基于超高效液相色谱-质谱法的肽段分析中非特异性吸附评估及通用型最小化策略

doi:10.3724/SP.J.1123.2021.12012

摘要/Abstract

摘要：

蛋白质组学技术在多肽和蛋白质类新型治疗药物的开发、临床诊断生物标志物的深入发掘中应用广泛。然而,多肽和蛋白质类大分子的非特异性吸附性质给分析方法的开发带来极大挑战,亟须一种通用型的策略去评估和降低非特异吸附对超高效液相色谱-质谱(UPLC-MS)大分子检测造成的负面影响。研究以牛血清白蛋白(BSA)为模型,探讨其酶解后多肽组理化性质与吸附程度之间的相关性;根据肽段的响应和吸附程度设计分级策略;针对高响应、强吸附的Class Ⅱ类肽段,从样品制备中离心管、进样瓶的选择,乃至液相色谱系统中色谱柱固定相、流速、梯度、柱温、洗针液的选择全过程设计试验,探讨非特异吸附的影响因素及其通用型最小化策略。结果显示,肽段的被吸附程度与其理化参数HPLC指数(HPLC Index)、肽段长度等显著相关(p<0.05),但仅凭上述参数仅能解释30%肽段的被吸附程度。改性的聚丙烯材料可使肽段溶液在储存或前处理过程中获得较高的回收率(24 h内回收率大于80%)。在对液相色谱条件的考察和优化过程中发现,C₈填料的色谱柱、高流速、缓梯度以及强洗针液,可使残留量降至最低(降低为原来的1/150)。柱温对残留的影响在肽段间存在较大个体差异,需要对不同的肽段具体分析以得到较少量的残留。研究以详实的数据考察并最小化模型肽段组在分析过程中的非特异吸附,提示了蛋白质类大分子药物分析方法建立中应重点关注的影响因素及其有效的解决方案。

关键词: 超高效液相色谱-质谱, 蛋白质组学, 非特异性吸附, 定量, 残留, 多肽

Abstract:

Proteomics technology is being increasingly used in the development of novel therapeutic peptides and protein drugs, and also in the intensive search for clinical biomacromolecule diagnostic biomarkers. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) is a rapid method to analyze peptides and proteins in low abundance. However, the nonspecific adsorption properties of peptides may lead to the loss or interference of the analytes throughout the analytical process. This unique nonspecific adsorption property is the main reason for the false negative and false positive results obtained through quantification, as well as for the poor precision, accuracy, linear range, and sensitivity, all of which impose significant challenges in the development of analytical methods.
Accordingly, a general strategy was established to evaluate and reduce the negative impact of the nonspecific adsorption of peptides on UPLC-MS analysis. In this study, bovine serum albumin (BSA) was used as a model protein to explore the correlation between the physicochemical properties of 50 peptides obtained by the enzymatic digestion of BSA, as well as the degree of nonspecific adsorption. First, these peptides were classified into four categories according to their response and the degree of adsorption in the pretreatment containers and LC system. Next, the factors influencing the adsorption of 12 Class Ⅱ peptides, which were highly responsive and susceptible to adsorption, were systematically studied in terms of several aspects, including: (1) time-dependent adsorption on centrifuge tubes of three kinds (Protein-LoBind polypropylene tube and two types of polypropylene tubes); (2) time-dependent adsorption on sample vials of three kinds (Protein-LoBind polypropylene vial, polypropylene vial, and glass vial); (3) carryovers on chromatographic columns with six different stationary phases (Polar C₁₈, Cortecs C₁₈⁺, PFP, BEH C₁₈, CSH C₁₈, and BEH C₈); (4) carryovers at different chromatographic gradients (2%B-30%B, 2%B-40%B, 2%B-50%B, and 2%B-60%B within 3 min), flow rates (0.2, 0.3, and 0.4 mL/min), and column temperatures (30, 40, 50, and 60 ℃); and (5) carryovers using different washing needle solutions (0.2% formic acid in 10% acetonitrile and 0.2% formic acid in 90% acetonitrile).
The results showed that parameters such as the HPLC index and amino acid length of peptides were significantly correlated with their degree of adsorption (p<0.05), However, the above parameters can only explain the adsorption degree of 30% of the peptides. The use of the modified polypropylene material resulted in higher recovery (recovery rate>80% within 24 h) of the peptide solution during storage or pretreatment. During protein/peptide pretreatment and storage, good overall recoveries (recovery rate>80% within 24 h) were obtained using centrifuge tubes and sample vials made of the modified polypropylene material. Analysis and optimization of the LC conditions revealed that the use of the C₈ chromatographic column, a high flow rate (0.4 mL/min), slow gradient (2%B-50%B within 3 min), and strong needle solution (0.2% formic acid in 90% acetonitrile) could minimize the carryover. However, the effect of the column temperature on the carryover varied considerably from peptide to peptide, and hence, requires further analysis for specific peptides. The combined optimization of the above experimental conditions resulted in minimal (approximately 1/150) or no adsorption of the Class Ⅱ peptides that were susceptible to adsorption in the analytical process.
In this study, a workflow was designed to standardize the procedures for evaluating and reducing peptide adsorption. Detailed data were collected to elucidate the key risk factors and corresponding general mechanism of nonspecific adsorption throughout the analysis. Thus, this study serves as a reference for the development of analytical methods for peptides and proteins with different physicochemical properties. In future work, the risk factors should be assessed during the development and validation of protein-based macromolecular analysis methods. In conclusion, it is important to implement adequate and appropriate measures to ensure risk elimination or minimization.

Key words: ultra-performance liquid chromatography-mass spectrometry (UPLC-MS), proteomics, nonspecific adsorption, quantification, carryover, peptides

中图分类号:

O658

张莹, 杨静, 马跃新, 曹玲, 黄青. 基于超高效液相色谱-质谱法的肽段分析中非特异性吸附评估及通用型最小化策略[J]. 色谱, 2022, 40(7): 616-624.

ZHANG Ying, YANG Jing, MA Yuexin, CAO Ling, HUANG Qing. Nonspecific adsorption evaluation and general minimization strategy in peptide analysis based on ultra-performance liquid chromatography-mass spectrometry[J]. Chinese Journal of Chromatography, 2022, 40(7): 616-624.

图/表 8

表 1 BSA酶解后Class Ⅱ类肽段的离子对信息及MRM参数

Table 1 Ion pair information and MRM parameters for Class Ⅱ peptides after enzymatic digestion of bovine serum albumin (BSA) hydrolysis

Number	Peptide	[M+H]⁺	Q1 (m/z)	Q3 (m/z)	Declustering potential/V	Collision energy/eV
P1	DAFLGSFLYEYSR	1567.743	784.38	1121.53	49.9	35.4
P2	GLVLIAFSQYLQQCPFDEHVK	2492.264	831.43	383.24	47.8	35.9
P3	ECCHGDLLECADDR	1749.662	583.89	765.28	58.7	28.0
P4	TVMENFVAFVDK	1399.693	700.35	1199.58	53.6	29.3
P5	LFTFHADICTLPDTEK	1907.921	636.65	589.28	56.4	30.6
P6	DDPHACYSTVFDK	1554.653	518.89	609.32	61.6	24.9
P7	MPCTEDYLSLILNR	1724.835	862.92	715.45	46.5	47.3
P8	LVNELTEFAK	1163.631	582.32	951.48	58.8	27.5
P9	SLHTLFGDELCK	1419.694	473.90	721.32	63.6	22.7
P10	VPQVSTPTLVEVSR	1511.843	756.43	900.52	51.1	42.1
P11	HLVDEPQNLIK	1305.716	653.36	1055.57	55.7	35.0
P12	LGEYGFQNALIVR	1479.795	740.40	1017.58	51.8	43.3

图 1 (a)肽段的分级策略及(b)非特异性吸附因素考察和优化流程

Fig. 1 (a)Grading strategy of peptides (b) nonspecific adsorption factor examination and optimization process

图 2 肽段溶液在不同样品容器中放置24 h后的回收率

Fig. 2 Peptides recoveries after 24 h storage in different sample containers Recoveries of Class Ⅱ peptide solutions at three levels (50, 500, 5000 ng/mL) stored in different centrifuge tubes (Fig. 2a) and sample vials (Fig. 2b) after 24 h. PP-LB(LB): lowbind polypropylene tubes or vials; PP-A(A) and PP-B(B): polypropylene tubes from company A and B; PP: polypropylene vials; Glass/G: glass vials; Average: average recovery, %; SD: standard deviation; Min: minimum; Count: the number of peptides solutions with recoveries less than 80%.

图 3 不同样品容器中低浓度肽段P7溶液的回收率变化趋势(n=3)

Fig. 3 Trends of the recovery of low concentrations of peptide P7 solutions in different sample containers (n=3)

图 4 (a)用于评估色谱柱上残留量的梯度洗脱程序及(b)残留肽段的典型色谱图

Fig. 4 (a) Gradient program for column carryover evaluation and (b) typical chromatogram for the carryover peptides

表 2 色谱柱参数

Table 2 Column characteristics

Stationary phase	Pore size/nm	Particle size/μm	Diameter/ mm	Length/ mm
Polar C₁₈	10	2.6	2.1	100
Cortecs C₁₈⁺	9	1.6	2.1	100
PFP	10	1.7	2.1	100
BEH C₁₈	30	1.7	2.1	100
CSH C₁₈	13	1.7	2.1	100
BEH C₈	13	1.7	2.1	100

图 5 固定相类型对肽段在色谱柱上残留的影响

Fig. 5 Effect of stationary phase types on peptide carryover on the chromatographic columns BEH-C8-opti is the carryover percentage obtained after optimization.

图 6 不同色谱条件对肽段溶液在色谱柱上肽段残留量的影响(n=3)

Fig. 6 Effect of different chromatographic conditions on the carryover of peptides on the columns (n=3) a. flow rate; b. gradient; c. percentage of formic acid (FA) in mobile phase; d. column temperature. G1-G4 were the gradients of 2-6 min: 2%B-30%B, 2%B-40%B, 2%B-50%B, and 2%B-60%B.

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