TY - JOUR
T1 - Mass Spectrometry-Compatible Subcellular Fractionation for Proteomics
AU - Masuda, Takeshi
AU - Sugiyama, Naoyuki
AU - Tomita, Masaru
AU - Ohtsuki, Sumio
AU - Ishihama, Yasushi
N1 - Funding Information:
We thank Yue Zhou of Toyama University and members of our research group for helpful discussions. This work was supported by research funds from Yamagata Prefecture and Tsuruoka City to Keio University and by Grant-in-Aid for Young Scientists (B) (Nos. 23710218 and 17K15042) and Grant-in-Aid for Scientific Research (C) (No. 19K05544) from Japan Society for the Promotion of Science to T.M.
PY - 2020/1/3
Y1 - 2020/1/3
N2 - We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry (MS)-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 105 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding proteomics were identified, which are potential candidate sites to regulate the protein subcellular localization, including serine 706 on CD44 and serine 22 on lamin A/C.
AB - We found that nuclear envelopes stabilize against surfactants in the presence of ethylene glycol (EG). We, therefore, developed a novel subcellular fractionation approach for proteomics using RIPA buffer containing EG and phase transfer surfactants. This method involves separating the cells into the cytoplasm, organelles, and nucleus, including intermediate filaments without ultracentrifugation. These fractions are directly applicable to sample preparation for shotgun proteomics as they have no mass spectrometry (MS)-incompatible chemicals, whereas those separated by traditional fractionation protocols require desalting. This protocol is successfully applied to subcellular fractionation with only 3.5 × 105 cells. Here, it was combined with phosphoproteomics and proteomics to identify phosphorylation sites regulating protein subcellular localization. In total, 59 phosphorylation sites on 42 phosphopeptides and 32 proteins showing different enrichment patterns between phosphoproteomics and the corresponding proteomics were identified, which are potential candidate sites to regulate the protein subcellular localization, including serine 706 on CD44 and serine 22 on lamin A/C.
KW - ethylene glycol
KW - mass spectrometry-compatible method
KW - phase transfer surfactant
KW - phosphoproteomics
KW - subcellular fractionation
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U2 - 10.1021/acs.jproteome.9b00347
DO - 10.1021/acs.jproteome.9b00347
M3 - Article
C2 - 31599158
AN - SCOPUS:85073823238
VL - 19
SP - 75
EP - 84
JO - Journal of Proteome Research
JF - Journal of Proteome Research
SN - 1535-3893
IS - 1
ER -