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1 生物工程学报 Chinese Journal of Biotechnology DOI: /j.cjb 王丹琪等 / 反相 C18 固相萃取小柱用于蛋白质组分析中少量样品预处理 November 25, 2015, 31(11): Chin J Biotech, All rights reserved 生物技术与方法 1643 C18 王丹琪, 孙伟, 邹丽莉, 王曌 ,,,. C18., 2015, 31(11): Wang DQ, Sun W, Zou LL, et al. Application of reverse phase C18 tip in pretreatment of small amount complex proteomic samples. Chin J Biotech, 2015, 31(11): 摘要 : 针对少量且复杂蛋白质组样品, 开发一种耗时短 操作简便的分离方法 以人的脑海马组织蛋白样品为研究对象, 采用反相 C18 固相萃取小柱, 采用不同乙腈浓度对酶切后样品进行梯度洗脱, 与反相高效液相色谱法对比分离效果 通过比较不同乙腈梯度洗脱方案所鉴定到的谱图数 非冗余多肽数 蛋白数和各洗脱组分间重复率分析, 确定一种样品量少 简单易行 分离效果好的实验方案 虽然反相 C18 固相萃取小柱法鉴定蛋白总数占高效液相色谱法的 85.5%, 但其操作简单, 耗时少 通过 4 种不同乙腈浓度方案比较, 确定乙腈洗脱浓度优化为 5% 15% 20% 和 90% 时, 分离 30 μg 人的海马多肽混合物可以得到较优的分离效果 其蛋白鉴定数为反相液相色谱法的 67.0%, 且重复性良好 该结果证实反相 C18 固相萃取小柱分离效果比反相高效液相色谱法稍差, 但此方法可以分离少量复杂蛋白质组样品 该方法分离样品充分, 操作易行, 耗时短, 是进行蛋白质组学分析中少量样品的一个简便预处理方法 : 反相色谱,C18 固相萃取小柱, 乙腈浓度, 多肽分离, 蛋白鉴定 Received: December 15, 2014; Accepted: January 21, 2015 Supported by: National Basic Research Program of China (973 Program) (Nos. 2013CB530805, 2014CBA02005), Key Basic Research Program of the Ministry of Science and Technology of China (No. 2013FY114100). Corresponding author: Wei Sun. Tel: ; Fax: ; sunwei1018@sina.com (973 ) (Nos. 2013CB530805, 2014CBA02005) (No. 2013FY114100)

2 1644 ISSN CN /Q Chin J Biotech November 25, 2015 Vol.31 No.11 Application of reverse phase C18 tip in pretreatment of small amount complex proteomic samples Danqi Wang, Wei Sun, Lili Zou, and Zhao Wang Core Facility of Instrument, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing , China Abstract: To develop a timesaving and easy operating Reverse Phase (RP) chromatography method, we adopted Thermo Pierce RP C18 Tip to separate small amount hippocampus peptide mixtures and to compare with high performance liquid chromatography (HPLC). According to the separation performance of 4 ACN gradient optimization methods, we determined the best ACN concentration gradient. The results showed that, the experiment took only 10 min by separating with eight ACN concentration gradient, which accounted 1/4 for HPLC. But as for the identified proteins, RP C18 Tip accounted 85.5% for HPLC. ACN gradient of 5%, 15%, 20% and 90% had best repeatability (P=0.429) and result for separating 30 μg peptides. This method is easy to operate, timesaving and has low cost. It could be used into pretreatment of small amount complex proteomic samples. Keywords: reverse phase chromatography, C18 tip, ACN concentration, peptide separation, protein identification (Bottom-up) [1-4] [1] ph ph [5-6] C18 Thermo Pierce C18 1 材料与方法 1.1 试剂与仪器 (Dithiothreitol DTT) (Iodoacetamide IAA) (Tris (hydroxymethyl)aminomethane Tris) Ⅱ Sigma Promega Merk C18 ( 80 μg 100 μl Thermo Pierce ) (Waters ) 1.2 样品制备 (7 mol/l 100 mmol/l

3 王丹琪等 / 反相 C18 固相萃取小柱用于蛋白质组分析中少量样品预处理 mmol/l DTT 50 mmol/l Tris) (Bradford ) mg [7] 20 mmol/l DTT 37 1 h 50 mmol/l IAA 45 min r/min 10 min 10 kda = min 37 Waters C18 SPD 0.1% BCA 1.3 方法 ph 10.0 ph (V/V) 2% 5% 10% 15% 20% 25% 30% 90% μl 50% (V/V) C18 50 μl 2% (V/V) C18 20 μl 2% (V/V) 15 C18 20 μl 2% (V/V) SPD LC-MS/MS C μl 0.1% C18 (75 μm 100 mm 3 μm) 5% 30% 0.1% 99.9% (ph 2.0) 0.3 μl/min LTQ Orbitrap amu (1 ) 20 ( 3 amu 35% 1 min) Mascot ( ) Scaffold ( 4.4.3) Swissprot ( 1% Da Carbamidomethy (C) 2 结果与分析 2.1 八个乙腈梯度多肽预分离 C μg ( ) 8 (2% 5% 10% 15% 20% 25% 30% 90% (V/V)) (ph 10.0) 1A % 10% C18 (5% 30%) cjb@im.ac.cn

4 1646 ISSN CN /Q Chin J Biotech November 25, 2015 Vol.31 No.11 表 1 反相 C18 固相萃取小柱及反相高效液相色谱分离鉴定谱图数 非冗余肽段数及蛋白数 Table 1 Spectrums, unique peptides and proteins identified in reverse phase (RP) C18 Tip and high performance liquid chromatography (HPLC) Reverse phase (RP) C18 Tip High performance liquid chromatography Fraction Spectra Unique peptide Protein Fraction Spectra Unique peptide Protein 2% % % % % % % % Total Total (ph 10.0) 8 1B 8 C18 ( C %) 10 min 2.2 梯度优化 ) 2% 20% 5% 25% 10% 30% 15% 90% 2) 90% 8 ( 2) 3 2% 10% 20% 90% 5% 15% 25% 90% 5% 15% 20% 90% % 表 2 反相 C18 固相萃取小柱各组分间多肽重复率 Table 2 Peptide repetition rate between each group in RP C18 Tip Fraction 2% 5% 10% 15% 20% 25% 30% 5% 31.2% 10% 28.9% 29.1% 15% 21.6% 17.5% 26.8% 20% 15.2% 10.0% 14.5% 23.0% 25% 11.5% 7.2% 9.9% 15.6% 27.0% 30% 13.1% 9.1% 11.8% 15.6% 23.2% 32.0% 90% 14.1% 10.0% 12.8% 16.4% 21.7% 26.0% 32.0%

5 王丹琪等 / 反相 C18 固相萃取小柱用于蛋白质组分析中少量样品预处理 1647 表 3 反相 C18 固相萃取小柱分离优化乙腈浓度 (%) 鉴定谱图数 非冗余肽段数及蛋白数 Table 3 Spectrums, unique peptides and proteins identified in optimized acetonitrile (ACN)% of RP C18 Tip Fraction 2% 20% 5% 25% 10% 30% 15% 90% Total Spectra Unique peptide Protein Fraction 2% 10% 20% 90% Total Spectra Unique peptide Protein Fraction 5% 15% 20% 90% Total Spectra Unique peptide Protein Fraction 5% 15% 25% 90% Total Spectra Unique peptide Protein % 4 (5% 15% 20% 90%) % 85.2% 78.3% % 4 4 ( n / (n=1, 2, 3, 4)) ( 1) % 56.0% 60.7% 61.7% 4 4 (5% 15% 20% 90%) 图 1 四种实验方案分离的多肽在 1 4 个组分中鉴定数量百分比 Fig. 1 Pie chart depict percentages of peptide identifications in four experiment approaches associated with one to four fractions. C18 5% 15% 20% 90% 2.3 重复性分析 C18 (5% 15% 20% 90%) 2A (5%) (15%) (20%) 972 (90%) 12% 1.2% % P (>0.05) 2B 60.3% (>50%) % [8] cjb@im.ac.cn

6 1648 ISSN CN /Q Chin J Biotech November 25, 2015 Vol.31 No.11 [5,11-12] ph ph [5,13] [13,16-17] C18 ph % C18 C18 85% μg 4 图 2 反相 C18 固相萃取小柱乙腈分离梯度优化后两次重复实验鉴定蛋白数 (A) 及蛋白重复率 (B) Fig. 2 Histogram distribution of proteins identified (A) and protein repetitive rate (B) in two optimized RP C18 Tip. 3 讨论 [5,9-10] [11-15] (5% 15% 20% 90% (V/V)) 61.70% % % 85.2% 78.3% 67.0% Thermo Pierce

7 王丹琪等 / 反相 C18 固相萃取小柱用于蛋白质组分析中少量样品预处理 1649 C18 C18 C18 4 [18-19] REFERENCES [1] Zhou H, Dai J, Sheng QH, et al. A fully automated 2-D LC-MS method utilizing online continuous ph and RP gradients for global proteome analysis. Electrophoresis, 2007, 28(23): [2] Dai J, Jin WH, Sheng QH, et al. Protein phosphorylation and expression profiling by Yin-Yang multidimensional liquid chromatography (Yin-yang MDLC) mass spectrometry. J Proteome Res, 2007, 6(1): [3] Zanivan S, Gnad F, Wickström SA, et al. Solid tumor proteome and phosphoproteome analysis by high resolution mass spectrometry. J Proteome Res, 2008, 7(12): [4] Qian WJ, Jacobs JM, Camp DG, et al. Comparative proteome analyses of human plasma following in vivo lipopolysaccharide administration using multidimensional separations coupled with tandem mass spectrometry. Proteomics, 2005, 5(2): [5] Wang YX, Yang F, Gritsenko MA, et al. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics, 2011, 11(10): [6] Phillips HL, Williamson JC, van Elburg KA, et al. Shotgun proteome analysis utilising mixed mode (reversed phase-anion exchange chromatography) in conjunction with reversed phase liquid chromatography mass spectrometry analysis. Proteomics, 2010, 10(16): [7] Wiśniewski JR, Zougman A, Nagaraj N, et al. Universal sample preparation method for proteome analysis. Nat Methods, 2009, 6(5): [8] Slebos RJC, Brock JWC, Winters NF, et al. Evaluation of strong cation exchange versus isoelectric focusing of peptides for multidimensional liquid chromatography-tandem mass spectrometry. J Proteome Res, 2008, 7(12): [9] Zhou F, Sikorski TW, Ficarro SB, et al. Online nanoflow reversed phase-strong anion exchangereversed phase liquid chromatography-tandem mass spectrometry platform for efficient and in-depth proteome sequence analysis of complex organisms. Anal Chem, 2011, 83(18): [10] Wang L, Zhou LL, Qian XH, et al. Optimization of conditions for the separation of peptide mixtures using strong cation exchange chromatography. Chin J Chromatogr, 2010, 28(4): (in Chinese).,,,.., 2010, 28(4): [11] Nakamura T, Kuromitsu J, Oda Y. Evaluation of comprehensive multidimensional separations using reversed-phase, reversed-phase liquid chromatography/mass spectrometry for shotgun proteomics. J Proteome Res, 2008, 7(3): [12] Delmotte N, Lasaosa M, Tholey A, et al. Two-dimensional reversed-phase x ion-pair reversed-phase HPLC: an alternative approach to high-resolution peptide separation for shotgun proteome analysis. J Proteome Res, 2007, 6(11): [13] Wang H, Hanash S. Multi-dimensional liquid phase based separations in proteomics. J Chromatogr B Analyt Technol Biomed Life Sci, 2003, 787(1): [14] Di Pala S, Hennrich ML, Heck AJ, et al. Recent advances in peptide separation by multidimensional cjb@im.ac.cn

8 1650 ISSN CN /Q Chin J Biotech November 25, 2015 Vol.31 No.11 liquid chromatography for proteome analysis. J Proteomics, 2012, 75(13): [15] Hong GF, Gao MX, Yan GQ, et al. Optimization of two-dimensional high performance liquid chromatographic columns for highly efficient separation of intact proteins. Chin J Chromatogr, 2010, 28(2): (in Chinese).,,,.., 2010, 28(2): [16] Sandra K, Moshir M, Dhondt F, et al. Highly efficient peptide separations in proteomics. Part 2: bi-and multidimensional liquid-based separation techniques. J Chromatogr B Analyt Technol Biomed Life Sci, 2009, 877(11/12): [17] Donato P, Cacciola F, Sommella E, et al. Online comprehensive RPLC RPLC with mass spectrometry detection for the analysis of proteome samples. Anal Chem, 2011, 83(7): [18] Wiśniewski JR, Zougman A, Mann M. Combination of FASP and StageTip-based fractionation allows in-depth analysis of the hippocampal membrane proteome. J Proteome Res, 2009, 8(12): [19] Dai J, Wang LS, Wu YB, et al. Fully automatic separation and identification of phosphopeptides by continuous ph-gradient anion exchange online coupled with reversed-phase liquid chromatography mass spectrometry. J Proteome Res, 2009, 8(1): ( 本文责编郝丽芳 )