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In-Depth Proteome Coverage by Improving Efficiency for Membrane Proteome Analysis Qun Zhao, Fei Fang, Yichu Shan, Zhigang Sui, Baofeng Zhao, Zhen Liang, Lihua Zhang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017
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Analytical Chemistry
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In-Depth Proteome Coverage by Improving Efficiency for Membrane
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Proteome Analysis
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Qun Zhaoa,1, Fei Fanga,b,1, Yichu Shana, Zhigang Suia, Baofeng Zhaoa, Zhen Lianga,
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Lihua Zhanga,*, Yukui Zhanga
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a
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Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese
Key Laboratory of Separation Science for Analytical Chemistry, National
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Academy of Science, Dalian 116023, China
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b
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*To whom correspondence should be addressed. E-mail:
[email protected].
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Phone& fax: +86-411-84379720.
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1
University of Chinese Academy of Sciences, Beijing 100039, China
These authors contributed equally to this study.
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Abstract
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Although great achievement has been made in the mapping of human proteome, the
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efficiency of sample preparation still needs to be improved, especially for membrane
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proteins. Herein, we presented a novel method to deepen proteome coverage by the
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sequential
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methylimidazoliumchloride (C12Im-Cl). With such a strategy, the commonly lost
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hydrophobic proteins by 8 M urea extraction could be further recovered by C12Im-Cl,
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as well as the suppression effect of high abundance soluble proteins could be
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decreased. Followed by the in-situ sample preparation and separation with different
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stationary phases, more than 9810 gene products could be identified, covering 8
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orders of magnitude in abundance, which was to the best of our knowledge, the
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largest dataset of HeLa cell proteome. Compared with previous work, not only the
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number of proteins identified was obviously increased, but also the analysis time was
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shortened to a few days. Therefore, we expect that such a strategy has great potential
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applications to achieve unprecedented coverage for proteome analysis.
extraction
of
proteins
using
urea
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and
1-dodecyl-3-
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Analytical Chemistry
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Introduction
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Mammalian cell lines are widely used in various studies, and become indispensable
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for pursuing biological insight.1-3 Mapping the entire cell proteome is of great
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significance to comprehensively elucidate molecular functions, which is in favor with
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the accurate diagnosis and treatment of diseases.4-6 Although great achievement has
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been witnessed in the large scale proteome analysis, great efforts still should be made
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to improve the identification of membrane proteins, with low abundance and high
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hydrophobicity.7,8
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Although chaotropic agents, such as urea and guanidine hydrochloride, are
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commonly used for cell lysis and protein extraction,9-15 the solubilization efficiency
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for membrane proteins is very limited.16 To solve this problem, sodium dodecyl
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sulfate (SDS) has been successfully applied to extract both hydrophilic and
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hydrophobic proteins.17-20 Unfortunately, the removal of such a detergent is difficult,
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and the residual SDS in proteome samples, even with low concentration, can suppress
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the subsequent enzymatic digestion and impede liquid chromatography-tandem mass
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spectrometry (LC-MS/MS) identification. Although by filter-aided sample preparation
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(FASP) strategy,21 SDS could be removed with high efficiency, and over 7000 gene
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products could be identified from mammalian cell line,22-26 the analysis of low
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abundance membrane proteins is still interfered by the co-existence of soluble
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proteins with high abundance.
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In our previous study, 1-dodecyl-3-methylimidazoliumchloride (C12Im-Cl) was
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successfully used to achieve the large-scale membrane proteome analysis,27 and
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demonstrated the superiority to other solubilizers, such as SDS and Rapigest. Herein,
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we developed a new strategy to deepen the coverage of proteome by improving the
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efficiency for membrane proteome analysis, with the combination of two-step
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sequential protein extraction with urea and C12Im-Cl, in-situ sample preparation in a
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filter, and RP-RPLC-MS/MS analysis with different stationary phase during
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separation at low pH, which demonstrated the great promise to achieve the deep
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coverage proteome analysis.
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Experimental Section
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Reagents and materials
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Protease inhibitor cocktail, urea, dithiothreitol (DTT), iodoacetamide (IAA), and
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trifluoroacetic acid (TFA) were purchased from Sigma (St. Louis, MO, USA). Trypsin 3
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was ordered from Promega (Madison, WI, USA). Acetonitrile (ACN, HPLC grade)
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was bought from Merck (Darmstadt, Germany). 1-Dodecyl-3-Methylimidazolium
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Chloride (C12Im-Cl) was obtained from Chengjie (Shanghai, China). Deionized
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water was purified by a Milli-Q system from Millipore (Milford, MA, USA). Other
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chemicals were of analytical grade.
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Durashell C8 particles and XBP C18 particles (5 µm, 100 Å pore) were obtained
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from Agela (Tianjin, China). Fused-silica capillaries (75 µm i.d./365 µm o.d.) were
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acquired from Sino Sumtech (Handan, Hebei, China), and Vivacon® 500 microcon
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filtration devices (molecular weight cutoff 10,000 Da) were purchased from Sartorius
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(Goettingen, Germany).
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Protein extraction
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HeLa cells (7x107) were first lysed and extracted with 4 mL of 8 M urea containing
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phosphate-buffered saline (1xPBS, pH 8.0) and 2% (v/v) cocktail. After centrifugation
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at 150,000 g for 40 min, the supernatant was collected and named as 8 M urea fraction.
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The residual pellet was further extracted with 400 µL of C12Im-Cl containing 50 mM
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NH4HCO3, and then clarified by centrifugation at 36,000 g for 20 min, named as
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C12Im-Cl fraction. Protein concentration was determined using a Bradford assay kit
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from Bio-Rad (Hercules, CA, USA) with BSA as a standard.
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Protein digestion and peptide fractionation
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The 8 M urea fraction was transferred to a filter device, and washed with 50 mM
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NH4HCO3, followed by centrifugation at 14,000 g for 15 min. Subsequently, proteins
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were denatured and reduced with 100 mM DTT at 95°C for 5 min, and washed with
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50 mM NH4HCO3. Then, proteins were subjected to alkylation with 20 mM IAA in
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the dark at the room temperature for 20 min, followed by centrifugation at 14,000 g
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for 15 min. Afterwards, proteins were digested with the trypsin/protein ratio (m/m) as
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1:25 at 37°C for 12 h. Finally, the peptides were collected by centrifugation at 14,000
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g for 15 min.
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The C12Im-Cl fraction was transferred to a filter device. Proteins were denatured
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and reduced with 100 mM DTT at 95°C for 5 min, and washed with 8 M urea in 50
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mM NH4HCO3. Subsequently, proteins were subjected to alkylation with 20 mM IAA
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containing 8 M urea in 50 mM NH4HCO3 in the dark at the room temperature for 20
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min, followed by centrifugation at 14,000 g for 15 min. Then proteins were washed
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with 50 mM NH4HCO3 for three times. Finally, proteins were digested with the
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trypsin/protein ratio (m/m) as 1:25 at 37°C for 12 h, and the peptides were collected 4
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by centrifugation at 14,000 g for 15 min.
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The tryptic peptides obtained from 8 M urea fraction and C12Im-Cl fraction were
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respectively fractionated by RPLC with a C18 column (5 µm, 100 Å, 250 mm×4.6
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mm i.d.). Mobile phases A (98% H2O, 2% acetonitrile, adjusted pH to 9.5 using
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NH3·H2O) and B (98% acetonitrile, 2% H2O, adjusted pH to 9.5 using NH3·H2O)
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were used to set the gradient as follows: 2%-8% B, 2 min; 8%-18% B, 17 min;
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18%-30% B, 14 min; 30%-35% B, 1 min; 35%-80% B, 1 min; 80%-80% B, 2 min.
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The flow rate was 1 mL/min. The eluent was collected every minute. A total of 36
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fractions were collected, and then concatenated to 18 fractions with equal time
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interval, vacuum dried and reconstituted in 0.1% (v/v) FA and 2% (v/v) ACN in water
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for subsequent analyses. Besides, for peptides from C12Im-Cl fraction, the
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non-retained peptides eluted during sample loading process were collected and
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individually analyzed.
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LC-ESI-MS/MS analysis
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The 19 peptide fractions from 8 M urea extraction (~1.8 µg of each fraction) and 18
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peptide fractions from C12Im-Cl extraction (~0.8 µg of each fraction) were further
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analyzed by nanoRPLC-ESI-MS/MS using a high-resolution Triple TOF 5600 mass
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spectrometer (AB SCIEX, Framingham, MA, USA). Peptides were separated on a
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capillary column (15cm×75µm i.d.) at the flow rate of 300 nL/min. The mobile phase
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used was composed of H2O with 2% ACN and 0.1% FA (A), and ACN with 2% H2O
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and 0.1% FA (B) with the gradient as 5%-22% B, 40 min; 22%-35% B, 15 min;
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35%-80% B, 5 min; 80%-80% B, 5 min. Mass spectrometry analysis was carried out
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in a data dependent manner with full scans (350-1,250 m/z). The top 60 precursor ions
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were selected in each MS scan for subsequent MS/MS scans with charge state of 2-5.
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MS scans were performed for 0.25s, and 60 MS/MS scans were subsequently
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performed for 0.04 s of each. The dynamic exclusion for MS/MS was set as 22 s. The
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CID energy was automatically adjusted by the rolling CID function of Analyst TF 1.6.
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Database searching
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Wiff files from Triple-TOF 5600 MS were firstly converted to Mascot generic
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format (mgf) files using Protein Pilot software (version 4.5). The mgf files were then
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delivered to Mascot software (version 2.4), and searched against the database IPI
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human 3.87 (91,464 entries, 22,200 Gene product) with the same parameters as
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followed: trypsin as the protease with a maximum of two missed cleavages allowed; the
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carbamidomethylation of cysteine was specified as a fixed modification; the oxidation 5
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of methionine and the acetylation of protein N termini were included as variable
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modifications; the mass error was set to 0.05 Da for precursor ions and 0.1 Da for
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fragment ions. After database searching, the results of 37 fractions were merged and
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filtered by pBuild28 to control the false discovery rate (FDR) at peptide spectral level
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at less than 1%, and reduce the apparent redundancy in protein identification. Besides,
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the minimum peptide length was specified to 6 amino acids.
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Bioinformatics analysis
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The grand average of hydropathicity (GRAVY) values of the identified peptides
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was calculated using ProtParam program (http://web.expasy.org/protparam/).29,30 The
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peptides with positive and negative GRAVY values were named as hydrophobic and
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hydrophilic peptides, respectively. The TMHMM (http://www.cbs.dtu.dk/services/
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TMHMM/) algorithm was used to predict the transmembrane domains (TMDs) of
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identified proteins.31 Proteins with at least one predicted TMD were considered as
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integral membrane proteins (IMPs). The topological structure of transmembrane
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proteins was displayed with TOPO2 software (http://www.sacs.ucsf.edu/cgi-bin/
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open-topo2.py). The cellular components and molecular functions based on Gene
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Ontology (GO) Consortium were assigned with GoMiner.32
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Results and discussion
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To discover more information of membrane proteins, which were easily lost during
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commonly used urea extraction method, and further generate the extensive proteome
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map of HeLa cell line, a two-step sequential extraction-based sample preparation
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strategy was established. As shown in Figure 1, we firstly extracted the cell lysates
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with 8 M urea, and then re-extracted the residual pellets with C12Im-Cl, which could
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supply stronger dissolving capability for the hydrophobic membrane proteins
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extraction.27 Subsequently, the obtained two protein fractions were respectively
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treated in situ by our previously developed imFASP method to improve the efficiency
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of protein digestion process.33 In order to further deepen the coverage of proteins,
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RPLC at high pH (pH 9.5) was employed to fractionate peptides to decrease the
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complexity of samples, followed by nanoRPLC at low pH (pH 3) for peptide
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separation. It should be pointed out that, the collected fractions of peptides from 8 M
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urea extraction and those from C12Im-Cl extraction were respectively separated on
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C18 and C8 capillary columns, since our previous study demonstrated that C8 column
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was beneficial to improve the separation efficiency of hydrophobic peptides.34 6
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Analytical Chemistry
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By such a protocol, the totally required MS/MS running time for the single-run
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analysis was ca. 34 h, much faster than that in previous work (12 days).23 In total,
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202,362±685 peptide-spectrum matches (PSMs) were obtained (FDR
