Optimization of Conditions for Blood Plasma Peptidome Analysis
- Авторлар: Ziganshin R.1, Ryabinin V.1, Azarkin I.1, Govorun V.1,2, Ivanov V.1
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Мекемелер:
- Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
- Federal Research and Clinical Center of Physical–Chemical Medicine
- Шығарылым: Том 44, № 3 (2018)
- Беттер: 289-295
- Бөлім: Article
- URL: https://journals.rcsi.science/1068-1620/article/view/228922
- DOI: https://doi.org/10.1134/S1068162018030159
- ID: 228922
Дәйексөз келтіру
Аннотация
The conditions for peptidome analysis of blood plasma were optimized and the efficacy of the proposed approach was compared with the methods described in the literature. The method implies solution of two main problems: inactivation of blood plasma proteases and dissociation of peptides from major blood plasma proteins, which they are quantitatively associated with. To solve these problems, we proposed a new method of sample preparation. The essence of the method is simultaneous denaturation of plasma proteins plus reduction and alkylation of thiol groups of Cys, which is achieved by heating a blood plasma sample (95°C) in the presence of sodium deoxycholate, tris(2-carboxyethyl)phosphine, and 2-chloroacetamide. After separation of peptides from proteins by ultrafiltration on microcentrifuge filters and removal of sodium deoxycholate, the peptides are identified by LC-MS/MS using a Q Exactive HF (Thermo Scientific) mass spectrometer. As a result of one LC-MS/MS run of the peptide mixture obtained from ~15 μL of blood plasma, 2257 peptide fragments of 867 proteins were identified, which is 1.5 times higher than the values achieved by using the generally accepted method of differential solubilization. Our immediate plans include the use of our approach for cataloguing human blood plasma peptides, as well as establishing the magnitude of individual variability and the features of the peptidome that are related to gender and age.
Негізгі сөздер
Авторлар туралы
R. Ziganshin
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
Хат алмасуға жауапты Автор.
Email: rustam.ziganshin@gmail.com
Ресей, Moscow, 117997
V. Ryabinin
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
Email: rustam.ziganshin@gmail.com
Ресей, Moscow, 117997
I. Azarkin
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
Email: rustam.ziganshin@gmail.com
Ресей, Moscow, 117997
V. Govorun
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry; Federal Research and Clinical Center of Physical–Chemical Medicine
Email: rustam.ziganshin@gmail.com
Ресей, Moscow, 117997; Moscow, 119435
V. Ivanov
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry
Email: rustam.ziganshin@gmail.com
Ресей, Moscow, 117997