Lee, Jae Won
(Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University)
,
Cho, Kyung Mi
(Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University)
,
Jung, Jae Hun
(Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University)
,
Tran, Quangdon
(Department of Pharmacology, Metabolic Diseases and Cell Signaling Laboratory, Research Institute for Medical Sciences, College of Medicine, Chungnam National University)
,
Jung, Woong
(Department of Emergency Medicine, School of Medicine, Kyung Hee University)
,
Park, Jongsun
(Department of Pharmacology, Metabolic Diseases and Cell Signaling Laboratory, Research Institute for Medical Sciences, College of Medicine, Chungnam National University)
,
Kim, Kwang Pyo
(Department of Applied Chemistry, The Institute of Natural Science, College of Applied Science, Kyung Hee University)
Matrix assisted laser desorption ionization (MALDI)-time of flight/mass spectrometry (TOF/MS) was applied to investigate alterations in phospholipids in mitophagic cancer cells. Several phospholipids, including phosphatidylcholines (PCs), sphingomyelins (SMs), and phosphatidylinositols (PIs), were s...
Matrix assisted laser desorption ionization (MALDI)-time of flight/mass spectrometry (TOF/MS) was applied to investigate alterations in phospholipids in mitophagic cancer cells. Several phospholipids, including phosphatidylcholines (PCs), sphingomyelins (SMs), and phosphatidylinositols (PIs), were successfully analyzed in control and mitophagy-induced H460 cells in the positive and negative ion modes. Principal component analysis was applied to differentiate the two groups. The upregulated and downregulated phospholipid species in the mitophagic cells were also represented in a heatmap. In the volcano plot (fold change > 1.3 and p value < 0.01), individual species of seven PCs, two SMs, and three PIs were selected as differentially regulated phospholipids. In particular, almost all the molecular species of PC, SM, and PI were downregulated in the mitophagic cells. Quantification of these lipids indicated that mitophagy induces altered metabolism of phospholipids. Therefore, phospholipid alterations during the mitophagic process of lung cancer cells were well characterized by MALDI-TOF/MS.
Matrix assisted laser desorption ionization (MALDI)-time of flight/mass spectrometry (TOF/MS) was applied to investigate alterations in phospholipids in mitophagic cancer cells. Several phospholipids, including phosphatidylcholines (PCs), sphingomyelins (SMs), and phosphatidylinositols (PIs), were successfully analyzed in control and mitophagy-induced H460 cells in the positive and negative ion modes. Principal component analysis was applied to differentiate the two groups. The upregulated and downregulated phospholipid species in the mitophagic cells were also represented in a heatmap. In the volcano plot (fold change > 1.3 and p value < 0.01), individual species of seven PCs, two SMs, and three PIs were selected as differentially regulated phospholipids. In particular, almost all the molecular species of PC, SM, and PI were downregulated in the mitophagic cells. Quantification of these lipids indicated that mitophagy induces altered metabolism of phospholipids. Therefore, phospholipid alterations during the mitophagic process of lung cancer cells were well characterized by MALDI-TOF/MS.
HPLC-grade methanol, chloroform, and water were purchased from Burdick and Jackson (USA). 2,5-Dihydroxybenzoic acid (DHB), a-cyano-4-hydroxycinnamic acid (CHCA), 9-aminoacridine, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich.
Anti-LC3B, anti-p62, and anti-actin antibodies were purchased from Sigma-Aldrich (USA). Anti-Beclin1 antibody was obtained from Aviva Systems Biology (USA) and anti-Fundc1 antibody was obtained from Novus Biologicals (Littleton, USA).
Anti-Beclin1 antibody was obtained from Aviva Systems Biology (USA) and anti-Fundc1 antibody was obtained from Novus Biologicals (Littleton, USA). Horseradish-peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from Komabiotech (Korea). CCCP was purchased from Sigma-Aldrich.
데이터처리
Features with signal-to-noise ratios greater than 5 were selected as peaks. Peaks affiliated with the same mass were aligned by the statistical regression-based approach using the identification of landmark peaks and estimation of a non-linear warping function.
To show the correlation between samples and features, hierarchical clustering of the differentially regulated phospholipids was performed using Euclidean distances and Ward linkage. The Pearson’s correlation coefficients of the features were calculated and clustered to identify correlating peaks.
이론/모형
Phospholipids were extracted from control and mitophagy-induced H460 cells using the Bligh and Dyer method [6]. H460 cells with or without CCCP treatment were collected from each culture dish and then the number of cells was counted by using a hemocytometer.
The spectrum background was evaluated using the statistics-sensitive non-linear iterative peak-clipping algorithm and used for baseline correction. The intensities of multiple spectra were normalized using the probabilistic quotient normalization method. Features with signal-to-noise ratios greater than 5 were selected as peaks.
The peak intensity of single spectra was transformed to a square root scale for variance stabilization and smoothed using a moving average algorithm. The spectrum background was evaluated using the statistics-sensitive non-linear iterative peak-clipping algorithm and used for baseline correction. The intensities of multiple spectra were normalized using the probabilistic quotient normalization method.
성능/효과
In conclusion, MALDI-TOF/MS was used to analyze alterations in phospholipids in control and mitophagy-induced H460 cells. Several phospholipids, including PC, SM, and PI, were successfully analyzed in the positive and negative ion modes.
3A). The MALDI spectra of phospholipids separated two groups with 69.8% total variances, including principal component (PC) 1 (55.3%) and PC 2 (14.5%). Furthermore, these two groups were also differentiated in the PCA score plot of negative-ion mode data with the variances of PC 1 (64.
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