Posted in: information

Nonspecific adsorption evaluation and universal minimization strategy in peptide analysis based on ultra high performance liquid chromatography mass spectrometry

In recent years, proteomics technology has been widely used in the vigorous development of new therapeutic drugs of peptides and proteins and the in-depth exploration of new clinical macromolecular biomarkers. The iteration of application mode puts forward higher requirements for the analysis technology of biological macromolecules.

Bottom up proteomics technology based on protein characteristic peptide detection is an existing researchProtein qualitative and quantitative methods with high sensitivity and resolution.The development of biological analysis methods for polypeptides is extremely challenging. In addition to the required low detection limit, the non-specific adsorption properties of polypeptides make it very easy to adsorb on the surface of materials in contact, which will lead to the loss or interference of substances to be measured in the whole process of analysis, and introduce huge risks to qualitative and quantitative analysis.

For example, in the mass spectrometry database search of proteomics research, even the loss or residue of micro peptides in the system may lead to false positive or false negative results. In the development of highly sensitive peptide quantitative methods, the non-specific adsorption of peptide segments has a negative impact on the linearity, accuracy and precision of quantitative analysis. The adsorption properties of low concentration peptide solutions will be more obvious, in the form of non-linearity of the standard curve, which eventually leads to the unnecessary increase of the limit of quantitation and poor repeatability of the method.

Some studies have explained this adsorption behavior at the molecular level, but little is known about its potential mechanism and interaction. Based on molecular dynamics simulation, eeltink et al. Proposed a three-phase molecular mechanism to explain the principle of peptide adsorption from solution to strongly interacting uncharged stationary phase. Kristensen et al. Studied the effect of the sample container on the adsorption of cationic peptides, when 1 μ After storing mol/l peptide solution in borosilicate or polypropylene bottle for 1 h, the recovery rate of peptide segment is only 10%- 20%. There are also studies to reduce this kind of adsorption by adding organic reagent, acid/alkaline solution, surfactant, adsorption competitor or adjusting the composition of mobile phase in the solvent. Most of these research papers study a specific group of peptides and/or surface materials, but they do not give the laws that can be used to predict the adsorption characteristics of peptides, nor do they give a general method to solve the adsorption.

In this study, bovine serum albumin (BSA) was selected as a model protein, and its enzymatically hydrolyzed peptides were used as”typical” polypeptide samples containing hydrophilic and hydrophobic peptides. Firstly, the correlation between the physical and chemical parameters of common peptides and the nonspecific adsorption degree of the above polypeptides was analyzed by ultra-high performance liquid chromatography high resolution mass spectrometry (uplc-hrms). Then, based on ultra-high performance liquid chromatography triple quadrupole mass spectrometry (uplc-qqq-ms/ms), an evaluation method for the degree of adsorption of strongly adsorbed peptides was established. From sample preparation to analysis, a whole process experimental design was established to investigate the effects of different materials’ preparation and storage consumables on peptide adsorption, as well as the effects of different chromatographic conditions on peptide residue. Finally, a general strategy to reduce non-specific adsorption in the whole process of peptide analysis was proposed.

01Sample preparation method

Dissolve 10 mg BSA in 10 ml water to prepare 1 mg/ml protein stock solution, and further dilute it with water to 100 μ G/ml working solution. Take 200 μ L the above working solution is put in a protein low adsorption centrifuge tube; Join 65 μ L 500 mmol/l ammonium bicarbonate and 60 μ L 50 mmol/l dithiothreitol, heated in a 60 ℃ water bath for 60 minutes to reduce protein; Cool to room temperature and add 120 μ L 50 mmol/l iodoacetamide, react in the dark for 30 minutes for alkylation; Add 100 μ G/ml trypsin 5 μ 50. Enzymolysis in 37 ℃ water bath for 8 hours, add formic acid for 20 minutes μ L terminate the reaction. After centrifuging 12000 g for 15 min, take 200 μ L supernatant is placed in the injection bottle with low protein adsorption as the mixed peptide solution to be tested.

02Parameters of ultra high performance liquid chromatography high resolution mass spectrometry method

Chromatographic conditions:the chromatographic column adopts waters acquisition premier peptide CSH C18 (100 mm × 2.1 mm, 1.7 μ m); The column temperature is 40 ℃; The flow rate is 0.25 ml/min; Mobile phase A and B are 0.1%formic acid aqueous solution and 0.1%formic acid acetonitrile solution respectively. Elution gradient is 0-1 min, 1%B; 1~13 min, 1%B~40%B; 13~13.1 min, 40%B~90%B; 13.1~16 min, 90%B; 16~16.1 min, 90%B~1%B; 16.1~20 min, 1%B。 Injector temperature 10 ℃; Injection volume 5 μ L。

Mass spectrometry conditions:capillary voltage 3 kV, conical hole voltage 30 V, ion source temperature 120 ℃, desolvent gas temperature 450 ℃, conical hole gas flow rate 25 l/h, desolvent gas flow rate 800 l/h. Electrospray ionization (ESI) source, determination in positive ion mode, MSE mode acquisition, scanning range m/z 50~2000; During data collection, leucine enkephalin correction solution is used for real-time quality correction to ensure the accuracy and repeatability of the collected mass number. The collected data is processed by Unifi software.

03Determination of relative residues and peptide classification strategy

After the above mixed peptide solution is collected under the above conditions and analyzed by Unifi software, the actual peptide composition of the peptide group after BSA enzymatic hydrolysis and the response value of each peptide area (test solution) can be obtained.

After injecting the above test solution, inject three needles of blank solvent continuously. The sum of the corresponding peptide responses detected in the three needles of blank solvent area (blank 1+blank 2+blank 3) is calculated as the total residue of the peptide. The relative residue of the peptide is the ratio of the total residue of the peptide to the response value of the peptide.

Based on the response and relative residue of the peptide, the peptide components after enzymatic hydrolysis of BSA can be divided into the following four categories:class I, the peptide with high response and no residue; Class II, high response but residual peptide; Class III and class IV are peptides with low response, no adsorption and adsorption, respectively. The response is measured by whether it is greater than the median, and whether there is residue is judged by whether there is detection in area (blank 1+blank 2+blank 3).

04Parameters of ultra high performance liquid chromatography triple quadrupole mass spectrometry method

Chromatographic conditions:the chromatographic column adopts waters acquity UPLC beh C8 (100 mm × 2.1 mm, 1.7 μ m); Column temperature 30 ℃; Flow rate:0.4 ml/min; Mobile phase A and B are 0.2%formic acid aqueous solution and 0.2%formic acid acetonitrile solution respectively. Elution gradient is 0~2 min, 2%B; 2~5 min, 2%B~60%B; 5~5.1 min, 60%B~90%B; 5.1~8 min, 90%B; 8~8.1 min, 90%B~2%B; 8.1~11 min, 2%B。 Injector temperature 10 ℃; Injection volume 5 μ L。 The needle washing solution is 90%acetonitrile aqueous solution (containing 0.2%formic acid).

Mass spectrum conditions:ionization voltage 5500 V; Air curtain pressure 0.14 MPa; Ion source temperature 500 ℃; The air pressure of spray gas and auxiliary heating is 0.38 MPa. ESI source was measured in positive ion mode and collected in multiple reaction monitoring (MRM) mode. The ion pairs, collision energy (CE) and de clustering voltage (DP) values of 12 class II peptides were optimized with the help of skyline software. The results are shown in Table 1 of the original text.

Article information

Chromatography, 2022, 40 (7):616-624DOI:10.3724/SP.J.1123.2021.12012

Zhang Ying 1,2, Yang Jing 1,2, Ma Yuexin 1,2, Cao Ling 2*, Huang Qing 2*

1. School of pharmacy, Nanjing University of traditional Chinese medicine, Nanjing, Jiangsu 210023

2. Jiangsu Institute of food and drug supervision and inspection, Key Laboratory of chemical impurity mass spectrometry, State Drug Administration, Nanjing, Jiangsu 210019