Enhancing Membrane Protein Identification Using a Simplified

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Enhancing Membrane Protein Identification Using a Simplified, Centrifugation and Detergent-based Membrane Extraction Approach Yanting Zhou, Jing Gao, Hongwen Zhu, Jingjing Xu, Han He, Lei Gu, Hui Wang, Jie Chen, Danjun Ma, Hu Zhou, and Jing Zheng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03710 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 27, 2018

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Analytical Chemistry

Enhancing Membrane Protein Identification Using a Simplified, Centrifugation and Detergent-based Membrane Extraction Approach Yanting Zhou12#, Jing Gao2#, Hongwen Zhu2, Jingjing Xu2, Han He2, Lei Gu1, Hui Wang1, Jie Chen2, Danjun Ma34*, Hu Zhou2* and Jing Zheng1* 1

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai, China, 200237

2

Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China, 201203 3

College of Mechanical Engineering, Dongguan University of Technology, Guangdong, China,

523808 4

Qingzi Biotechnology (Shenzhen) LLC., 4026 Shen Nan Middle Rd, Shenzhen, Guangdong,

China, 518039 KEYWORDS: Membrane proteins, enrichment, identification, mass spectrometry, sequence coverage ABSTRACT: Membrane proteins may act as transporters, receptors, enzymes and adhesionanchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of userfriendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture

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membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY >0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly-identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456. INTRODUCTION Broadly divided into integral membrane proteins and membrane-associated proteins, membrane proteins possess diverse cellular functions, including cell signal transduction, molecular transportation, protein secretion, and maintenance of cell homeostasis besides their basic roles in holding structural integrity of membranes. They may act as transporters, receptors, enzymes and adhesion-anchors1, accounting for nearly 70% of pharmaceutical drug targets2 due to their ligand binding domains. Although modern analytical methods and strategies have dramatically accelerated the characterization of structure, modification and interactions of membrane proteins, difficulties in efficient enrichment, extraction and solubilization still exist because of their relatively low abundance and poor solubility. Centrifugation-based methods enable separation of membrane proteins according to density, shape and size3. J. Schindler associated differential centrifugation with high-salt and high pH washes to separate membrane and organelle fraction, resulting in high enrichment of membrane proteins4. Different centrifugal forces have been applied for subcellular fractionation. Synaptosomal membrane fraction was isolated by centrifugation at 25,000×g5. Crude nuclear fraction and mitochondria can be obtained with 1,000×g and 15,000×g centrifugal forces, respectively6. Solubilization of membrane proteins is a critical process as lipid bilayer has to be disrupted to solubilize integral membrane proteins, while intrinsic structural and physical properties should be retained. This could be realized by organic solvents but more commonly by appropriate detergents via binding to the hydrophobic parts of the protein on one side and interacting with the aqueous parts on the other7. For instance, advantages of sodium dodecyl sulfate (SDS) in lysing membranes and solubilizing hydrophobic membrane proteins and sodium deoxycholate (SDC) in improving protein solubilization was highlighted in rat liver membrane proteomes8.


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Mass spectrometry (MS) has already been applied to analyze membrane proteins in terms of elucidating dynamic structure9, authenticating interactions among intact membrane proteinligand complexes10, demonstrating post-translational modifications, or quantifying proteins. However, efficiency of digestion could influence the proteome coverage particularly in bottomup proteomic workflows where digestion of protein into peptides with suitable m/z for MS analysis is essential. Waas et al11 found that addition of guanidine and acetonitrile improved sequence coverage of transmembrane (TM) proteins by improving digestion efficiency and specificity, and a new open-source software tool, PeptideEclispe, was simultaneously developed for specific assessment of TM domain sequence coverage. Additionally, detergents retained in protein solution may likewise suppress the signal and interfere with MS analysis12. Distler et al13 isolated the resident membranes to reduce sample complexity, and used liquid chromatography coupled with MS to simplify the digestion products, providing high coverage of proteins without involvement of detergents. Herein, we developed an extraction method with good repeatability and significant effectiveness for better identification of membrane proteins. The current method is a simplified version of

a reported one14. Membrane proteins extracting based on ultra-high speed

centrifugation requires specific centrifuges and continuous gradients of either sucrose or Percoll.Considering that not all research institutions are equipped with an ultra-high speed centrifuge with a maximum 900,000×g centrifugal force, we reduced the rotational speed to 35,000×g, offering a more convenient option without rigorous requirement on experimental equipment. Another advantage was its speediness. Almost 3 h-centrifugation was required in centrifugal proteomic reactor (CPR) for streamlined protein extraction (20 min at 11,000×g, 90 min at 100,000×g, 30 min at 541,000×g for two times)15, while 20 min at 35,000×g for three times were needed in our approach. The method was also able to identify higher proportion of membrane proteins, integral proteins and transmembrane proteins, and tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains, and successfully provided for better identification of membrane proteins with higher sequence coverage. EXPERIMENTAL PROCEDURES Cell Culture and Sample Preparation


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Mouse Neuro 2a (N2a) cells (American Type Culture Collection) were cultured in DMEM supplemented with high glucose, 200 mM glutamineand 10% FBS (Invitrogen, Carlsbad, CA) and maintained in a humidified incubator with 5% CO2 at 37 °C. For membrane sample preparation, 5 of 15 cm dishes cells at 80% confluence were harvested, washed and homogenized with high salt buffer and centrifuged at 35,000×g for 20 min subsequently. The resulted pellet was twice washed with Na2CO3 (pH11.3) to open vesicles and centrifuged at 35,000×g again for 20 min. Pellet was twice washed with 6 M Urea to dissociate membrane-associated proteins and centrifuged at 35,000×g for 20 min. The generated pellet was washed with Tris buffer and solubilized with SDT buffer (2% SDS (m/v), 100 mM DTT, 100 mM Tris, pH=7.6), and the ultimate supernatant was the membrane fraction. For total cell lysate preparation, a total of N2a cells were washed with ice-cold PBS buffer for three times and harvested in SDT lysis buffer. The lysate was homogenized by sonication and centrifuged at 15,000×g for 30 min and the acquired supernatant was the total cell lysate. The same membrane protein extracting method was applied to rat brain cortex tissues, and membrane proteins preparation based on Percoll density gradient according to a previously described method14 was also performed for comparison. Briefly, rat brain cortex tissue were prepared and fractionated by Percoll density gradient using 900,000×g centrifugation force (Himac CS150GX Micro Ultracentrifuge with a S140AT-0017 Rotor, Hitachi), and the resulted Fraction 9 & Fraction 10 was collected as the membrane protein fractions. LC-MS/MS An online liquid chromatography-tandem mass spectrometry (LC-MS/MS) setup consisting of an Easynano-LC system and a Q-Exactive mass spectrometer (Thermo, Bremen, Germany) equipped with a nanoelectrospray ion source was used for all LC-MS/MS experiments. The tryptic digested peptides were loaded on a 75 µm × 200 mm fused silica column packed in-house with 3-µm ReproSil-Pur C18 beads (Dr. Maisch GmbH, Ammerbuch, Germany) and separated with a 90-min gradient at a flow rate of 300 nL/min. Solvent A contained 100% H2O and 0.1% formic acid; Solvent B contained 100% acetonitrile and 0.1% formic acid. The gradient was: 25% B, 2 min; 5%-28% B, 74 min; 28%-90% B, 1 min; 90% B, 4 min; 90%-0% B, 1min; 0% B, 8 min. The mass spectrometry instrument parameters were: The temperature of the heated capillary was set at 320 °C and the source voltage was set at 2.4 kV; MS1 full scan resolution, 70,000 @ m/z 200; automatic gain control target, 3×106; maximum injection time, 30 ms. MS2 scan


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resolution 17,500 @ m/z 200; automatic gain control target, 1×105; maximum injection time, 100 ms. The precursor ions were fragmented by higher energy collisional dissociation (HCD) with a normalized collision energy of 27%. Data Analysis The MS data was analyzed via software MaxQuant16 (http://maxquant.org/, version 1.5.1.0 or 1.6.0.1). Carbamidomethyl (C) was set as a fixed modification, while oxidation (M, + 15.99492 Da) and protein N-term acetylation (+42.01056 Da) as variable modifications. The MS/MS spectra from Mouse Neuro 2a (N2a) cells and rat cortex were searched against the Mouse UniProt FASTA database (July 2016, containing 51418 entries) and Rat UniProt FASTA database (December 2017, containing 29971 entries) respectively, and Trypsin/P was selected as the digestive enzyme with two potential missed cleavages. The false discovery rate (FDR) for peptides and proteins was rigorously controlled 0, and this figure in total cell lysate was 495 (14.6%) and 302 (8.9%) (Fig. 2B). As for TM segments, 488 out of 2106 (23.1%) in membrane fraction, and 225 out of 3370 (6.7%) in total cell lysate had TMs ≥2 (Fig. 2C). It seemed that the extraction strategy tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains. GO Enrichment Analysis in Proteome - GO enrichment analysis was conducted to further assess the difference of proteins identified in membrane fraction and total cell lysate. Among 2111 and 3377 protein groups identified, 676 were found only in membrane fraction and 1942


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only in total cell lysate, with an overlap value of 1435 (Fig. S-2A). The barplots represented cellular component classification of unique proteins identified only in total cell lysate or membrane fraction. Membrane-related proteins were found to be significantly enriched in membrane fraction, while nuclear-related and cytoplasmic proteins were more enriched in total cell lysate. Cellular component classification of commonly-identified proteins in both membrane fraction and total cell lysate was also displayed in Fig. S3. All the proteins identified in two cell samples were then ranked according to abundance and were divided into three sections. The position of GO terms along the horizontal axis belonged to the respective protein abundance section. Different patterns in cellular components were observed (Fig. S-2B), and several membrane proteins with potential to be the biomarkers were identified only in membrane fraction. Good Repeatability and Improved Identification of Membrane Proteins - Correlation plots of peak intensity of all the identified peptides (Fig. S-5A) and peptides from the annotated membrane proteins by DAVID (Fig. S-5B), as well as overlap analysis of identified membrane proteins across all the LC-MS/MS runs (Fig. S-5C) were calculated to evaluate repeatability. Sequence coverage of membrane proteins identified in both two cell samples was plotted, and it was revealed that sequence coverage in four parallel sample loadings was higher in membrane fraction than that in total cell lysate (Fig. S-4A). Subsequently, sequence coverage acquired in four parallel sample loadings was averaged with X-axis representing that value of membrane fraction and Y-axis of total cell lysate (Fig. S-4B). Dots underneath the green line represented commonly-identified membrane proteins with higher sequence coverage in membrane fraction than in total cell lysate, and the percentage was 60.6%, which indicated the effectiveness of the extraction method. Additionally, sequence coverage of identified membrane proteins across all the LC-MS/MS runs showed good correlation (Fig. S-5D). To figure out why some membrane proteins have better sequence coverage in the total lysate than that in membrane fraction, the 332 and 606 membrane proteins with higher sequence coverage respectively in total lysate and membrane fraction were used to do a TMHMM analysis. We found that 11 out of 332 (3.3%) membrane proteins in total lysate had transmembrane domains ≥2, while 168 out of 606 (27.7%) membrane proteins in membrane fraction had transmembrane domains ≥2. Fisher’s exact test showed an odds ratio of 0.0895 with p-value 74% in these two membrane isolation methods, and were significantly higher than that of the total lysate (61%); and the percentage of transmembrane proteins (TMs>=2) in total identified proteins was >20% in these two membrane isolation methods, and were significantly higher than that of the total lysate (11%). Both methods demonstrated very similar capability for enrichment and identification of membrane proteins, and outperformed the total lysate of rat cortex without any membrane purification. Hierarchical cluster analysis was performed on the intensities of membrane proteins from the total lysate and the two membrane isolation methods to figure out the identification preference of these different methods, resulting in five distinct protein clusters (Fig. S-6D). For examples, extracellular vesicle proteins showed higher protein intensity in the total lysate, while the intensities of the proteins involved in intrinsic component of membrane and plasma membrane represented similar patterns between our method and the reported method and were higher than the total lysate. Figure S-7 showed the comparison of the physiochemical properties of the identified proteins from different methods. As shown in Fig. S7A-F, the two membrane isolation methods outperformed the total lysate for membrane identification in grand average hydrophobicity index (GRAVY>0) and transmembrane domains (TMs>=2). The Venn diagram showed very high overlap in the total identified proteins (Fig. S-7G) and the identified membrane proteins (Fig. S-7H) between the two membrane isolation methods. Moreover, the identified proteins by the reported and our method showed similar physiochemical characteristics


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in grand average hydrophobicity (GRAVY) index, molecular weight (MW) (Fig. S-7I-K), transmembrane domains (TMs) and isoelectric point (pI) (Fig.S-7L-N). Although we performed our experiments with reduced centrifugal force (35,000×g), the efficacy of membrane protein enrichment and identification of our method is very similar to the reported method.

CONCLUSION Application of detergent and centrifugation has been a frequently-used strategy for extraction of membrane proteins. Detergents are surfactants commonly with a hydrophilic polar head group and a hydrophobic non-polar tail group being able to decrease the interfacial tension between two immiscible liquids. The extraction method combining centrifugation and detergent along with LC-MS/MS developed herein was able to enhance identification of membrane proteins. It had advantages of high efficiency and speediness with minimal residue of detergent and without rigorous requirement on experimental equipment, resulting in identification of higher proportion of membrane proteins, integral proteins and transmembrane proteins, and several with potential to be the biomarkers were found only in membrane fraction. It also tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains, which may further promote the research on structure and function of transmembrane and hydrophobic membrane proteins. It successfully provided for better identification of membrane proteins via improving sequence coverage. This method was also used for membrane protein isolation from the rat brain cortex tissue, and demonstrate good membrane enrichment capability as well as the reported method. The simplified extraction method may offer some reference for fast, convenient, and effective enrichment and identification of membrane proteins. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +86-21-50806706. *E-mail: [email protected] Phone: +86-21-64250608


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*E-mail: [email protected] Phone: +86- 769-22861122. Author Contributions #

These authors contributed equally.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported by Grant No. 2017YFC1700204 of the National Key Research and Development Program from the Ministry of Science and Technology of China, by Grant No. 21375138 and 31570830 from National Natural Science Foundation of China, by the Strategic Priority Research Program of the Chinese Academy of Science, “Personalized MedicinesMolecular Signature-based Drug Discovery and Development” (No. XDA12030203), by the Innovation Project of Instrument and Equipment Function Development (Grant No. YZ201542 etc.), and the Bureau of Goods, Chinese Academy of Sciences and by Key Laboratory of Receptor Research, Chinese Academy of Sciences. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE18 partner repository with the dataset identifier PXD008456. ABBREVIATIONS MS, mass spectrometry; N2a, Mouse Neuro 2a; FASP, Filter-Aided Sample Preparation; IAA, iodoacetamide; ABC, ammonium bicarbonate; LC-MS/MS, liquid chromatography-tandem mass spectrometry; FDR, false discovery rate; GRAVY, grand average hydrophobicity; pI, isoelectric point; TMs, transmembrane domains REFERENCES

(1) Savas, J. N.; Stein, B. D.; Wu, C. C.; Yates, J. R., 3rd. Trends in biochemical sciences 2011, 36, 388-396. (2) Wu, C. C.; Yates, J. R., 3rd. Nature biotechnology 2003, 21, 262-267. (3) Mathias, R. A.; Chen, Y. S.; Kapp, E. A.; Greening, D. W.; Mathivanan, S.; Simpson, R. J. Methods (San Diego, Calif.) 2011, 54, 396-406. (4) Schindler, J.; Jung, S.; Niedner-Schatteburg, G.; Friauf, E.; Nothwang, H. G. Journal of neural transmission (Vienna, Austria : 1996) 2006, 113, 995-1013.


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(5) Bermejo, M. K.; Milenkovic, M.; Salahpour, A.; Ramsey, A. J. Journal of visualized experiments : JoVE 2014, e51896. (6) Jiang, X. S.; Zhou, H.; Zhang, L.; Sheng, Q. H.; Li, S. J.; Li, L.; Hao, P.; Li, Y. X.; Xia, Q. C.; Wu, J. R.; Zeng, R. Molecular & cellular proteomics : MCP 2004, 3, 441-455. (7) Stansfeld, P. J.; Goose, J. E.; Caffrey, M.; Carpenter, E. P.; Parker, J. L.; Newstead, S.; Sansom, M. S. Structure (London, England : 1993) 2015, 23, 1350-1361. (8) Lin, Y.; Liu, H.; Liu, Z.; Liu, Y.; He, Q.; Chen, P.; Wang, X.; Liang, S. Analytical biochemistry 2013, 432, 41-48. (9) Konijnenberg, A.; van Dyck, J. F.; Kailing, L. L.; Sobott, F. Biological chemistry 2015, 396, 991-1002. (10) Gault, J.; Donlan, J. A.; Liko, I.; Hopper, J. T.; Gupta, K.; Housden, N. G.; Struwe, W. B.; Marty, M. T.; Mize, T.; Bechara, C.; Zhu, Y.; Wu, B.; Kleanthous, C.; Belov, M.; Damoc, E.; Makarov, A.; Robinson, C. V. Nature methods 2016, 13, 333-336. (11) Waas, M.; Bhattacharya, S.; Chuppa, S.; Wu, X.; Jensen, D. R.; Omasits, U.; Wollscheid, B.; Volkman, B. F.; Noon, K. R.; Gundry, R. L. Analytical chemistry 2014, 86, 1551-1559. (12) Zhang, N.; Li, L. Analytical chemistry 2002, 74, 1729-1736. (13) Distler, A. M.; Kerner, J.; Peterman, S. M.; Hoppel, C. L. Analytical biochemistry 2006, 356, 18-29. (14) Nielsen, P. A.; Olsen, J. V.; Podtelejnikov, A. V.; Andersen, J. R.; Mann, M.; Wisniewski, J. R. Molecular & cellular proteomics : MCP 2005, 4, 402-408. (15) Zhou, H.; Wang, F.; Wang, Y.; Ning, Z.; Hou, W.; Wright, T. G.; Sundaram, M.; Zhong, S.; Yao, Z.; Figeys, D. Molecular & cellular proteomics : MCP 2011, 10, O111.008425. (16) Cox, J.; Matic, I.; Hilger, M.; Nagaraj, N.; Selbach, M.; Olsen, J. V.; Mann, M. Nature protocols 2009, 4, 698-705. (17) Cox, J.; Hein, M. Y.; Luber, C. A.; Paron, I.; Nagaraj, N.; Mann, M. Molecular & cellular proteomics : MCP 2014, 13, 2513-2526. (18) Cox, J.; Neuhauser, N.; Michalski, A.; Scheltema, R. A.; Olsen, J. V.; Mann, M. Journal of proteome research 2011, 10, 1794-1805. (19) Vizcaino, J. A.; Csordas, A.; del-Toro, N.; Dianes, J. A.; Griss, J.; Lavidas, I.; Mayer, G.; Perez-Riverol, Y.; Reisinger, F.; Ternent, T.; Xu, Q. W.; Wang, R.; Hermjakob, H. Nucleic acids research 2016, 44, D447-456.

Figure Legend Table 1. Peptides, unique peptides, protein groups and predicted membrane proteins identified from membrane fraction and total cell lysate. Samples

N2A-Membrane

N2A-Total

Unique Peptides

13478

21683

Protein Groups

2111

3377


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Membrane Proteins

1618(76.6%)

1405(41.6%)

Integral to Membrane

1016(48.1%)

555(16.4%)

Proteins with TMs

858(40.6%)

456(13.5%)

Figure 1. Work flow of extraction for membrane proteins and four parallel runs of membrane fraction and total cell lysate. Work flow of extraction for membrane proteins (A). Membrane proteins and other proteins identified in four parallel runs of two samples (B). Figure 2. Biological process classifications and characteristics of identified proteins. Cellular component classification of proteins identified in membrane fraction and total cell lysate (A). Identified proteins were assigned to DAVID GO terms for cellular component, and selected GO terms were assessed by Fisher’s exact test with “*” representing P-value

Enhancing Membrane Protein Identification Using a Simplified

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