Qualitative and quantitative characterization of elastin-like polypeptides combining low-PH/low-temperature bottom-up and intact protein liquid chromatography mass spectrometric analysis

IF 1.8 3区化学 Q4 BIOCHEMICAL RESEARCH METHODS Rapid Communications in Mass Spectrometry Pub Date : 2024-09-02 DOI:10.1002/rcm.9905

André Vente, Marija Mladic, Olaf Schouten, Jens Thies, Rob van der Hoeven

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Abstract

Rationale

Elastin-like polypeptides (ELPs) are elastic and thermoresponsive biopolymers composed of VPGXG repeats (X can be any amino acid except proline), used in biomedical applications, for example, tissue engineering and drug delivery. As different variants of ELP are mostly produced fermentatively, there is a need for the development of analysis methods that allow for absolute protein quantification in both complex matrices and purified samples and MW determination of the final products.

Methods

ELPs were intracellularly expressed in Escherichia coli quantified after cell lysis and enzymatic digestion using a proline-specific protease ProAlanase (Promega) at acidic conditions. Resulting peptides were separated by liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using an Orbitrap mass spectrometer. The addition of a stable isotopically labeled internal standard enabled quantification in complex matrices. Prior to intact mass analysis, ELPs were purified from fermentation broth by inverse temperature cycling. Intact protein analysis was performed using reversed-phase liquid chromatography, and mass spectrometry analysis was conducted by electrospray ionization high-resolution mass spectrometry using a time-of-flight mass spectrometer.

Results

Absolute quantification of ELPs was achieved by utilizing ELP-specific properties, that is, proline-rich, soluble at low pH and low temperature. The repetitive nature of ELPs allows for sensitivity increase and use of higher dilution factors to minimize the matrix effects. Despite the lack of amino acids with charged side chains (Arg, His, Lys, Asp, and Glu) in ELP, we demonstrated successful intact protein analysis using reversed-phase LC coupled to electrospray ionization TOF MS. Moreover, truncated protein forms could be chromatographically separated and characterized as well as N-terminal modifications.

Conclusions

Both methods combined enabled quantitative and qualitative characterization of fermentatively produced ELPs.

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结合低压/低温自下而上分析法和完整蛋白质液相色谱质谱分析法，对弹性蛋白样多肽进行定性和定量表征。

理论依据：弹性蛋白样多肽（ELPs）是由 VPGXG 重复序列（X 可以是除脯氨酸以外的任何氨基酸）组成的具有弹性和热塌缩性的生物聚合物，可用于组织工程和药物输送等生物医学应用领域。由于 ELP 的不同变体大多通过发酵产生，因此需要开发分析方法，以便对复杂基质和纯化样品中的蛋白质进行绝对定量，并对最终产品进行分子量测定：方法：在酸性条件下，使用脯氨酸特异性蛋白酶 ProAlanase（Promega 公司）对细胞进行裂解和酶解后，在大肠杆菌中对细胞内表达的 ELPs 进行定量。用液相色谱法分离得到的肽，并使用 Orbitrap 质谱仪进行电喷雾电离高分辨质谱分析。加入稳定的同位素标记内标，可对复杂基质进行定量分析。在进行完整质量分析之前，先通过反向温度循环从发酵液中纯化 ELPs。使用反相液相色谱法进行完整蛋白质分析，并使用飞行时间质谱仪通过电喷雾离子化高分辨率质谱进行质谱分析：结果：利用ELP的特异性，即富含脯氨酸、可溶于低pH值和低温，实现了ELP的绝对定量。ELPs 的重复性使灵敏度得以提高，并可使用较高的稀释因子将基质效应降至最低。尽管 ELP 中缺乏带电侧链的氨基酸（Arg、His、Lys、Asp 和 Glu），但我们还是利用反相液相色谱结合电喷雾电离 TOF MS 成功地分析了完整的蛋白质。此外，我们还利用色谱法分离和鉴定了截短蛋白形式以及 N 端修饰：结论：这两种方法结合使用可对发酵产生的 ELP 进行定量和定性表征。

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来源期刊

Rapid Communications in Mass Spectrometry 化学-分析化学

CiteScore

4.10

自引率

5.00%

发文量

219

审稿时长

2.6 months

期刊介绍： Rapid Communications in Mass Spectrometry is a journal whose aim is the rapid publication of original research results and ideas on all aspects of the science of gas-phase ions; it covers all the associated scientific disciplines. There is no formal limit on paper length ("rapid" is not synonymous with "brief"), but papers should be of a length that is commensurate with the importance and complexity of the results being reported. Contributions may be theoretical or practical in nature; they may deal with methods, techniques and applications, or with the interpretation of results; they may cover any area in science that depends directly on measurements made upon gaseous ions or that is associated with such measurements.