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Improved in‐solution trypsin digestion method for methanol–chloroform precipitated cellular proteomics sample
Author(s) -
Shahinuzzaman A. D. A.,
Chakrabarty Jayanta K.,
Fang Zixiang,
Smith David,
Kamal Abu Hena Mostafa,
Chowdhury Saiful M.
Publication year - 2020
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201901273
Subject(s) - chromatography , chemistry , chloroform , tandem mass spectrometry , sample preparation , deoxycholic acid , mass spectrometry , methanol , liquid chromatography–mass spectrometry , digestion (alchemy) , sodium dodecyl sulfate , lysis , protein precipitation , proteomics , bile acid , biochemistry , organic chemistry , gene
Methanol–chloroform based protein precipitation is an essential step in many liquid chromatography–tandem mass spectrometry‐based cellular proteomics applications. However, re‐solubilization of the total protein precipitate is difficult using regular in‐solution digestion protocol. Sodium deoxycholate is reported as an efficient surfactant for re‐solubilization of membrane fractions. In this study, we demonstrated an application combining methanol–chloroform based protein precipitations and deoxycholic acid assisted re‐solubilization of pellets to evaluate the improvement of protein identifications in mass spectrometry‐based bottom‐up proteomics. We evaluated the modified method using an equal amount of Raw 264.7 mouse macrophage cell lysate. Detailed in‐solution trypsin digestion studies were presented on methanol–chloroform precipitated samples with or without deoxycholic acid treatments and compared with popular sample digestion methods. A mass spectrometric analysis confirmed an 82% increase in protein identification in deoxycholic acid‐treated samples compared to other established methods. Furthermore, liquid chromatography–tandem mass spectrometry analysis of an equal amount of proteins from methanol–chloroform precipitated, and methanol–chloroform/deoxycholic acid‐treated macrophage cell lysate showed a 14% increase and 27% unique protein identifications. We believe this improved digestion method could be a complementary or alternative method for mammalian cell sample preparations where sodium dodecyl sulfate based lysis buffer is frequently used.