metal-Organic Framework Nanosheets for Serum Profiling of Diabetes and Phosphoproteomics Analysis Shishu Yang, Yu-Jie Chang, Hao Zhang, Xizhong Yu, Wenbin Shang, Gui-Quan Chen, David D. Y. Chen, and Zhi-Yuan Gu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04417 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 29, 2018

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Analytical Chemistry

Enrichment of Phosphorylated Peptides with Metal-Organic Framework Nanosheets for Serum Profiling of Diabetes and Phosphoproteomics Analysis Shi-Shu Yang1, Yu-Jie Chang1, Hao Zhang2, Xizhong Yu2, Wenbin Shang2, Gui-Quan Chen3, David Da Yong Chen1,4,*, and Zhi-Yuan Gu1,* 1Jiangsu

Key Laboratory of Biofunctional Materials, Jiangsu Collaborative Innovation

Center of Biomedical Functional Materials, College of Chemistry and Materials Science, Nanjing Normal University, Nanjing 210023, China 2Key

Laboratory for Metabolic Diseases in Chinese Medicine, First College of Clinical

Medicine, Nanjing University of Chinese Medicine, Nanjing 210023, China 3State

Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of

Model Animal for Disease Study, Model Animal Research Center, Nanjing University, 12 Xuefu Avenue, Nanjing, 210061, China 4Department

of Chemistry, University of British Columbia, Vancouver, BC, Canada,

V6T 1Z1

Corresponding authors' E-mails: [email protected]; [email protected]

*

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ABSTRACT Capturing phosphopeptides from complicated biological samples is essential for the discovery of new post-translational modification sites and disease diagnostics. Although several two-dimensional (2-D) materials have been used for phosphopeptides capturing, metal-organic frameworks (MOF) nanosheets has not been reported. The Ti-based MOF nanosheets have well-defined 2-D morphology, high density of active sites, large surface area and ultrathin structure. Phosphopeptides can be efficiently extracted and superior detection limits of 0.1 fmol µL-1 can be achieved even for extremely low molar ratio of phosphoprotein/nonphosphoprotein (1:10000) mixtures. The selectivity over nonphosphopeptides can be enhanced further by pretreatment with a 10 mM salt solution (βglycerophosphate disodium, NaCl or KCl). The performance of 2-D Ti-based MOF nanosheets is much better than Zr-based MOF (Zr-BTB) nanosheets or any other Ti-based 3-D MOF counterpart, such as MIL-125 and NH2-MIL-125. The nanosheets were used for in situ isotope labeling for abnormally-regulated phosphopeptides analysis from serum samples of Type 2 diabetes patients. The relative quantitative results showed three of the phosphorylated

fibrinogen

peptides

A

(FPA,

DpSGEGDFLAEGGGV,

DpSGEGDFLAEGGGVR, ADpSGEGDFLAEGGGVR) were down-regulated while the other isoform (ADpSGEGDFLAEGGGV) was up-regulated in the serum samples of Type 2 diabetes patient compared with those of healthy volunteers. Finally, proteomics analysis showed selective enrichment of phosphopeptides with 2-D Ti-based MOF nanosheets from real samples including tryptic digests of mouse brain neocortex lysate, mouse spinal cord 2


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lysate and mouse testis lysate followed by LC-MS/MS analysis. Total numbers of 2601, 3208 and 2866 phosphopeptides were successfully identified from the three sample, respectively. The 2-D Ti-based MOF nanosheets significantly improved sample preparation for mass spectrometric analysis in phosphopeptides and phosphoproteomics research.

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INTRODUCTION Metal-organic frameworks (MOFs) are porous crystalline materials that have been used for gas adsorption, separation, catalysis, energy storage and carbon dioxide capture due to their unique properties such as high specific surface areas, ultrahigh porosity and diverse functionalities.1-7 Two-dimensional (2-D) MOF nanosheets are often the subjects of research,8-14 but their successful applications were limited to a few areas, such as energy storage, membrane separation and sensing.15-17 The advantages of 2-D MOF nanosheets, such as well-defined morphology, large active surface areas and good dispersibility, can be significant for the selective enrichment of compounds with unique functional groups such as phosphopeptides. Reversible protein phosphorylation plays crucial regulatory in many cellular processes and significantly alters protein activities and functions.18,19 Shotgun proteomics based on mass

spectrometry

(MS)

is

a

mainstream

method

for

detecting

phosphoproteins/phosphopeptides.20 However, this type of analysis are difficult due to the extremely low concentrations (less than 1×10-9 M)21 and background peaks interference from the abundance of nonphosphopeptides.22-27 To tackle these issues, highly efficient enrichment of phosphopeptides is the prerequisite to investigate their functions and relevant biochemical process which has attracted tremendous attention in proteomics research. Different strategies have been used for the selective enrichment of phosphopeptides, including

ion-exchange

chromatography,28-30

immunoprecipitation,24,31

4


chemical

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modification strategies,32 immobilized metal affinity chromatography (IMAC)33-36 and metal oxide affinity chromatography (MOAC).37-40 IMAC relies on chelation and electrostatic interactions between the phosphate group and the immobilized metal cations,41 such as Al3+, Ti4+, Zr4+, which sometimes required complicated synthesis procedures. MOAC bases on bridging bidentate binding between the phosphate group and the surface of the metal oxide,42 such as Al2O3, TiO2, ZrO2 with fair affinity. Because the phosphate groups in phosphopeptides are hard bases, the hard acids such as high-valence metal cations or their oxides are usually chosen for phosphopeptides enrichment according to the hard and soft acids and bases (HSAB) theory.43 Thus, simple and robust materials with significant selectivity and affinity with high-valence metal, such as Ti and Zr, should be highly desirable for selectively enrich low abundance phosphopeptides from the complex biological samples. Herein, four water-stable MOFs and MOF nanosheets, including Ti-based 3-D MOFs (MIL-125 and NH2-MIL-125), Ti-based 2-D MOF nanosheets and Zr-based 2-D nanosheets were used as the enrichment materials. Due to the well-defined 2-D morphology, nanometer scaled thickness and high surface area with abundant Ti, the nanosheet can provide sufficient binding sites for phosphopeptides, which is different from previous reports,44-46 the Ti-based MOF nanosheets demonstrated the best selectivity, sensitivity and capacity in the enrichment of standard phosphopeptides. Furthermore, the Ti-based MOF nanosheets were used with in situ isotope labeling in the analysis of the

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dysregulated phosphopeptides in serum samples from Type 2 diabetes patients and enrichment of phosphopeptides from three tryptic digest of mouse tissues lysate. EXPERIMENTAL SECTION Materials and Chemicals. Acetonitrile (ACN), Trifluoroacetic (TFA), α-casein (from bovine milk), β-casein (from bovine milk), bovine serum albumin (BSA, from bovine milk), trypsin (TPCK treated), ammonium bicarbonate (NH4HCO3), 2,5-dihydroxybenzoic acid (DHB) and 2,5-dihydroxyterephtalic acid (H4DOBDC) were purchased from SigmaAldrich (St. Louis, MO). Ammonia solution (ACS, 28.0-30.0% NH3 basis) and βglycerophosphate disodium salt hydrate were obtained from Aladdin (Shanghai, China). Nonfat milk was purchased from a local grocery store. 1,3,5-benzenetribenzoic acid (H3BTB) was obtained from TCI (Shanghai, China). N,N-diethylformamide (DEF, 99%) was obtained from Alfa Aesar (Ward Hill, USA). The ultrapure water (18.4 MΩ·cm) was prepared by an ELGA purification system (Veolia Water Solutions & Technologies, UK). Characterization and Measurements. X-ray diffraction (XRD) pattern was obtained using a Rigaku D/MAX-2500 (Tokyo, Japan) diffractometer with a CuKα radiation (1.54056Å). Transmission electron microscopy (TEM) images were collected on a H7650 (state manufactuer and its location) operated at an accelerating voltage of 120 kV. Atomic force microscopy (AFM) measurement was performed with a PicoPlus in tapping mode (Agilent, US). The scanning electron microscopy (SEM) images were collected on a JSM7600F (JEOL Ltd). The N2 adsorption-desorption experiment was measured in a Micromeritics ASAP 2050 system at 77 K. 6


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Sample Preparation. Proteins (bovine α-casein, β-casein or BSA) were dissolved in a solution of ammonium bicarbonate (50 mM, pH~10) with a final concentration of 1 μg·μL-1 for α-casein or β-casein, and 10 μg·μL-1 for BSA, then digested with trypsin. The mixture with concentration ratio of 50:1 (protein versus trypsin) was incubated in a 37 oC water bath for 20 h. For nonfat milk, 30 μL of the sample was diluted in 970 μL 50 mM NH4HCO3 aqueous buffer solution. The mixed solution was centrifugated at 14000 rpm for 25 min to remove precipitate. The supernatant was denatured at 100 oC for 5 min, then was mixed with 30 μg of trypsin to incubate at 37 oC for 20 h. Finally, all the samples were lyophilized and stored at -80 oC. Enrichment of Phosphopeptides from Tryptic Digest of Standard Proteins. The Tibased MOF nanosheets were synthesized according to previous reported procedures.17,47 The Ti-based MOF nanosheets (1 mg) were first incubated with 200 µL 10 mM βglycerophosphate disodium for 20 min and then washed with 200 µL deionized water 3 times. The phosphopeptides enrichment procedures were similar to the previously described strategies.48 Briefly, 1 µL peptides mixture was incubated with Ti-based MOF nanosheets for 30 min in a sample loading buffer (40% ACN (v/v) and 3% TFA (v/v)). Then, the unadsorbed peptides were washed away using Washing Buffer 1 (50% ACN (v/v) and 4% TFA (v/v) and 200 mM NaCl) and Washing Buffer 2 (30% ACN (v/v) and 0.1% TFA (v/v)) sequentially. Finally, the adsorbed peptides were eluted from the nanosheets with 10 µL of 10% NH3·H2O and analyzed by MALDI-TOF MS.

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MALDI-TOF MS Analysis. A 1 μL eluate was deposited on the MALDI plate and then covered with 1 μL of 2,5-DHB matrix aqueous solution (20 mg·mL-1 DHB in 1% H3PO4 aqueous solution). Phosphopeptides were analyzed by MALDI-TOF MS (Bruker Daltonics, Germany) in a positive-ion mode with SmartbeamTM-II laser technology. The measurements were made in the reflection mode with the shots number of 500 and the acceleration voltage of +25 kV. In situ Isotope Dimethyl Labeling of Phosphopeptides. In situ isotope labeling was performed after phosphopeptides capturing and nonphosphopeptides washing steps and before the elution step. At that time, the 2-D Ti-based MOF nanosheets with adsorbed phosphopeptides were dispersed into a 200 µL CH3COONa buffer solution (100 mM, pH~5.8) and added with 8 µL of 4% (v/v) CD2O or CH2O and 8 µL NaBH3CN (0.6 M). After vortexing for 40 min, 10 µL of 88% formic acid (FA) was added to terminate the labeling reaction. Rinsing with 200 µL washing buffer (50% ACN (v/v) and 0.1% TFA (v/v)) was performed to remove the excess labeling reagents. Finally, the captured phosphopeptides on the 2-D Ti-based MOF nanosheets and labeled in situ with CD2O or CH2O were eluted by adding 10 µL of 10% NH3·H2O. Relative Quantification of Serum Phosphopeptides. Human serum samples were collected from 3 patients with diabetes mellitus Type 2 (also known as Type 2 diabetes) and 3 healthy volunteers in Jiangsu Province Hospital of TCM (Affiliated Hospital of Nanjing University of Chinese Medicine) according to their standard clinical pocedures. All of the human serum samples were obtained with consent of both patients and healthy 8


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volunteers, and the handling of human serum samples were performed in observance with the relevant laws and institutional guidelines. Also, we have obtained the ethical approval from the institutional committee. Typically, 5 µL of raw serum was added to 100 µL loading buffer and shaken using vortex mixer with 1.0 mg of Ti-based MOF nanosheets for 30 min. The phosphopeptides enrichment, loading, washing and eluting process were the same as the above experiments. For relative quantification, a total volume of 1 µL with different ratios of 1:4, 1:1 and 4:1 was pipetted and mixed from the eluents of healthy volunteers (CH2O light-labeled) and patients (CD2O heavy-labeled), respectively. Finally, a 1 µL mixture was deposited onto the MALDI target. Protein Extraction from Mouse Tissues. Mouse brain neocortex tissues, mouse spinal cord tissues and mouse testis lysate were first dissected on ice. A half of them were homogenized in 500 μL RIPA buffer (20 mM Tris pH 7.5; 1% Nonidet P-40; 150 mM NaCl; 0.5% Sodium Deoxycholate; 0.1% SDS; 1 mM EDTA) under ice bath and diluted to 750 μL. The obtained samples were cooled on dry ice then stored at -80 oC. The samples were then thawed at 4 oC and sonicated for 15 s twice under ice bath. Thereafter, about 700 μL of supernatant was obtained by centrifugation with 12,000 G for 15 min. Finally, the samples were stored at -20 oC. All animal experiments were under the regulation of local authority with permissions. MS Detection. Enriched phosphopeptides were desalted with StageTip C18 columns. Then, RPLC-ESI-MS/MS was used to analyze the samples. LC-MS/MS detection was carried out on a hybrid quadrupole-TOF LC/MS/MS mass spectrometer (TripleTOF 5600+, AB Sciex) 9



equipped with a nanospray source. Peptides were first loaded onto a C18 trap column (5 μm, 5×0.3 mm, Agilent Technologies) and then eluted into a C18 analytical column (75 μm×150 mm, 3 μm particle size, 100 Å pore size, Eksigent). Mobile phase A (3% DMSO, 97% H2O, 0.1% formic acid) and mobile phase B (3% DMSO, 97% ACN, 0.1% formic acid) were used to establish a 100 min gradient, which comprised of: 0 min in 5% B, 65 min of 5-23% B, 20 min of 23-52% B, 1 min of 52-80% B, the gradient was maintained in 80% B for 4 min, followed by 0.1 min of 80-5% B, and a final step in 5% B for 10 min. A constant flow rate was set at 300 nL min-1. MS scans were conducted from 350 to 1500 amu, with a 250 ms time span. For MS/MS analysis, each scan cycle consisted of one fullscan mass spectrum (with m/z ranging from 350 to 1500 and charge states from 2 to 5) followed by 40 MS/MS events. The threshold count was set to 120 to activate MS/MS accumulation and former target ion exclusion was set for 18 s. Database Searching. Raw data from TripleTOF 5600+ were analyzed with ProteinPilot Software. Data were searched against the Uniprot mouse reference proteome database using the following parameters: Sample Type, Identification; Cys Alkylation, Iodoacetamide; Digestion, Trypsin; Special Factors, Phosphorylation Emphasis. Search Effort was set to Rapid ID. RESULTS AND DISCUSSION Materials Characterization. In brief, Ti-based MOF, Ti2(HDOBDC)2(H2DOBDC), (H4DOBDC=2,5-dihydroxyterephtalic acid) was synthesized from titanium isopropoxide and H4DOBDC with solvothermal reaction in DEF.47 The 2-D Ti-based MOF nanosheets 10


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were obtained via a liquid ultrasonication exfoliation.17 The Zr-BTB nanosheets49 and other two Ti-MOFs (MIL-12550 and NH2-MIL-12551) were synthesized under solvothermal conditions and used for comparison. The detailed synthesis procedures were shown in the Supporting Information (SI). The microstructure and morphology of the Ti-based MOF was characterized by XRD and SEM. The SEM image of the Ti-based MOF displayed a clear layered crystalline structure (Figure 1a). The XRD pattern illustrated that the Ti-based MOF was successfully synthesized (Figure 1e). The solvothermal synthetic method with DEF was chosen because it was simple and reproducible compared to other methods, although it resulted in low signal-to-noise ratio of the XRD spectrum which was discussed in a previous report.47 The 2-D nanosheets were obtained by exfoliating Ti-based MOF in isopropanol for two days, and were subsequently characterized by TEM, AFM and XRD. The 2-D Ti-based MOF nanosheets were observed to be uniform with TEM and AFM (Figure 1b-d). The thickness of these nanosheets was 5 nm and in accordance to the nine layers thickness with a single layer of 0.56 nm (Figure 1f). Moreover, the XRD data showed an almost straight line due to its ultrathin thickness (Figure 1e). The above structural and mophology characterization of ultrathin 2-D Ti-based MOF nanosheets proved the successful preparation of 2-D Tibased MOF nanosheets with high density of active sites although the measured BrunauerEmmett-Teller (BET) surface area was 0.4 m2 g-1 calculated from the N2 adsorptiondesorption isotherms, which was probably due to the stacking of MOF nanosheets in solid phase other than in the aqueous solution. 11



The MIL-125, NH2-MIL-125 and Zr-BTB nanosheets were similarly characterized. The SEM images and XRD patterns of MIL-125 and NH2-MIL-125 were presented in the SI with the crystal structures of MIL-125 and NH2-MIL-125, respectively. (Figure S2 and S3). The consistency between the experimental and simulation XRD revealed that the MIL-125 and NH2-MIL-125 were successfully prepared. While the Zr-BTB nanosheets were characterized by TEM, showing a uniform 2-D morphology. The XRD pattern of the ZrBTB nanosheets was similar to the simulated one (Figure S1).

Scheme 1. Schematic illustration of the phosphopeptides enrichment from biological samples using 2-D Ti-based MOF nanosheets.

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Figure 1. The characterization of the Ti-based MOF and Ti-based MOF nanosheets. (a) SEM image of Ti-based MOF. (b) TEM image of Ti-based MOF nanosheets. (c) AFM image of the Ti-based MOF nanosheets. (d) Height outlined along the solid line (5 nm). (e) Powder XRD data for simulated Ti-based MOF, as-synthesized Ti-based MOF and Ti-based MOF nanosheets. (f) The crystal structure of a single layer of Ti-based MOF nanosheet. The Ti, C, and O atoms are shown in blue, grey, and red, respectively.

Selective Enrichment of Phosphopeptides. A tryptic digest of bovine β-casein (4×10-6 M) was used to assess the performance of the Ti-based MOF nanosheets. The tryptic digest of the protein was mixed and incubated with Ti-based MOF nanosheets for 0.5 h. Then the Ti-based MOF nanosheets were washed with Washing Buffer 1 and Washing Buffer 2, respectively. Finally, the captured phosphopeptides were eluted using an aqueous solution

13



of 10% NH3 ·H2O (pH ~ 10). One mg of Zr-BTB nanosheets, other two Ti-MOFs (MIL125 and NH2-MIL-125) and commercial TiO2 nanoparticles were also investigated in this work for comparison, respectively. As shown in Figure S4a, 4c and 4d, four phosphopeptides from the tryptic digest of β-casein (m/z=2061.6(β1), 2556.4(β2), 2965.7(β3), 3122.1(β4)) were successfully detected with the samples enriched with Tibased MOF nanosheets, MIL-125 and NH2-MIL-125. After enrichment by Zr-BTB nanosheets (Figure S4b), only one phosphopeptide (β1) was detected and the signal was very low compared to others. The results showed that the 2-D Ti-based MOF nanosheets had exceptional enrichment selectivity and efficiency, demonstrating very rare capability of the 2-D MOF nanosheets for the selective enrichment of phosphopeptides.

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Figure 2. Analysis of 4×10-6 M tryptic digest of β-casein with and without enrichment with different materials. (a) direct analysis, and (b) enrichment with Ti-based MOF nanosheets, (c) enrichment with Zr-BTB nanosheets, (d) enrichment with MIL-125 and (e) enrichment with NH2-MIL-125 (#: dephosphorylated residue of corresponding peptides (m/z: [M+H]+-80); all unmarked peaks are from non-phosphopeptides; β1: No. 1 phosphopeptide from β-casein; NL: Normalized Level). The detected phosphopeptides with m/z value and amino acid sequences are listed in Table S1.

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In order to further increase the enrichment selectivity and efficiency of 2-D Ti-based MOF nanosheets, we pretreated the materials with β-glycerophosphate disodium before the enrichment. This pretreatment established a surface modification by specific coordination between β-glycerophosphate and Ti(IV) of 2-D Ti-based MOF nanosheets52. It should enhance the selectivity of phosphopeptides over non-phosphopeptides by their competitive substitution for β-glycerophosphate on the surface of nanosheets, which was reported previously.52 For 2-D Ti-based MOF nanosheets, after pretreated by β-glycerophosphate disodium (10 mM), the non-phosphopeptides signal was significantly reduced, demonstrating successful surface modification (Figure 2a). At the same time, the MS signal of phosphopeptides was increased remarkably with a cleaner background. It was worth mentioning that although the dephosphorylated peptides were nonphosphopeptides, they were still detected in these conditions. This observation suggested that dephosphorylation did not happen during the enrichment process but rather happened in the final elution step under the basic conditions with 10% NH3·H2O. After pretreatment with β-glycerophosphate disodium for MIL-125 (Figure 2c) and NH2-MIL-125 (Figure 2d), most non-phosphopeptides were also suppressed and the intensity of the phosphopeptides were increased. The NH2-MIL-125 showed better selectivity of phosphopeptides over non-phosphopeptides than its isostructural MIL-125, which was the first time that evaluated in the enrichment of phosphopeptides. At the same time, similar results were observed in commercial TiO2 nanoparticles (Figure S5a and S5b). However, the pretreatment strategy demonstrated no significant difference for Zr-BTB 16


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nanosheets (Figure S4b and Figure 2b). In contrast, the analysis of untreated samples showed that non-phosphopeptides dominate in the spectrum, while the signal intensities of the phosphopeptides were low (Figure 2e). These results showed that after pretreated by βglycerophosphate disodium, the enrichment selectivity and efficiency were further increased and the 2-D Ti-based MOF nanosheets exhibited the best enrichment results with an excellent reproducibility (Figure S6a) and high recovery (84.56%, Figure S6b) that compared to Zr-BTB nanosheets, Ti-based two MOF materials (MIL-125 and NH2-MIL125) and commercial TiO2 nanoparticles (Table S2 and S3). The exceptional enrichment selectivity and efficiency of the 2-D Ti-based MOF nanosheets was mainly because the well dispersion of the nanosheets in the loading buffer and the abundant binding sites on the surface of the Ti-based nanosheets towards phosphopeptides. At the same time, similar results were observed after pretreated by other salts, such as 10 mM NaCl and 10 mM KCl (Figure S7). Thereafter, two samples of nonfat milk (Figure S8b and S8c) and human serum (Figure S9b and S9c) were investigated which exhibited the similar enrichment efficiency. The procedure for the phosphopeptides enrichment is illustrated in Scheme 1. The structures of phosphopeptides were simulated by PEPstrMOD.53,54

Ti-based MOF Nanosheets for Phosphopeptides Enrichment. To investigate the efficiency and selectivity of 2-D Ti-based MOF nanosheets for phosphopeptides enrichment, tryptic α-casein and β-casein digests were mixed and enriched at a molar ratio of 1:1 in different concentrations (1×10-7 M to 1×10-10 M) (Figure 3). As shown in Figure 17



3a and 3b, phosphopeptides in 1×10-7 M and 1×10-8 M of α-casein and β-casein digests were easily detected and the dephosphorylated fragments were observed. When the concentration of the mixed digests was decreased to 1×10-9 M, two phosphopeptides derived from α-casein and four phosphopeptides derived from β-casein could still be identified (Figure 3c). Moreover, three phosphopeptides together with three dephosphorylated peptides were detected when the concentration of α-casein and β-casein digests were as low as 1×10-10 M (2.4 pg μL-1) (Figure 3d), which was better than the previously reported results in

that only two phosphopeptides were observed.55 The result

further demonstrated that the Ti-based MOF nanosheets exhibited high enrichment capacity towards phosphopeptides with lower concentration.

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Figure 3. MALDI mass spectra of phosphopeptides enriched by Ti-based MOF nanosheets derived from a peptide mixture of α-casein and β-casein at a molar ratio of 1 : 1 with final concentrations of (a) 1×10-7 M. (b) 1×10-8 M. (c) 1×10-9 M. (d) 1×10-10 M (#: dephosphorylated residue of peptides (m/z: [M+H]+-80); all unmarked peaks are from nonphosphopeptides; α2: No. 2 phosphopeptides from α-casein; β1: No. 1 phosphopeptides from β-casein; NL: Normalized Level).

To simulate a complex biological sample and to further evaluate the selectivity of the Tibased MOF nanosheets toward phosphopeptides, we added the tryptic digest of bovine 19



serum albumin (BSA) to the tryptic digest of β-casein with different molar ratios of βcasein : BSA from 1 : 10 to 1 : 10000 (Figure 4). As shown in Figure 4a-d, after enrichment with the Ti-based MOF nanosheets, the phosphopeptides from β-casein (β1, β2, β3 and β4) can be observed in the mass spectrum with a clear background. When the molar ratios decreased to 1 : 5000 (Figure 4e) and 1 : 10000 (Figure 4f), three phosphopeptides from βcasein were still observed with high signal to noise (S/N) ratios and dephosphorylated fragments were also detected. A few non-phosphopeptides were observed because of the presence of high number of non-phosphopeptides in BSA. These results indicated that a trace amount of phosphopeptides can be effectively enriched by the 2-D Ti-based MOF nanosheets, even in the presence of exceedingly more interfering peptides. We then chose two samples of nonfat milk and human serum to examine the selectivity of the 2-D Ti-based MOF nanosheets in phosphopeptides enrichment. As shown in Figure S8, some abundant non-phosphopeptides were detected in direct analysis of untreated samples and only three phosphopeptides with low signals were observed (Figure S8a). In contrast, after enriched by Ti-based MOF nanosheets (Figure S8c), five phosphopeptides were detected with high intensity that derived from -casein and β-casein. As to human serum sample (Figure S9), the enrichment results were similar to nonfat milk. As shown in Figure S9c, after enriched by 2-D Ti-based MOF nanosheets, a better enrichment efficiency with a high phosphopeptides signal intensity and a clean background are obtained that compared to direct analysis (Figure S9a). The results further confirmed the ability to enrichment phosphopeptides. 20


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Figure 4. MALDI mass spectra of phosphopeptides enriched by Ti-based MOF nanosheets from a peptide mixture of β-casein and BSA at a different molar ratio of (a) 1 : 10. (b) 1 : 100. (c) 1 : 500. (d) 1 : 1000 (e) 1 : 5000 and (f) 1 : 10000 (#: dephosphorylated residue of peptides (m/z: [M+H]+-80); all unmarked peaks are from non-phosphopeptides; β1: No. 1 phosphopeptides from β-casein; NL: Normalized Level).

Relative Quantification of Phosphopeptides in Serum Samples from Patients with Type 2 Diabetes. Serum is one of the easiest sample to obtain in clinical assays and it contains a library of potential biomarkers.56,57 The human serum phosphopeptides are an informative subclass of compounds in pathology and physiology, and are potentially reflective of the development of various diseases, including cancer, hypertension and Type 21



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2 diabetes.58-60 Endogenous phosphopeptides in serum can be markers of many diseases, and the difference in expression of these diseases are modulated by a cascade of endogenous proteases, with highly different activities between healthy and diseased states.61 For relative quantification of serum phosphopeptides from patients and healthy volunteers, an in situ isotope dimethyl labeling strategy was implemented after pretreatment with 2-D Ti-based MOF nanosheets. Stable isotope dimethyl labeling is a dependable and cost-effective method for quantitative proteomics,62 in which the ε-amino group of lysine and N-terminus of peptides go through reductive amination with deuterated formaldehyde (CD2O) or formaldehyde (CH2O) to generate a mass difference of +32 or +28 Da per amino group (Figure S10).63 Table 1. Sequence information of endogenous phosphopeptides enriched from human serum by Ti-based MOF nanosheets and labeled by CH2O and CD2O with the in situ isotope dimethyl labeling strategy. Peak

Peptide sequences

No.

No. of

Original

Labeled

Labeled

phosphorylation

(m/z)

by CH2O

by CD2O

(m/z)

(m/z)

HS1

DpSGEGDFLAEGGGV

1

1389.6

1417.5

1421.5

HS2

ADpSGEGDFLAEGGGV

1

1460.7

1488.5

1492.5

HS3

DpSGEGDFLAEGGGVR

1

1545.7

1573.6

1577.6

HS4

ADpSGEGDFLAEGGGVR

1

1616.8

1644.6

1648.6

pS : phosphorylated serine

Herein, we used an in situ labeling strategy based on 2-D Ti-based MOF nanosheets to characterize the levels of serum phosphopeptides. The serum samples were collected from 22


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3 healthy volunteers and 3 patients with Type 2 diabetes, enriched by 2-D Ti-based MOF nanosheets and labeled in situ with CD2O, while healthy volunteers serum was labeled with CH2O as a control. As depicted in Figure S10, 4 endogenous phosphopeptides which degraded from phosphorylated fibrinogen were captured from diabetes patients (heavy isotope-labeled) and healthy volunteers (light isotope-labeled), in accordance with the mass shift of 32 and 28 Da (Figure 5a) (The m/z value of detected phosphopeptides and amino acid sequences are listed in Table 1). To demonstrate the reliability of in situ labeling for quantitative results of serum phosphopeptides, the peak area of the isotope cluster from each phosphopeptides was recorded (Table S4, S5 and S6). As presented in the bar chart, 3 of 4 phosphopeptides HS1, HS3 and HS4 in the serum drawn from diabetes persons exhibited a significant down-regulation compared to the healthy volunteers. However, as calculated from the isotope cluster peak areas, the phosphopeptides HS2 underwent an upregulation from diabetes persons that compared to healthy volunteers (Figure 5b). The statistical test results P-Value were obtained from a T-Score calculator. As for HS1, HS2, HS3 and HS4, the P

Enrichment of Phosphorylated Peptides with Metal-Organic

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