Identification of Palmitoylated Transitional Endoplasmic Reticulum

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Identification of palmitoylated transitional endoplasmic reticulum ATPase by proteomic technique and pan anti-palmitoylation antibody Caiyun Fang, Xiaoqin Zhang, Lei Zhang, Xing Gao, Pengyuan Yang, and Haojie Lu J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00979 • Publication Date (Web): 11 Feb 2016 Downloaded from http://pubs.acs.org on February 12, 2016

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Journal of Proteome Research

Article Identification of palmitoylated transitional endoplasmic reticulum ATPase by proteomic technique and pan anti-palmitoylation antibody

Caiyun Fang#, Xiaoqin Zhang#, Lei Zhang, Xing Gao, Pengyuan Yang, Haojie Lu*

Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, China # These authors contributed equally to this work. * Corresponding author: Lu Haojie, Department of Chemistry and Institutes of Biomedical Sciences, Fudan University, Shanghai 200433, P. R. China; Phone: +86-21-54237618; Fax: +86-21-54237618; Email: [email protected].

1

ACS Paragon Plus Environment


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ABSTRACT: Protein palmitoylation plays a significant role in a wide range of biological processes such as cell signal transduction, metabolism, apoptosis and carcinogenesis. For high throughput analysis of protein palmitoylation, approaches based on the acyl-biotin exchange or metabolic labelling of azide / alkynylpalmitate analogs were commonly used. No palmitoylation antibody has been reported. Here, the palmitoylated proteome of human colon cancer cell lines SW480 was analyzed via TS-6B-based method. Totally, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Except for 3 known palmitoylated transmembrane proteins ATP1A1, ZDHHC5 and PLP2, some important proteins including kinases, ion channels, receptors, and cytoskeletal proteins were also identified, such as CLIC1, PGK1, PPIA, FKBP4 and Exportin-2, etc. More importantly, the pan anti-palmitoylation antibody was developed and verified for the first time. Our homemade pan anti-palmitoylation antiserum could differentiate well protein palmitoylation from mouse brain membrane fraction and SW480 cells, which affords a new technique to analyze protein palmitoylation by detecting the palmitic acid moiety directly. Furthermore, the candidate protein transitional endoplasmic reticulum ATPase (VCP) identified in SW480 cells was validated to be palmitoylated by

western

blotting

with

anti-VCP

antibody

and

the

homemade

pan

anti-palmitoylation antibody. KEYWORDS:

transitional

endoplasmic

reticulum

proteomic analysis, pan anti-palmitoylation antibody

2


ATPase,

palmitoylation,

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INTRODUCTION

Protein post-translational modifications (PTMs) can modulate the activity of most proteins and control many biological processes by covalently attaching such functional groups as phosphate, acetate, methyl groups, and so on.1 Quantitative and qualitative analysis of protein PTMs will contribute to better understanding protein functions and the mechanisms of cell regulation.2,3 Protein palmitoylation, also known as S-acylation, is a major PTM involving the covalent addition of long-chain fatty acids (predominantly the 16-carbon palmitic acid) to specific cysteine residues through thioester linkages.4 Unlike other lipid modification such as myristoylation and prenylation, palmitoylation is reversible and dynamic. Palmitoylation can modulate activities and stability of proteins, protein-protein interactions and protein’s membrane association, so it plays a significant role in a wide range of biological processes such as cell signal transduction, apoptosis, and carcinogenesis.5 To further understand the molecular mechanisms and dynamics of protein palmitoylation, it is of great significance to profile the palmitoylated proteome qualitatively and quantitatively. Conventionally, the candidate cysteine (Cys) residue is mutated to Ser or Ala, and site-directed mutagenesis approach is used. Another well-established method involves metabolic labelling with radioactive 3H- or 125

I-palmitate, followed by autoradiography to visualize the degree of isotopic

incorporation. These methods contribute to our understanding greatly, but they are tedious, even hazardous and can’t realize high throughput analysis of 3



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palmitoylated proteins/sites. Now two types of approaches have been widely used for large-scale profiling of protein palmitoylation together with mass spectrometry (MS). In the “palmitate-centric” approaches, azide- or alkynylpalmitate analogs are incorporated metabolically into cellular proteins, and then those proteins with palmitate analogs are selectively dectected by using modified biotin or fluorophore via a Staudinger ligation6 or a click reaction7. Kostiuk et al.8 obtained 21 palmitoylated proteins from rat liver mitochondria. Zhang et al.9 monitored simultaneously palmitate cycling rates and protein turnover by using a tandem labelling and fluorescence imaging method. Yount et al.10 detected selectively 157 palmitoylated proteins in mouse dendritic cell line. Martin et al.11 identified 125 high-confidence and about 200 medium-confidence palmitoylated proteins from Jurkat T cells by using commercially available palmitic acid analog 17-octadecynoic acid. They also analyzed quantitatively more than 400 palmitoylated proteins in mouse T-cell hybridoma cells, and distinguished stably palmitoylated proteins from those that turned over rapidly.12 For the “Cysteine-centric” approaches (commonly known as acyl-biotin exchange, ABE), after all free thiols were modified by N-ethylmaleimide (NEM) or iodoacetamide (IAA), thioester bond between palmitic acid and protein was selectively cleaved by hydroxylamine (NH2OH) to obtain new free sulfhydryl groups. And then the target proteins were purified via avidin/streptavidin-biotin interactions. Wan et al.13 identified 50 palmitoyl proteins in yeast including many SNARE proteins, amino acid permeases and 4


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signaling proteins. Kang et al.14 identified 68 known and >200 candidate S-acylated proteins from whole rat brain, purified rat synaptosomes and cultured embryonic rat neurons. Yang et al.15 identified 67 known and 331 novel candidate S-acylated proteins as well as the localization of 25 known and 143 novel candidate palmitoylation sites. Zhang et al.16 screened substrates of human DHHC2 in HeLa cells and a total of 57 sites were identified from 50 proteins. To simplify the procedure, Forrester et al.17 used thiopropyl sepharose as support to capture nascent thiols after the thioester linkages in proteins were cleaved by NH2OH. In total, 93 putative sites on 88 peptides were identified in HEK293 cells. It was rapid and the entire procedure can be completed in several hours. To our knowledge, no studies have been reported using pan anti-palmitoylation antibody to detect palmitoylated proteome. Herein, we analyzed the palmitoylated proteome of SW480 cells using thiopropyl sepharose based enrichment method. Totally, 151 putative palmitoylated sites on 92 proteins, including 100 novel sites, were identified. Furthermore, pan anti-palmitoylation antibody was first developed and verified. At the same time, the candidate protein, transitional endoplasmic reticulum ATPase (VCP), was confirmed to be palmitoylated using western blotting analysis with anti-VCP antibody and homemade pan anti-palmitoylation antibody. EXPERIMENTAL SECTION Materials and Reagents NH4HCO3, TPCK treated trypsin (E.G 2.4.21.4), bovine serum albumin (BSA), 5



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dithiothreitol (DTT), IAA, urea, tris(2-carboxyethyl)phosphine (TCEP) and NEM were from Sigma-Aldrich (St. Louis, USA). Acetonitrile (ACN, 99.9%) and trifluoroacetic acid (TFA, 99.8%) were purchased from Merck (Darmstadt, Germany). Sep-Pak C18 columns were from Waters (Milford, MA). Dried thiopropyl Sepharose 6B (TS-6B) was from GE Healthcare (Pittsburgh, PA). Deionized water used in experiments was from a Milli-Q system (Millipore). Other chemical reagents such as HEPES, EDTA and hydroxylamine hydrochloride were of analytical grade and from Shanghai Chemical Reagent Company, Ltd.. Cell Culture and Sample Preparation from SW480 Cells The human colon cancer cell lines SW480 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (Gibco, invitrogen) 105 U/L penicillin, 100 mg/L streptomycin, 2mM glutamine, 25 mM HEPES solution, 1 mM sodium pyruvate, and 250 mg/L G418 (Geneticin; Gibco, Ivitrogen) at 37 °C under 5% CO2. SW480 cells were harvested and rinsed three times in cold PBS. The cell pellets were lysed in 0.05% SDS Buffer (0.05% SDS, 50 mM Tris, 5 mM EDTA, 6 M urea, pH7.5), as well as 1 × protease inhibitor cocktail (EDTA Free, Roche Diagnostics) on ice. The lysates were centrifuged at 16 000 × g for 30 min at 4 °C to collect proteins. Samples were reduced with 20 mM TCEP for 30 min and alkylated with 50 mM NEM for 2.5 hrs at RT with end-over-end rotation. To block free thiols present in proteins completely, the reduction and alkylation procedure was carried out twice. Excess NEM was removed with two sequential acetone precipitations. Protein pellets were re-dissolved with 0.05% SDS Buffer. Protein concentration was determined by Bradford assay. Prior to 6


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digestion, the solution was diluted with 50 mM Tris buffer (pH 7.5) until the final concentration of urea was less than 1.5 M. Trypsin was added according to the enzyme-to-substrate ratio of 1:50 (w/w) and hydrolyzed for 16 hrs at 37 °C, under gentle shaking. Preparation of Mouse Brain Crude Membrane Fraction The fresh adult mouse brains were excised and homogenized in homogenization buffer (150 mM NaCl, 50 mM Tris, 5 mM EDTA, pH 7.5) containing 1 × protease inhibitor cocktail (EDTA Free, Roche Diagnostics) 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 resuspended in homogenization buffer, quantified by BCA assay and stored at -80 °C prior to use. Generation of Pan Anti-palmitoylation Antibody To generate pan anti-palmitoylation antibody, a short peptide only consisting of two cysteines (C-C(pal)) (98.5%), in which one cysteine was palmitoylated by using the method previously reported,18 was synthesized and used as the hapten. Then the synthesized peptide C-C(pal) was coupled to Keyhole Limpet Hemocyanin (KLH) as antigen to immunize rabbits. Antiserum was collected after three doses of immunization. Prior to use, the antiserum was purified by using ammonium sulfate precipitation method. SDS-PAGE and Western blotting Analysis 7



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Protein samples were separated on 12% polyacrylamide gel electrophoresis in a Laemmli buffer system,19 transferred to a polyvinylidene fluoride (PVDF) membranes (Immobilon; Millipore) and blocked by 5% skim milk in tris-buffered saline with Tween 20 (TBST) buffer for 1 hr at room temperature (RT). The PVDF membranes were incubated with the following primary antibodies: rabbit monoclonal anti-transitional endoplasmic reticulum ATPase (VCP) (1:10000 dilution, Abcam), anti-β-actin (1:5000, Cell Signaling Technology) or pan anti-palmitoylation antiserum (1:20, homemade). Then the PVDF were washed with TBST three times and primary antibodies were detected with goat anti-rabbit IgG antibodies conjugated with horseradish peroxidase (1:8000, Thermo Scientific). The target proteins were detected using ECL system (Thermo Scientific) and the image was scanned by ImageQuant ECL Imager (GE Healthcare Life Sciences). For dot-blotting analysis, the samples were directly spotted on the PVDF membrane, incubated with antibodies and detected by ImageQuant ECL Imager. On PVDF Membrane Protein Digestion The PVDF membrane was stained with Ponceau S dye after electroblotting, the interested bands were excised and destained. After the PVDF bands were washed at least 6 times with Milli-Q water, protein digestion procedure was carried out similarly as previously described,20 including reduction, alkylation, protein digestion and peptide extraction. Selective Enrichment of Palmitoylated Peptides by Thiol-Reactive Resin The tryptic digests from SW480 cells were dissolved in incubation buffer and divided 8


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equally into two portions. TS-6B resin together with 2 M NH2OH or 2 M NaCl were added to each portion (+ NH2OH or - NH2OH), respectively. Samples were incubated at RT for 2 hrs with end-over-end rotation. Resins were washed as mentioned above, and then peptides were eluted, alkylated with IAA, desalted by Sep-Pak C18 columns and lyophilized for further LC-MS/MS. Mass Spectrometry (MS) Analysis The nano-LC MS/MS analysis was performed on a nano Acquity UPLC system (Waters, USA) connected to a LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Germany) equipped with an online nano-electrospray ion source (Michrom Bioresources, USA). The peptides were re-suspended with buffer A (5% ACN containing 0.1% FA) and injected into a CAPTRAP column (0.5 × 2 mm, MICHROM Bioresources, CA) in 5 min with a flow rate of 20 µL/min. Subsequently, samples were separated on a Magic C18AQ reverse phase column (100 µm id × 15 cm, Michrom Bioresources, USA) with a linear gradient from 5 to 45% buffer B (90% ACN in 1% FA) in 70 min at a flow rate of 500 nL/min. The separated samples were introduced into the mass spectrometer via an ADVANCE 30 µm silica tip (MICHROM Bioresources, CA). The spray voltage was 1.4 kV, and capillary temperature was 180 °C. The mass spectrometer was operated in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Survey full-scan MS spectra with one microscan (400-1800 Da) was acquired in the Orbitrap with a mass resolution of 100,000 at m/z 400, followed by MS/MS of the 8 most-intense peptide ions in the LTQ analyzer. The automatic gain control (AGC) was set to 1000 9



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000 ions, with maximum accumulation time of 500 ms. For MS/MS, an isolation window of 2 m/z was used and the AGC of LTQ was set to 20 000 ions, with maximum accumulation time of 120 ms. Single charge state was rejected and dynamic exclusion was used with two microscans in 10 s and 90 s exclusion duration. For MS/MS, precursor ions were activated using 35% normalized collision energy at the default activation q of 0.25 and an activation time of 30 ms. The system control and data collection were achieved via Xcalibur (version 2.0.7) software. MS Data Processing and Database Searching Database search was performed using Mascot v2.3.2 (Matrix Science, UK) as previously described with minor modifications.21 Briefly, peak lists were generated from raw files with ProteoWizard v3.0 with default settings.22 Mascot Daemon (Version 2.3.2) was used for database searching against a composite database, including original and reversed protein database assuming the digestion enzyme trypsin. A maximum of 2 missed cleavages was allowed. Mass value was set as monoisotopic, and peptide charges 2+, 3+ and 4+ were taken into account. Precursor ion mass tolerance was set to 20 ppm and product ion tolerance to 1.0 Da. The Searching database contained 20,272 human protein entries extracted from UniProtKB/Swiss-Prot database with an in-house perl script. For the protein identification from PVDF membrane, oxidation (M), acetylation (protein N-term) and carbamidomethyl (C) were set as variable modifications. For analysis of palmitoylation proteome, all samples were searched with oxidation (M), acetylation (protein N-term), carbamidomethyl (C) and nethylmaleimide modification (C) as 10


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variable modifications. In addition, the results were further filtered by Scaffold (Proteome Software, V4.2.0). An additional database search by X! Tandem was performed with parameters similar to Mascot except that Glu->pyro-Glu, ammonia-loss and Gln->pyro-Glu of the N-terminus were specified additionally. Peptide identifications by X! Tandem were accepted with >95.0% probability assigned by the PeptideProphet algorithm.23 Probabilities of Mascot identifications were assigned by the Scaffold Local false discovery rate algorithm. Protein identifications with >99.0% probability by the ProteinProphet algorithm24 and ≥ 1 unique peptides were accepted.25 The abundance of each peptide was represented by the normalized Total Ion Current (TIC). The normalization was performed by multiplying TIC across samples so that the total number was the same across all samples and categories. The average TIC of the same peptide was added in the + NH2OH and - NH2OH group, respectively. To calculate the fold changes, the average TIC in the + NH2OH dataset was divided by that of the same peptide in - NH2OH dataset. Peptides with consistent changes by > 2 were accepted as significant differential peptides, that is to say, the differential peptides were assigned as putative palmitoylated peptides, and otherwise, the peptide was defined as contaminant peptide. Bioinformatic Analysis IPA software (http://www.ingenuity.com) was used to complete subcellular localization, molecular function and network analysis. TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) was used to predict transmembrane 11



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helices (TMHs) of proteins. RESULTS AND DISCUSSION Validation of Pan Anti-palmitoylation Antibody To evaluate sensitivity and specificity of the homemade pan anti-palmitoylation antiserum, the synthesized peptide C-C(pal) was coupled to BSA (pal-CC-BSA) as the model protein firstly. In the short peptide C-C(pal), only one cysteine residue was palmitoylated. And the peptide C-C was also attached to BSA (CC-BSA) as a control, in which C was not modified. CC-BSA and different amounts of pal-CC-BSA were loaded onto PVDF membrane, incubated with pan anti-palmitoylation antiserum and analyzed by dot-blotting. As shown in Figure 1, 10 µg of CC-BSA exhibited no signal, while the same amount of pal-CC-BSA showed significant signal due to the attachment of palmitoyl groups. With increasing of pal-CC-BSA amount (from 0 to 10 µg), the signal intensities were enhanced gradually. This results indicated that the homemade antiserum were directed against palmitoyl moieties and the immune signals enhanced with the increasing of amounts of palmitoyl groups. We further investigated the efficiency of homemade pan anti-palmitoylation antibody by using two complex samples. Firstly, the mouse brain crude membrane fraction was used because protein palmitoylation was widely distributed in nervous system.5 5 mg of crude membrane fraction proteins from mouse brains were lysated with 2% SDS Buffer (2% SDS, 50 mM Tris, 5 mM EDTA, pH 7.5), reduced with TCEP and alkylated with NEM. The Methanol/Chloroform protein precipitation was performed to remove the excess of TCEP and NEM. Then the protein samples were 12


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divided equally into two portions, treated with NH2OH (pH 7.5) or Tris (pH 7.5) buffer, respectively. After being incubated at room temperature for 2-3 hrs, 100 µg of each samples were separated by 12% SDS-PAGE, transferred onto PVDF membranes and incubated with the homemade purified antiserum (1:20 dilution). The palmitate moiety could be cleaved from proteins in NH2OH buffer (pH 7.5), while it was stable in Tris buffer. As expected, when the sample loadings were approximately equal, the sample without NH2OH treatment (M-B-M-NH2OH) exhibited a significant signal after being incubated against the homemade antiserum, while the sample treated with NH2OH (M-B-M+NH2OH) appeared almost no signal (as shown in Figure 2(A)). Furthermore, 100 µg of whole SW480 cell lysate (without NH2OH treatment) was separated and western blotting analysis with the homemade antiserum. As shown in Figure 2(E), there were significant immunosignals in the right lane. To further verify the results, the band indicated with an arrowhead was cut, digested and analyzed by LC-MS. In this band, several palmitoylated proteins indicated in the Uniprot database, including Apolipoprotein B-100(APOB)26, Excitatory amino acid transporter 2 (SLC1A2), Tyrosine-protein kinase Fyn (FYN) and Ras-related protein R-Ras2 (RRAS2), were identified unambiguously. Thus it can be seen that the homemade antiserum could well differentiate palmitoylated proteins from complex biological samples. Analysis of Palmitoylated Proteome from SW480 Cell Lysate The tryptic digests of SW480 cells were divided equally into two portions, and subjected to the enrichment procedure in presence and absence of NH2OH, 13



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respectively. The resulting eluates from both conditions were analyzed by LC-MS/MS. In order to reduce false negative results caused by free thiols present in proteins and additional hydroxylamine-insensitive modifications, the reduction and alkylation procedure by TCEP and NEM was carried out twice, label free method was used to quantity the identified peptides. Peptides with consistent changes (+ NH2OH /NH2OH) by > 2 were designated to be palmitoylated. Totally 151 putative sites on 92 proteins were identified, including a number of sites previously known to undergo palmitoylation (Supplemental Table), among which 51 candidate sites15-17 and 81 proteins8,11,12,14-17,27-29 had been reported in the peer-reviewed literatures. Palmitoylation can take place not only in soluble proteins but also in integral membrane proteins, and many important proteins have been identified as its targets including ion channels, receptors, cytoskeletal proteins and kinases.30,31 Here, we used IPA software to analyze protein subcellular localization, function and pathway (shown in Supplemental Figure 1). Three transmembrane proteins were identified to be palmitoylated including Sodium/potassium-transporting ATPase subunit alpha-1 (ATP1A1), Palmitoyltransferase ZDHHC5 (ZDHHC5) and Proteolipid protein 2 (PLP2). ATP1A1, which contains 10 TMHs, is the catalytic component of the active enzyme and catalyzes hydrolysis of ATP coupled with the exchange of sodium and potassium

ions

across

plasma

membrane.

It

was

also

reported

to

be

palmitoylated.11,12,14,15,17,27,32 ZDHHC5, which is an integral membrane protein with 4 TMHs, was identified to be palmitoylated on three Cys residues, which were reported in ref.11,14,15. Proteolipid protein (PLP) is a highly conserved hydrophobic protein 14


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which contains four transmembrane segments, and contains covalently bound fatty acids via labile thioester linkages, predominantly palmitic acid.33,34 In our study, PLP2 was also palmitoylated as reported.15,32 Interestingly, 23 nuclear proteins were identified to be palmitoylated including 12 ribosomal proteins, suggesting that these proteins may be targeted to membrane fractions by palmitoylation, which was in accordance with those reported previously.15 In parallel, some important proteins including kinases, ion channels, and receptors were also identified to be palmitoylated in

our

experiment,

such

as

Chloride

intracellular

channel

1

(CLIC1),

Phosphoglycerate kinase 1 (PGK1), nucleoside diphosphate kinase B (NME2), Exportin-2, and so on. Transitional endoplasmic reticulum ATPase (also called Valosin-containing protein, VCP or p97) belongs to family of AAA (ATPases associated with diverse cellular activities)-type ATPases and plays various important roles in cells such as ubiquitin-dependent processes.35,36 It was reported that VCP was highly modified via acetylation,37 phosphorylation38,39 and lysine methylation40 to modulate its biological functions. In addition, VCP was also reported to be a candidate palmitoylated protein using proteomic technique.15,27,29 In our study, VCP was also identified to be palmitoylated, and the representative mass spectrum of potential modified peptide LGDVISIQPcPDVK was shown in Figure 3. It should be mentioned that many abundant proteins including metabolic, structural and ribosomal proteins were mainly identified in our dataset, because total SW480 cell lysate, but not the purified membrane fraction (as commonly used in previous 15



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reports), was directly used to enrich the palmitoylated proteins. However, many known palmitoylated proteins such as signaling and scaffolding proteins are lower abundant, or sub-stoichiometric palmitoylation takes place in these proteins in vivo, which makes these proteins very difficult to be identified without prefractionation. Verification of Transitional Endoplasmic Reticulum ATPase as a Palmitoylated Protein Although VCP has been reported to be palmitoylated in previous proteomic analysis,15,27,29 but the proteomic data about VCP palmitoylation usually did not be verified by another technique, except that Wilson et al.27 conducted an immunoblot analysis against VCP antibody to confirm mass spectrometric identification. To confirm that VCP is a palmitoylated protein, two different immunoblotting analyses were performed here. In the first strategy, the putative palmitoylated proteins from SW480 cells were first purified via TS-6B resin and subjected to western blotting against anti-VCP antibody. As shown in Figure 4, VCP in NH2OH-treated samples could be purified by TS-6B resin due to the cleavage of thioester bonds between proteins and palmitoyl moieties, while VCP in the control samples could not be enriched by TS-6B resin. This result showed that VCP was susceptible to the treatment of NH2OH and likely a palmitoylated protein. In the second strategy, VCP was firstly immunoprecipitated from SW480 cell lysate (named as IP fraction) by using monoclonal anti-VCP antibody. After the total lysate and IP fraction was separated by SDS-PAGE and transferred onto a PVDF membrane, the PVDF was sequentially incubated with the homemade purified pan anti16


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palmitoylation antiserum (as shown in Figure 5(B)) and anti-VCP antibody (as shown in Figure 5(A)). Compared with those of IP fractions, the amount of VCP in total lysate was actually very low and thus exhibited very poor signal. After being incubated with pan anti-palmitoylation antiserum, IP-1 and IP-2 had apparent immunosignals corresponding to the protein band of VCP. Interestingly, there was another band with very strong immunosignals. So the proteins in Band 1 and Band 2 were further analyzed by LC-MS. As expected, VCP was identified unambiguously in Band 2. In addition, we identified myelin proteolipid protein (PLP1) from Band 1, which was a known highly palmitoylated protein reported in Uniprot database (Cys-6, 7, 10, 109, 139 and 141 were palmitoylated) and ref41. According to the IPA analysis (shown in Supplemental Figure 2), PLP1 could interact with VCP by indirect protein interaction and was thus pulled down. To further validate the palmitoylation of VCP, the immune precipitates formed with anti-VCP antibody was treated with NH2OH, analyzed by western blotting analysis against homemade antiserum. Compared with samples without NH2OH treatment, no immunosignal could be detected after NH2OH treatment (shown in Figure 5(C)), which indicated that the modification attached to VCP was in a hydroxylamine-dependent manner. Furthermore, the palmitoylation of VCP could be regulated by 2-bromopalmitate (2BP, a widely used palmitoylation inhibitor) as shown in Figure 5(D), in which the sample treated with 2-BP exhibited weaker immune signals than that treated with 0.1% DMSO. CONCLUSIONS Protein palmitoylation plays a significant role in a wide range of biological 17



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processes

such

as

cell

signal

transduction,

Page 18 of 24

metabolism,

apoptosis,

and carcinogenesis. Here, the palmitoylated proteome of SW480 cells were analyzed via TS-6B-based enrichment method. A total of 81 known and 11 novel candidate palmitoylated proteins as well as 51 known and 100 novel candidate palmitoylation sites in SW480 cells. Except for 3 known palmitoylated transmembrane proteins ATP1A1, ZDHHC5 and PLP2, many important proteins including kinases, ion channels, and receptors were also identified as palmitoylation, such as CLIC1, PGK1, PPIA, FKBP4, and so on. In particular, we firstly developed and confirmed effectiveness of the pan anti-palmitoylation antiserum. Furthermore, the palmitoylation state of VCP was confirmed by using two different western blotting strategies based on anti-VCP antibody and pan anti-palmitoylation antibody. It is the first time to verify protein palmitoylation using a pan anti-palmitoylation antibody by detecting the palmitic acid moiety directly. ASSOCIATED CONTENT Supporting Information Figure 1 demonstrated the analysis of protein subcellular localization and molecular function for identified candidates; Figure 2 exhibited protein interactions between VCP and PLP1 by IPA analysis; Table 1 listed the identified candidate palmitoylated proteins and peptides. These materials are available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION 18


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Corresponding author * Lu Haojie: tel, +86-21-54237618; Fax, +86-21-54237618; e-mail, [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The work was supported by National Science and Technology Key Project of China (2012CB910103), the National Science Foundation of China (21205018 and 21335002), Shanghai Pujiang Program (No. 13PJD003), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry.

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Figures Fig 1. Evaluation of homemade pan anti-palmitoylation antiserum. (A) 10 µg of pal-CC-BSA and CC-BSA were directly loaded onto PVDF membrane and dot-blotting analysis was carried out against pan anti-palmitoylation antibody. pal-CC-BSA and CC-BSA denoted BSA coupled with the synthesized peptide C-C(pal) and C-C, respectively. (B) 2 µL of pal-CC-BSA samples with different concentrations (5, 2.5, 1.25, 0.625, 0.313 and 0 µg/µL, respectively) were loaded onto PVDF

membrane

and

analyzed

by

dot-blotting

technique

against

pan

anti-palmitoylation antiserum.

Fig 2. Western blotting of mouse brain crude membrane fraction and the SW480 cell lysate against homemade anti-palmitoylation antiserum. M-B-M-NH2OH and M-B-M+NH2OH denoted samples without and with NH2OH treatment, respectively. The PVDF was firstly stained with Ponceau S dye (C), and incubated against antiserum (A). β-actin was used as an internal standard (D). After being transferred, the gel was stained with Coomassie Brilliant Blue (B). Marker denoted molecular mass markers. The SW480 cell lysate were analyzed by western blotting against homemade antiserum (E), in which the band indicated with an arrowhead was cut and subjected to on PVDF membrane digestion and LC-MS analysis.

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Fig 3. Representative tandem mass spectrum of potential palmitoylated peptide LGDVISIQPcPDVK of VCP protein.

Fig 4. Western blotting of VCP against anti-VCP antibody. + NH2OH and - NH2OH denoted that the samples was treated with NH2OH or not, respectively. Total lysate denoted the whole proteins extracted from SW480 were analyzed directly as the loading reference. As shown in the right panel, the proteins were first purified via TS-6B resin and subjected to western blotting.

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Fig 5. Western blotting of VCP against anti-VCP antibody and pan anti-palmitoylation antiserum. Total lysate denoted whole proteins extracted from SW480. IP-1 and IP-2 were two replicates in which VCP was first immunoprecipitated by monoclonal anti-VCP antibody. The PVDF membrane was incubated with pan anti-palmitoylation antiserum (B) and anti-VCP antibody (A), sequentially. The immune precipitates treated with (+) or without (-) NH2OH were analyzed using western blotting against homemade antiserum (C). In (D), SW480 cells were firstly treated with 2-BP (100 µM final concentration) or 0.1% DMSO (Control) for 18 hrs after serum starvation for 24 hrs. The cells were harvested and proteins were extracted. After being separated by SDS-PAGE, the proteins pulled down by anti-VCP antibody were immune-analyzed against our homemade antiserum or anti-VCP antibody, successively.

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Identification of Palmitoylated Transitional Endoplasmic Reticulum

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