Fluorous Solid-Phase Extraction Technique Based on Nanographite

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A Novel Fluorous Solid-phase Extraction Technique Based on Nanographite Fluoride Cheng Zhang, Tao Tao, Wenjuan Yuan, Lei Zhang, Xiaoqin Zhang, Jun Yao, Ying Zhang, and Haojie Lu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05071 • Publication Date (Web): 29 Mar 2017 Downloaded from http://pubs.acs.org on March 29, 2017

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Analytical Chemistry

A Novel Fluorous Solid-phase Extraction Technique Based on Nanographite Fluoride Cheng Zhang,a,b Tao Tao,a,b Wenjuan Yuan,a,b Lei Zhang,a Xiaoqin Zhang,a,b Jun Yao,a Ying Zhang*,a and Haojie Lu*,a,b,c a. Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai, 200032, P.R.China. b. Department of Chemistry, Fudan University, Shanghai, 200433, P.R.China. c. Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai, 200032, P.R.China ABSTRACT: Fluorous solid-phase extraction (FSPE) has been employed to isolate target compounds from complex chemical or biological samples in many research areas. However, the lack of efficient and economical available perfluorinated materials impeded its development. In this study, we present a novel nanographite fluoride-based fluorous solid-phase extraction (GF-FSPE) as a replacement of commercially available cartridge-based FSPE, which showed remarkable selectivity, low LOD, high post enrichment recovery and large enrichment capacity. To demonstrate the potency of GF-FSPE, it was designed and successfully applied to isolate cysteine-containing peptide subsets from complex protein samples to improve the proteome coverage. Additionally, since graphite fluoride was inexpensive and highly commercialized, this study was expected to promote the popularization of FSPE in both chemical and biological separations, as well as encourage the synthesis and broaden the application of highly fluorinated carbon fluoride in material science.

Fluorous solid-phase extraction (FSPE) has been employed to isolate compounds of interest from complex samples in organic chemistry and heterogeneous catalysis for a long time.1-5 It relies on the derivatization of target analytes with compounds bearing highly fluorinated portion (called F-tag) and subsequent separation with the aid of perfluorinated solid phases. The unique ability of fluorinated molecules to recognize other molecules bearing a F-tag is termed “fluorous affinity” or “fluorophilicity”.5-8 Recently, this approach has been extended into many “omics” researches, such as proteomics,9-14 glycomics15-17 and metabolomics,18 owning to its better compatibility with mass spectrometry (MS) compared with the classical biotin-tag separation strategy, as the F-tag not only enables the selective enrichment of target analytes, but also avoids unexpected dissociation of MS tag during MS process. What’s more, fluorous derivatization can promote the ionization of analytes during MS process due to the hydrophobicity of Fportion, which makes FSPE more suitable for analysis of analytes of low abundance and low ionization efficiency.19 The diversity in the highly fluorinated compound family makes a great contribution to the popularization of FSPE and endows it with bright prospects in bio-separation. Owning to the blooming of fluorine chemistry, so far, a lot of highly fluorinated derivatization compounds have been synthesized that are equipped with chemical reactivity with native biomolecules (e.g. protein, peptide, glycan and metabolite).20-24 On the contrary, the choice of perfluorinated materials for FSPE is relative limited, which hinders the development of FSPE. Commercially available FSPE cartridges are mostly elaborated with fluorous silica, which is composed of highly pure silica and fluorous slilyl reagents, therefore raising the cost of FSPE and demanding sophisticated techniques to ensure the high fluorine content and large surface area of the

solid phase.6-7 Some home-made perfluorinated materials were also designed for FSPE.13,17,25,26 However, the expensive raw materials and complex production processes impeded their popularization. Considering the pervasive application in chemical and biological separations, there is still a demand for perfluorinated materials that is less expensive and more common for FSPE. Graphite fluoride (GF) is a unique species of carbon fluoride in which fluorine atoms are attached to carbon atoms by covalent C-F bonds. It was different from fluorine-graphite intercalation compounds (GICs) as every carbon atom of GF takes a chair-type structure with sp3-hybridization instead of sp2.27,28 It was first reported in 1934 and has attracted much attention in the past few decades due to its potential for industrial application as a solid lubricant and a cathode of primary lithium battery.29-31 The most widely used route to get graphite fluoride is the solid/gas reaction between carbon and molecular fluorine, either pure or diluted with inert gas. Thanks to indepth studies about different synthesize strategies, GF is highly commercialized with low price and can be designed to nanoscale nowadays.27,29,32,33 Compared with other forms of carbon (e.g. graphene), graphite is proposed more easy to fluorinate, which leads to the high fluorine content and low cost of fluorination.34,35 However, in spite of the application of GF in many other areas, there has been no research about using it as a perfluorinated material for FSPE. Since the surface of GF is covered with fluorine atoms, we suppose that it can be used to recognize the F-tag in fluorous derivative by “fluorous affinity”. Importantly, considering the highly fluorinated level, GF, as a silica-free perfluorinated material with lower price and high surface area, is supposed to perform the specific task as capable as, or even better than other commercially available materials.

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In this work, we explored a novel fluorous solid-phase extraction technique using nanographite fluoride (nGF) as a perfluorinated solid phase (GF-FSPE). To demonstrate the application prospects of GF-FSPE, we employed it to isolate cysteine-containing peptides from protein digestion samples to simply the complex protein sample and reduce the dynamic range of proteins, in order to improve proteome coverage, which was supposed to provide a useful tool for in-depth proteomics investigation and benefit the researches about proteogenomics.36-39 A systematic comparison with fluorous NuTipsTM (a commercially available material of FSPE) was conducted to evaluate performance of nGF as a novel FSPE substrate. Based on the comparison, a brief exploration about the interactions between the nGF and peptides was discussed to confirm the “fluorous affinity” mechanism. At last, the designed workflow was applied to selectively isolate cysteinecontaining peptide subset from complex protein samples, and improved proteome coverage was successfully achieved, which demonstrated the application prospects of GF-FSPE in chemical and biological separations.

EXPERIMENTAL SECTION Materials and Chemicals. Nanographite fluoride (nGF) was purchased from Xianfeng Nanotech. (Nanjing, JS, China). Synthetic peptide (sequence: AGVHGCNGLR, purity ≥95%) was purchased from Top-peptide Co. (Shanghai, China). N[(3-Perfluorooctyl)propyl]iodoacetamide (FIAM), ammonium bicarbonate (ABC), DL-Dithiothreitol (DTT), Iodoacetamide (IAA), urea, myoglobin (proteomics grade), trypsin (proteomics grade), sodium dodecyl sulfate (SDS), sodium chloride (NaCl) and Tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Bovine albumin (RIA Grade) was purchased from Amresco (Fountain Parkway Solon, OH, U.S.A.). Fluorous NuTipsTM were obtained from Fluorous Technologies Inc. (Pittsburgh, PA, U.S.A.). HPLC-grade methanol and acetonitrile (ACN) were purchased from Merck (Darmstadt, Germany). Analyticalgrade ethanol and acetone was purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Distilled water was purified by a Milli-Q system (Milford, MA, U.S.A.). Fluoroalkylation of cysteine-containing peptide. Synthetic peptide was dissolved in H2O (final concentration: 1 nmol/µL) as a stock solution, while FIAM was dissolved in ACN (final concentration: 10 µg/µL) as a stock solution. Before fluoroalkylation, the peptide was pretreated with TCEP (final concentration: 0.5 mM) in 100 mM ABC buffer for 30 min at 37 oC, and then adjusted to the designed reaction system with the addition of myoglobin peptides, FIAM stock solution, ACN, 1 M ABC Buffer and H2O. The volume of the whole reaction system was 200 µL containing 2 nmol synthetic peptide. The derivatization reaction was conducted in a ThermoMixer under certain condition (see supporting information). After the reaction, 1 µL of the product was used for matrix-assisted laser desorption/ionization mass spectrometric (MALDI-MS) analysis and the rest was lyophilized for further use if necessary. Isolation of fluoroalkylated peptide with nGF. Graphite fluoride was washed with CH3OH and coupling buffer (discussed below) sequentially before using. 1 mg nGF was added to a coupling system of 200 µL each time. The mixture was incubated at r. t. for 5 min, and the supernatant was collected after centrifugation at 10000 rpm for 2 min. The resulting nGF was washed twice with 100 µL coupling buffer and the fluoro-

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alkylated peptides were eluted with 40 µL CH3OH at r.t. for 5 min. 1 µL of supernatant or elution was used for MALDI-MS analysis and the rest was lyophilized for further use if necessary. Isolation of cysteine-containing peptides from BSA sample. 200 µg BSA was dissolved in 100 µL denaturing solution which contained 50 mM ABC and 8 M urea. The mixture was treated with DTT (final concentration: 10 mM) at 55 °C for 30 min. The excess DTT and urea was removed by buffer exchanges in a 10 kDa filter with 100 mM ABC twice. The resulting solution was adjusted with H2O and ACN to 100 µL 50 mM ABC/50% ACN (v/v) for fluoroalkylation with FIAM (final concentration: 1 µg/µL). The fluoroalkylation reaction was conducted in the same filter at 55 oC for 2 h in the dark, and the excess FIAM was removed by buffer exchange with 50 mM ABC twice. Then trypsin was added to the same filter with a mass ratio of 1:40 (trypsin: BSA). The mixture was incubated at 37 oC for 16 h and the tryptic digestion were collected by ultrafiltration. The resulting peptide mixture was adjusted with CH3OH to 200 µL 25 mM ABC/50% CH3OH (v/v) followed by the addition of 4 mg nGF. After GF-FSPE, the supernatant and washing solution were combined together, while the elution was collected separately. Both fractions were lyophilized for further use. Isolation of cysteine-containing peptides from Human Umbilical Vein Endothelial Cell (HUVEC) sample. HUVECs were harvested and lysed in lysis buffer containing 7 M urea and 2 M thiourea. Protein concentration was determined by Bradford protein assay (Bio-Rad), and the protein concentration was adjusted to 2 µg/µL with lysis buffer. 400 µg protein was treated with DTT (final concentration: 10 mM) at 55 °C for 30 min. The excess DTT, urea and thiourea were removed by buffer exchanges in a 10 kDa filter with 100 mM ABC twice. The resulting solution was adjusted with H2O and ACN to 400 µL 50 mM ABC/50% ACN (v/v) for fluoroalkylation with FIAM (final concentration: 1 µg/µL). The fluoroalkylation reaction was conducted in the same filter at 55 oC for 2 h in the dark, and the excess FIAM was removed by buffer exchange with 50 mM ABC twice. Then trypsin was added to the same filter with a mass ratio of 1:40 (trypsin: BSA). The mixture was incubated at 37 oC for 4 h and an equal amount of trypsin was added. After incubation for another 12 h, the tryptic digestion were collected by ultrafiltration. The resulting peptide mixture was adjusted with CH3OH to 200 µL 25 mM ABC/50% CH3OH (v/v) followed by the addition of 4 mg nGF. After GF-FSPE, the supernatant and washing solution were combined together, while the elution was collected separately. Both fractions were lyophilized for further use.

RESULTS AND DISCUSSION Brief of the analytical workflow. The entire process for isolation of cysteine-containing peptide subset with GF-FSPE consisted of five essential parts, 1) reduction of protein disulfide bonds with dithiothreitol (DTT), 2) fluoroalkylation of protein cysteine residues combined with the filter-aided sample preparation (FASP) switch,40 3) protease digestion, 4) isolation of cysteine-containing peptides with GF-FSPE to divide the peptide mixture into “FW fraction” (combination of flow though and washing fraction) and “EL fraction” (eluted fraction), and 5) parallel LC-MS/MS analysis of the obtained peptide fractions (Scheme 1). Based on this workflow, we carefully investigated the fluoroalkylation condition and isolation condition of GF-FSPE.

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Analytical Chemistry Scheme 1. The illustration of the analytical workflow.

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Figure 1. Changes of RI-F value in different aqueous solutions: (a) influence of organic constituent (with the dilution radio of 1:1 (v/v)) and (b) content of methanol.

As for nGF, the characterization data demonstrated its large surface area, and the proper size made it easy to separate from different buffers by centrifugation (Figure S1, Table S1 and S2). The Fourier transform infrared spectroscopy (FT-IR) data clearly showed the C-F bond in nGF (Figure S2).41 And the energy dispersive spectroscopy (EDS) data showed that the nGF had an F/C ratio close to 1 (Figure S3 and Table S3), which indicated the layered structure was closed to (CF)n type.27,28,42 This highly fluorous level was expected to ensure the good properties of nGF as a perfluorinated material. Fluoroalkylation of cysteine residues. We first investigated the fluoroalkylation condition of cysteine residues. FIAM was chosen to be the fluorous derivatization reagent because of the good chemical reactive of iodoacetyl amino with thiol groups.43,44 The reaction are usually accomplished in the alkalescency environment (e.g. ABC buffer), to improve the chemical reactivity of thiol groups while avoid the unexpected influences on protein backbone or other side chains (Scheme S1). Additionally, owning to the poor solubility of FIAM in pure water,45 organic solvent owning good miscibility with water, mostly tetrahydrofuran (THF), was required. In the present work, we chose ACN as a substitution of THF because of its lower volatility and toxicity. We investigated the derivatization condition using a synthetic cysteine-containing peptide (sequence: AGVHGCNGLR, MW: 983.1 Da) as a model. After the reaction, the product mixture was analyzed by MALDI-MS. Derivatization efficiency was calculated by dividing the peak area of fluoroalkylated peptide by the total peak area of native and fluoroalkylated peptide in the MS spectrum. After the investigation (see supporting information), the derivatization condition was decided to be 1 µg/µL FIAM at 55 oC for 2 h in 50 mM ABC/50% ACN (v/v) aqueous solution. The signal-promoting effect of F-tag mentioned in previous works was confirm (Figure S4), which was supposed to benefit the analysis of cysteine-containing peptides of low ionization efficiency and abundance.14-16,19 Investigation of isolation condition of GF-FSPE. Investigation of the isolation condition for GF-FSPE was conducted in a system, in which synthetic cysteinecontaining peptide was mixed with myoglobin tryptic digestion at a molecule ratio of 1:5 (synthetic peptide :

tryptic digest of myoglobin, the same below). After GFFSPE, the initial solution and flow through were both analyzed by MALDI-MS. A defined parameter “RI” was applied to evaluate the loading efficiency: (1) RI = (Iinitial-Iflow)/Iinitial Iinitial was the relative intensity of certain peptide in the MS spectrum of initial solution, and Iflow was the relative intensity of the same peptide in the MS spectrum of flow through after enrichment. In particular, for fluorous labeled peptide, “RI” was typed as “RI-F”. Since myoglobin peptide VEADIAGHGQEVLIR (MW: 1606.80 Da) was the donor of base peak of either initial solution or flow through, it could be used as an internal reference. Larger the “RI” value was, easier the peptide was to hook on the nGF. Ethanol, methanol, ACN and acetone were selected as candidates of organic composition in coupling buffer according to the advice from Fluorous Technologies. Methanol aqueous system performed the highest efficiency (Figure 1a) and 50% (v/v) was the best content (Figure 1b). The “RI-F” value was as high as 0.98 for this system, which indicated the high loading efficiency. According to the further investigation of different washing and eluting conditions (data not shown), two candidates of the entire GF-FSPE process were provided (Table S4). Since the analytical workflow we established aimed to divide the peptide sample into two factions, it was necessary to balance the peptide quantity. So we chose condition 1 for the analysis of complex samples at last, as it showed higher post enrichment recovery though some losses in selectivity. Comparisons between graphite fluoride and fluorous NuTipsTM. To further demonstrate the unique property of nGF, it was compared with a widely used and commercially available FSPE cartridges (fluorous NuTipsTM) in terms of their enrichment performance besides the experimental cost (Table S5). The synthetic cysteine-containing peptide mixed with myoglobin tryptic digestion at a certain molecule ratio was sent to enrichment using these two different types of fluorous supports. The isolation conditions for both strategies are listed in Table S4. For the fluorous NuTipsTM, we conducted a protocol applied in previous works of other groups and recommended by Fluorous Technologies.43 For GF-FSPE, we used condition 1. The result showed that when the molecule ratio was 1:1, both materials showed a good selectivity (Figure 2b, c) and signals of myoglobin peptides were greatly eliminated. However, when the molecule ratio reached 1:10, GF-FSPE began to show its advantage over FSPE with fluorous NuTipsTM (Figure 2e, f). When applying condition 2 for GF-FSPE, we found that even at a molecule ration of

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Figure 2. MALDI-MS spectra of synthetic peptide and myoglobin peptides mixed at different molecular ratios (a, b, c of 1: 1 and b, d, f of 1:10): (a, d) before enrichment, (b, e) after enrichment with fluorous NuTipsTM and (c, f) after enrichment with nGF. Signals of fluoroalkylated synthetic peptide were marked with “#”.

1:100, the fluoroalkylated peptide could be clearly detected (Figure S5). This result showed a remarkable selectivity of nGF compared with fluorous NuTipsTM. The limit of detection (LOD) and post enrichment recovery of either method, as well as enrichment capacity of nGF, influences of salt and detergent on GF-FSPE were also evaluated (see supporting information). Using S/N>3 as a cutoff, it could speculate that the LOD of GF-FSPE (approximately 10-9~10-8 M) was lower than FSPE with NuTipsTM (approximately 10-8~10-7 M). In the mean time, GF-FSPE showed higher post enrichment recovery (around 90%) than FSPE with fluorous NuTipsTM (around 73%) measured by the dimethylation quantification method.46 The enrichment capacity of nGF could reach 50 µg synthetic peptide/mg. Considering that cysteine-containing peptides only occupied a small proportion in protein digestion, the loading quantity of protein sample was supposed to be much larger than this value. According to our data, GF-FSPE could endure the high salt concentration as same as FSPE with other fluorous support.14,15 Previous work also reported that “fluorous affinity” could endure detergents.47 However, our data failed to provide more evidences because detergents were hard to remove from the sample owning to the buffers used during FSPE, which would greatly affact the MS signals of target analysts. Considering that detergents also affacted the LC system, depletion of detergents was preferred before GF-FSPE in “omics” researches with LC-MS/MS.

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level and high surface area. Unlike silica-based perfluorinated materials, the fluorine content on the surface of nGF did not suffer from incomplete silanization that was supposed to reduce the “fluorous affinity” property. Additionally, highly purified nGF seldom contained heteroatoms except carbon and fluorine atoms, the extremely high fluorous level ensured the good “fluorous affinity” property of nGF. Exploration about the “fluorous affinity” and nonspecific interactions on the surface of nGF. In spite of “fluorous affinity”, nGF might adsorb peptides via hydrophobic interaction.28 And functional materials with hydrophobic surface were usually employed for the enrichment of low abundant proteins/peptides.48,49 Considering the fact that fluoroalkylation would increase the hydrophobic property of target peptide, which would strengthen the hydrophobic interaction between nGF and target peptide, it was important to prove that the selectivity of enrichment was mainly resulted from “fluorous affinity”, regardless of hydrophobic interaction. To achieve this goal, adsorption behaviors of fluorous labeled synthetic peptide and unlabeled myoglobin peptides were all evaluated according to their “RI” values, and a brief exploration about the changes of “RI” values between different coupling conditions was conducted to trace the nonspecific interactions on the surface of nGF (see supporting information). It was concluded that adsorption of fluorous labeled peptide was the result of a specific interaction different from hydrophobic-hydrophilic interaction and the impact of hydrophobic interaction decreased with the increase of organic solvent content in the coupling buffer. The nonspecific adsorption of peptides under the coupling condition we used was more likely to result from hydrophilic interaction rather than hydrophobic interaction. To provide more evidences for our conclusion, we used the dimethylation strategy to increase the hydrophobicity of myoglobin peptides artificially.50 Peptides and their dimethylated forms were mixed at a molecular ratio of 1:1 and sent to incubation with nGF under different conditions. The initial solution and flow through were both analyzed by MALDI-MS. The adsorption behaviors of certain peptide and its dimethylated form were evaluated with a defined parameter “RA”: RA = (Adi, initial/Ainitial - Adi, flow/Aflow) / (Adi, initial/Ainitial) (2) Ainitial and Adi, initial meant the peak areas of certain peptide and its dimethylated form in the MS spectrum of initial solution while Aflow and Adi, flow meant the corresponding ones in the MS spectrum of flow through. It was obvious that higher the “RA” value was, easier the dimethylated form (with higher hydrophobicity) was to retain on the nGF. The experimental result showed that the rising content of CH3OH would result in decreasing “RA” values, and original peptides (with lower hydrophobicity) were easier to adsorb on the surface of nGF under the condition of 50% CH3OH (v/v) (see supporting information). This result supported our previous conclusion and further confirmed the unique “fluorous affinity” property of nGF. We also believed that a detailed comparison between nGF and classical fluorous support in the form of fluorous liquid chromatography was needed to truly understand the fluorous nature of nGF and our system in the future.51-54 Isolation of cysteine-containing peptides from bovine serum albumin (BSA) sample. To assess the feasibility of the entire analytical workflow, we further introduced GF-FSPE to isolate cysteine-containing peptides from BSA. Samples either

We speculated the remarkable “fluorous affinity” performance of nGF could be attributed to its highly fluorous

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Analytical Chemistry

Figure 3. BSA sequence coverage analysis for data of different pre-treatment methods.

undergoing classic reductive alkylation with IAA or not were applied as controls for the comparison with the analytical workflow we established (see supporting information). The sequence coverage of BSA was well summarized for each of these three protocols (Figure 3). Direct analysis of BSA sample could only get the sequence coverage of 37.9%. With the help of classic reductive alkylation, the sequence coverage could reach to 84.3%, which confirmed that the classic method did benefit the analysis of proteins rich in disulfide bonds. Withe GF-FSPE, the combination of FW and EL fractions could further increase the sequence coverage to 92.4%. All the cysteine residues could be located with both the classic protocol and the newly established workflow with GF-FSPE while no cysteine residue could be located without reductive alkylation. It was noticed that the our strategy seemed to benefit the coverage of “short” sequences (e.g. F157-K160, L221-R222, L372K374) compared with chassic protocol (see supporting information). These sequences were identified in peptides with at least one or more missed cleavage sites (MCSs). The phenomenon might indicate that fluoroalkylation would affact the protease digestion efficiency. But since peptides with MCSs more than 2 were not identified in the sample undergoing either classic or the newly established protocol and the protease digestion efficiency was reported to relate with the charge state around the cleavage sites,55 we believed this phenomenon was more likely to result from the samplesimplifying effect. Because peptides carring MCSs were supposed to be of lower abundance compared with these without MCSs, and they were easy to “escape” from the m/z trap in shotgun proteomics analysis.56 The simplification of peptide sample with GF-FSPE might help reveal the signals of these peptides. On the other hand, the qualitative result also showed the newly established workflow greatly benefit the identification of cysteine-containing peptides with MCSs (see supporting information). We believed it was resulted from the signal-promoting effect of F-tag as mentioned above.

Figure 4. Repeatability and overlap analysis of HUVEC samples: identified proteins in samples undergoing (a) classic protocol and (b,c) newly established workflow ((b) for FW fraction, (c) for EL fraction); (d) totally identified peptides in the newly established workflow (left part for FW fraction and right part for EL fraction); (e) totally identified proteins in samples undergoing classic protocol (left part) and newly established workflow (right part).

Isolation of cysteine-containing peptides from HUVEC sample. Finally, we decided to examine the our workflow to a more challenging sample, the HUVEC lysis solution. Sample undergoing classic reductive alkylation with IAA was again applied as a control to evaluate the performance of our strategy. Totally, 1121 proteins were identified with classic protocol while 1655 ones were identified through the newly established workflow (Figure 4e). The result showed even EL fraction alone could cover more proteins than classic method (Figure 4a,c). Owning to the sample-simplifying effect, the qualitative result of either the FW or the EL fraction had a better repeatability than that of the sample undergoing classic protocol according to the overlaps between each replicate (Figure 4a-c). Nearly 90% of the peptides identified through the newly established workflow were unique to either FW or EL fraction, which indicated that peptides in either fraction had significant differences from those in the other one after GF-FSPE (Figure 4d). 0.11% of the 6545 peptides unique to FW fraction were cysteine-containing peptides while 47.65% of the 2002 peptides unique to EL fraction had cysteine residues (Figure 4d). And only 12.03% of the peptides identified through classic method were cysteine-containing peptides, which showed a great loss in selectivity compared with GF-FSPE (32.13% in EL fraction) (Figure S6). These results revealed that GF-FSPE did help the isolation of cysteine-containing peptides from the complex protein digestion. The overlap of proteins identified through different methods indicated the new method contributed to the identification of great number of proteins that could not be revealed through classic method (Figure 4e). A considerable increase of protein sequence coverage was also accomplished when appling the new strategy (Figure S7). These results clearly illustrated that our method could improve the proteome coverage and showed a good example of the application of GF-FSPE.

CONCLUSIONS It was the first time that carbon fluoride was introduced to the field of FSPE, and GF-FSPE had many advantages over

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the commercially available FSPE cartridge, as it showed better selectivity, lower LOD and higher post enrichment recovery, as well as large enrichment capacity. We believed the analytical workflow we established not only showed application prospects of GF-FSPE, but also provided a useful technique for in-depth proteomics investigation. In the meantime, as technologies for isolation of cysteine-containing peptide subset were usually needed in researches about cysteine post-translational modifications (PTMs),57,58 this technique also provided a optional method in these research areas. Additionally, we speculated that GF-FSPE could be introduced to other “omics” researches based on FSPE, as well as to organic chemistry and heterogeneous catalysis, because nGF was highly commercialized, inexperience and stable in most chemical buffers. What’s more, considering the potential imperfection of commercial available nGF, this study might encourage the researches about synthesis and broaden the application of nGF or other highly fluorinated carbon fluoride in material science.

ASSOCIATED CONTENT Supporting Information (Word Style “TE_Supporting_Information”). A listing of the contents of each file supplied as Supporting Information should be included. For instructions on what should be included in the Supporting Information as well as how to prepare this material for publication, refer to the journal’s Instructions for Authors.

AUTHOR INFORMATION Corresponding Author *Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected]. *Phone: +86 21 54237618. Fax: +86 21 54237961. E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was supported by the NST (Grants 2016YFA0501303), NSF (Grants 21335002, 21375026 and 31670835), the Ph.D. Programs Foundation of Ministry of Education of China (20130071110034) and Shanghai Projects (Eastern Scholar, Shanghai Rising-star 15QA1400600, 15JC1400700 and B109).

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