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Ultra-deep palmitoylomics enabled by a dithiodipyridine functionalized magnetic nanoparticles Xiaoqin Zhang, Yuting Zhang, Caiyun Fang, Lei Zhang, Pengyuan Yang, Changchun Wang, and Haojie Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00534 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018
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Analytical Chemistry
Ultra-Deep Palmitoylomics Enabled by a Dithiodipyridine Functionalized Magnetic Nanoparticles Xiaoqin Zhang, ‡
‡, §
Yuting Zhang,
†, §
Caiyun Fang,
*, ‡
‡
‡
*, †
Lei Zhang, Pengyuan Yang, Changchun Wang
and Haojie Lu
*, ‡
Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China.
†
State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Laboratory of Advanced Materials, Fudan University, Shanghai 200433, China.
ABSTRACT: Palmitoylation, a type of fatty acylation, has vital roles in many biological processes. For ultra-deep identification of protein palmitoylation, an enrichment approach based on a novel magnetic microsphere modified with 2,2'-dithiodipyridine (Fe3O4/SiO2-SSPy microsphere) was presented in this study. The Fe3O4/SiO2-SSPy microspheres were synthesized by directly coating thiol-containing silane coupling agent onto the magnetic supraparticles in aqueous solution at room temperature. Due to the intrinsic magnetic properties, high surface-to-volume ratios and abundant reactive functional groups on surface, these microspheres enabled direct capture of palmitoylated targets and convenient isolation, contributing to remarkable enrichment selectivity (purifying palmitoylated peptides from mixtures with non-palmitoylated peptides even at a 1:500 molar ratio) and sensitivity (the detection limit was at fmol level), thus enables a global annotation of protein palmitoylation for complex biological samples. We had successfully identified 1304 putative palmitoylated proteins from mouse brain tissues by using this method, which is the largest mouse palmitoylome dataset to date. Except for those known members, many new proteins and pathways were also found to be regulated by palmitoylation.
Reversible and dynamic palmitoylation is one of the most important protein post-translational modifications (PTMs) involving covalent attachment of long-chain fatty acids (predominantly the 16-carbon palmitic acid) on specific cysteine residues via thioester linkages, which plays vital roles in a wide range of biological processes including cell signal transduction, apoptosis, carcinogenesis, etc1. Dysregulated palmitoylation can result in a number of neurological disorders, cancers and other diseases2. Therefore, it is of great significance to identify the palmitoylated proteins and their modified sites for further understanding their biological functions, while complete characterization of palmitoylated proteins faces substantial analytical challenges due to its low abundance and stoichiometry, absence of a well-defined consensus sequence, no commercial antibodies available, and severe signal suppression from abundant non-palmitoylated peptides in mass spectrometry (MS) analysis. Thus, the selective enrichment approach based on chemical strategy prior to MS analysis becomes a prerequisite for in-depth palmitoylome studies. To analyze palmitoylated proteins and their modification sites on a large scale, two kinds of MS-based proteomic strategies (“palmitate centric” and “cysteine centric” approaches) have been developed and widely used. In the “palmitate centric” approaches3,4, which are most suited for cell samples, are based on the metabolic incorporation of bioorthogonal lipid probes such as the commercially available 17-octadecynoic acid into proteins. With the use of Staudinger ligation or “click” chemistry, target proteins can be captured and isolated from protein mixtures via the alkyne or azide groups in lipid probes. While the “cysteine centric” approaches exploit the exposure of a reactive cysteine after neutral hydroxylamine (NH2OH) cleavage of the cysteine-acyl thioester bond. The candidates can be purified by using reaction between the newly exposed
cysteine thiol and cysteine-reactive groups [such as biotinHPDP used in the acyl-biotin exchange (ABE) method5,6 or thiopropyl sepharose resin used in resin-assisted capture method (Acyl-RAC)7]. We also analyzed the palmitoylated proteins in SW480 cells using Acyl-RAC method and developed the first pan antipalmitoylation antiserum8. Many groups have artfully employed these methods to carry out palmitoylomic analysis and greatly contributed to the understanding of protein palmitoylation. However, it remains a challenge to characterize palmitoylome in depth coverage. In the overwhelming majority of current methods, the putative palmitoylated proteins are pulled down using agarose/sepharose beads via biotin-streptavidin/avidin interaction, in which multi-step procedures (e.g. biotin derivatization and then avidin pull down) are involved, inevitably influencing both sensitivity and selectivity of these methods. Meanwhile, the properties of support materials, including support matrix type, pore size, particle size and amount of ligands, can greatly affect the performance of selectivity and specificity to the target proteins/peptides9. As we know, the agarose or sepharose beads are porous and not visible in tubes, easily leading to higher background and high probability of bead loss during handling. It was reported that nearly one-third of the identified proteins were designated as false positives in ABE approach due to the enrichment procedure and the inherent background observed with streptavidin bead enrichments3. Nanoparticles, especially functionalized magnetic nanoparticles (FMNs), have exhibited outstanding performance and been widely used in MS-based proteomic studies due to its easy surface modification, easy operation and high extraction efficiency10. Therefore, we here described a simple and robust alternative to ABE method based on a dithiodipyridine FMNs, in which the putative palmitoylated proteins were directly captured and isolated in lieu of biotinylation followed by avidin pull down via
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agarose/sepharose beads, contributing to an ultra-deep identification conveniently. A model palmitoylated peptide and mouse brain samples were used for proof-of-principle demonstrations. The experiment results demonstrated that the magnetic microspheres modified with 2,2'dithiodipyridine (Fe3O4/SiO2-SSPy microspheres) possessed remarkable selectivity for the model palmitoylated peptide (GDFCpalmIQVGR) even at a very low molar ratio of palmitoylated/non-palmitoylated peptides (1:500), extreme sensitivity (the detection limit was at the fmol level) and excellent speed. Totally, 1304 putative palmitoylated proteins from mouse brain, including many well known members such as SNAP25 and PSD-95, were successfully identified, in which many proteins or pathways were first found to be regulated by palmitoylation in our study, greatly enhancing data scale compared to previous studies. MATERIALS AND EXPERIMENTAL PROCEDURE Materials and Chemicals. Mercaptopropyltrimethoxysilane (MPTMS) and 2,2'-dithiodipyridine (DDPy) were purchased from Aladdin. The model palmitoylated peptide GDFCpalmIQVGR (Purity>95%) was synthesized by SynPeptide Co., Ltd., China, in which the Cys residue was modified by palmitoylation. Ovalbumin, ammonium bicarbonate, dithiothreitol (DTT), iodoacetamide (IAA), tris (2-carboxyethyl) phosphine (TCEP), N-Ethylmaleimide (NEM), and MALDI matrix (α-cyano-4-hydroxycinnamic acid, CHCA) were from Sigma-Aldrich (St. Louis, USA). Acetonitrile (ACN, 99.9%), formic acid (FA) and trifluoroacetic acid (TFA, 99.8%) were purchased from Merck (Darmstadt, Germany). Commercial thiopropyl sepharose 6B resins were from GE Healthcare, OPSS-PEGOPSS was from Seebio and Activated Thiol-Sepharose® 4B was from Sigma. Protease inhibitor cocktail (EDTA Free) was from Roche Diagnostics. Micro BCA protein assay kit was from Pierce. MS grade trypsin was from Promega. Deionized water (18.2 MΩ cm) used for all experiments was obtained from a Milli-Q system (Millipore, Bedford, MA).
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Other chemicals such as EDTA, NaOH and hydroxylamine hydrochloride were of analytical grade and from Shanghai Chemical Reagent Company, Ltd. The hydroxylamine solution (NH2OH) was freshly prepared in H2O from hydroxylamine hydrochloride and brought to pH 7.5 with concentrated NaOH in this study. GSK-3α (D80E6) Rabbit mAb, GSK-3β (27C10) Rabbit mAb were purchased from Cell Signaling Technology. Calnexin (Canx) Rabbit pAb, Flotillin 1 (Flot1) Rabbit pAb, Transitional endoplasmic reticulum ATPase (VCP) (EPR3307(2)) Rabbit mAb, Heterogeneous nuclear ribonucleoprotein L (HnRNPL) (4D11) Mouse mAb and RRAS2 Rabbit pAb were purchased from Abcam. Goat anti-rabbit/anti-mouse IgG antibodies conjugated with horseradish peroxidase secondary antibodies were from Thermo Scientific. Synthesis of Dithiodipyridine Functionalized Magnetic Microspheres. The synthesis route of Fe3O4/SiO2-SSPy microspheres is illustrated in Scheme 1a. Fe3O4 magnetic supraparticles (MSPs) were prepared by a modified solvothermal reaction11,12. The core/shell Fe3O4/SiO2-SH microspheres were synthesized by directly coating a layer of MPTMS onto the surface of MSPs via a new sol-gel reaction in aqueous solution at room temperature (RT). Specifically, 10 mg Fe3O4 supraparticles were dispersed in 10 mL water, and then 40 µL MPTMS was added and stirred for 5 min, after that 30 µL NH3·H2O was added and the reaction ended after 1 hr at RT. To obtain the optimal performance (e.g. appropriate shell thickness and abundant reactive groups), the dosage of MPTMS was investigated from 10, 20, 40 and 80 µL. The Fe3O4/SiO2-SSPy microspheres were then fabricated through disulfide exchange reaction with DDPy13. Typically, 10 mg Fe3O4/SiO2-SH microspheres were dispersed in 10 mL methanol, then after bubbling nitrogen for 0.5 hr, 2 mL DDPy methanol solution (5 mg/mL) was added and the reaction ended after 10 hr. The product was collected by magnetic separation, washed with methanol and deionized water for several times and stored for further use.
Scheme 1. Schematic illustration of the fabrication procedures of Fe3O4/SiO2-SSPy microspheres (a) and selective enrichment strategy for palmitoylated peptides or proteins based on the microspheres (b).
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Analytical Chemistry
Characterization of Fe3O4/SiO2-SSPy Microspheres. Highresolution transmission electron microscopy (TEM) images were taken on a JEM-2100F transmission electron microscope at an accelerating voltage of 200 kV. Samples dispersed at an appropriate concentration were cast onto a carbon-coated copper grid. Field-emission scanning electron microscopy (FE-SEM) was performed on a Hitachi S-4800 Scanning electron microscope at an accelerating voltage of 20 kV. Sample dispersed at an appropriate concentration was cast onto a glass sheet at RT and sputter-coated with gold. Hydrodynamic diameter (Dh) measurements were carried out by dynamic light scattering (DLS) with a ZEN3600 (Malvern, UK) Nano ZS instrument using He-Ne laser at a wavelength of 632.8 nm. Thermogravimetric analysis (TGA) measurements were performed on a Pyris 1 TGA instrument. All measurements were taken under a constant flow of air of 40 mL/min. The temperature was first increased from RT to 100 oC and held until constant weight, and then increased from 100 to 800 oC at a rate of 20 oC/min. Magnetic characterization was tested on a vibrating sample magnetometer (VSM) on a Model 6000 physical property measurement system (Quantum, USA) at 300 K. Fourier transform infrared spectra (FT-IR) were conducted on a Magna-550 (Nicolet, USA) spectrometer. Spectra were scanned over the range of 400-4000 cm-1. All of the dried samples were mixed with KBr and then compressed to form pellets. X-ray photoelectron spectrum (XPS) was conducted using an RBD upgraded PHI-5000C (Perkin-Elmer, USA) ESCA system with Mg Kα radiation (hν = 1253.6 eV) at 250 W and 14.0 kV with a detection angle at 54°. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) measurement was taken on a P-4010 instrument. The S contents were calculated by using inductively coupled plasmaatomic emission spectrometry to test the amount of Si. Preparation of Tryptic Digests of Model Protein. To obtain standard model tryptic peptides, ovalbumin was dissolved in 50 mM Tris-HCl (pH 7.5) and denatured by incubating at 100 °C for 10 min. The mixture was treated with 10 mM DTT at 56 °C for 30 min and alkylated with 30 mM IAA at RT for 30 min in the dark. The digestion procedure was proceeded at 37 °C overnight at a trypsin-to-substrate ratio of 1:50 (w/w). After being purified by C18 column, the eluent was concentrated by vacuum evaporation and stored at -80 °C until further processing. Selective Enrichment of Palmitoylated Peptides. The procedure of palmitoyl-peptide enrichment is illustrated in Scheme 1b. Typically, peptide mixtures were firstly reduced to open disulfide bonds and alkylated to block free cysteine residues. The samples dissolved in 100 µL of loading buffer (50 mM Tris-HCl, pH 7.5) were treated with neutral NH2OH solution (pH 7.5) that the final concentration was 1 M to selectively cleave thioester linkage and produce new free thiol group at the palmitoylated site, followed by being incubated with the Fe3O4/SiO2-SSPy microspheres. Afterward, the microspheres with captured fresh thiol-containing peptides were separated from the solution using an external magnetic field, and washed for several times to remove non-specifically adsorbed peptides and other impurities. The targets were
released from the microspheres with 60 mM freshly prepared DTT for 30 min at RT, purified by ZipTip C18 Pepette tips and eluted with 5 µL 50% ACN containing 0.1% TFA. Preparation and Purification of Palmitoylated Proteins from Mouse Brain Tissues. The fresh adult mouse brains were excised and homogenized into homogenization buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, pH 7.5) containing 1 × protease inhibitor cocktail (EDTA Free) by using a glass Teflon Homogenizer. The resulting homogenate was centrifuged at 450 × g for 10 min. Then the supernatants were centrifuged at 200,000 × g for 30 min, giving rise to a crude membrane pellet. Homogenization and subsequent fractionations were all performed at 0-4 °C. The pellets were dissolved with 4SB buffer (4% SDS, 50 mM Tris, 5 mM EDTA, pH 7.5). Following dilution with 3 volumes of homogenization buffer, samples were reduced with a final concentration of 10 mM fresh TCEP for 30 min and alkylated with a final concentration of 50 mM fresh NEM for 2.5 hr in the dark at RT with end-over-end rotation. Excess NEM was removed by chloroform/methanol precipitation. To block free thiols present in proteins completely, the entire procedure was repeated twice more. Finally, two sequential chloroform/methanol precipitations were performed to remove excess NEM followed by being redissolved in 4SB buffer. Protein concentration was determined using the Micro BCA protein assay. Samples were divided equally into two portions. The functionalized nanoparticles were washed five times with LB buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, pH 7.5). The equal amount of nanoparticles were added to each protein portion together with 1 M NH2OH (EXP) or 50 mM Tris-HCl (CON). After samples were incubated at RT for 4 hr with end-over-end rotation, the nanoparticles were collected by an external magnetic field, and washed with LB buffer for 5 times. Bound proteins were eluted by incubating nanoparticles with 60 mM fresh DTT for 30 min at RT with end-over-end rotation. The elution step was repeated one more time and both eluted samples were combined. The eluted proteins were resolved by 12% polyacrylamide gel electrophoresis (SDS-PAGE), and stained with silver staining. Each lane was cut into six gel slices, destained with a buffer including 15 mM ferricyanide potassium and 50 mM sodium hyposulfite, reduced with 10 mM DTT in 50 mM ammonium bicarbonate for 45 min, and alkylated with 55 mM iodoacetamide in 50 mM ammonium bicarbonate for 45 min in the dark. Afterwards, proteins were in-gel digested by 12.5 ng/µL MS grade trypsin in 25 mM ammonium bicarbonate at 37 °C overnight. Tryptic peptides were extracted with 50% ACN in 0.1% TFA. Mass Spectrometric Analysis. All MALDI-TOF MS experiments were carried out on a 5800 Proteomics analyzer (Applied Biosystems) in the positive ion mode with a pulsed Nd/YAG (Neodymium-doped yttrium aluminum garnet) laser at 355 nm. The matrix was 10 mg/mL CHCA dissolved in 50% ACN (v/v) containing 0.1% TFA. A 1 µL aliquot of the eluate and 1 µL of the CHCA matrix were sequentially dropped onto the MALDI plate for MS analysis. The acquired mass spectra were interpreted manually using Data Explorer V4.5 (Applied Biosystems).
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The LC-MS analysis was performed on Triple TOF 4600 mass spectrometer (AB Sciex, CA) in information-dependent data acquisition mode to switch automatically between MS and MS/MS acquisition. Electrospray voltage of 2.5 kV versus the inlet of the mass spectrometer was used. MS spectra were acquired across the mass range of 350-1250 m/z using 250 ms accumulation time per spectrum. Tandem mass spectral scanned from 100-1250 m/z in high sensitivity mode with rolling collision energy. The 25 most intense precursors were selected for fragmentation per cycle with dynamic exclusion time of 25 s. For LC-MS/MS analysis, the samples were dissolved in buffer A (5% ACN containing 0.1% FA) and separated on a reverse phase analytical column (Eksigent, C18, 3 µm, 150 mm × 75 µm) by nano-HPLC (Eksigent Technologies). Peptides were eluted using the following gradient conditions with phase B (98% ACN with 0.1% FA) from 5 to 45% B in 60 min and total flow rate was maintained at 300 nL/min. The sample was analyzed three times, each with 5 µL. All of the MS/MS spectra in the *.wiff files were converted to single *.mgf files using AB_SCIEX_MS_Data_Converter Tools (Version beta 1.3, https://sciex.com/softwaresupport/software-downloads). The *.mgf files were searched using Mascot Daemon software (Version 2.3.2, Matrix Science, UK) against a composite database, including original and reversed protein database assuming the digestion enzyme trypsin. The searching database contained 76089 mouse protein entries extracted from UniProtKB/Swiss-Prot database. A maximum of 2 missed cleavages was allowed. Mass value was set as monoisotopic, and peptide charges 2+ and 3+ were taken into account. All samples were searched with oxidation (M), acetylation (protein N-term), carbamidomethyl (C) and nethylmaleimide modification (C) as variable modifications. Parent mass tolerance was set to 25 ppm and fragment tolerance to 0.1 Da. The results were further filtered by Scaffold (Proteome Software, V4.2.0). Probabilities of Mascot identifications were assigned by the Scaffold Local false discovery rate algorithm. Protein identifications with 1.0% peptide FDR, 2.0% protein FDR and ≥ 2 unique peptides were accepted. Spectral Count-Based Identification of Palmitoyl-Protein Candidates. The relative protein abundance changes between the paired EXP and CON samples were determined using a label free spectral counting approach6,14,15 with some modifications. The spectral counts for each protein were merged over all biological/technical replicates and normalized by Scaffold. Subsequently, statistical analysis was performed on the merged data sets. All identified proteins were plotted on x,y-scatter plots by merged, normalized + NH2OH sample spectral counts (x-coordinate) versus merged, normalized NH2OH sample spectral counts (y-coordinate). The new candidate palmitoyl-proteins were defined as those proteins that showed NH2OH-dependent purification, co-clustering along the x-axis. All zeros for spectral counts were replaced with 0.5 and the EXP/CON ratios were calculated. Based upon this graphical analysis, candidates were sub-divided into three subsets based upon strength of the spectral count-based support for palmitoylation - Subset I with strong support
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(EXP/CON >8), Subset II with medium support (5< EXP/CON
