Control or CARM1 knockout A1847 cells were washed with cold PBS, scraped into cold methanol, and spiked with EquiSPLASH mix (Avanti Polar Lipids). Lipids were extracted using a modified Folch extraction (2:1:1 chloroform:methanol:0.88% sodium chloride) and analyzed by LC/MS-MS. LipidSearch 4.2 software (Thermo Fisher Scientific) detected lipid species based on MS-MS spectra with product ion mass tolerances and 5 ppm precursor. The identification of lipid species was filtered on the basis of the expected identification quality and main adduct. The peak areas were utilized for quantification and were adjusted by EquiSPLASH lipids to represent the respective classes. In addition, the values were normalized to the protein content in each sample. The quantification of lipid classes was performed by summing peak areas of all species in the same class. Regarding the saturation analysis, the FAs incorporated into lipids with high-confidence identifications (grades A and B from LipidSearch) were classified in accordance with the number of carbon double-bonds as saturated (0), monounsaturated (1 (link)), or polyunsaturated (>1). The quantification of each lipid species with FA level identification was weighted by the number of FAs of each type present in the specie.
Lipidsearch 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
LipidSearch 4.2 is a comprehensive software solution for lipid analysis. It provides automated lipid identification and quantification from liquid chromatography-mass spectrometry (LC-MS) data. The software features advanced algorithms for peak detection, lipid annotation, and data visualization.
Automatically generated - may contain errors
Lab products found in correlation
Xcalibur 4, by Thermo Fisher Scientific (3 mentions)
Acquity beh c8 column, by Waters Corporation (3 mentions)
Freestyle 1, by Thermo Fisher Scientific (3 mentions)
Equisplash mix, by Avanti Polar Lipids (2 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Triversa nanomate, by Advion (2 mentions)
Ultimate 3000 rslcnano lc system, by Thermo Fisher Scientific (1 mentions)
Orbitrap eclipse tribrid mass, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap 100 c18, by Thermo Fisher Scientific (1 mentions)
Orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions)
1100 quaternary pump hplc with room temperature autosampler, by Agilent Technologies (1 mentions)
Q exactive plus orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive plus orbitrap, by Thermo Fisher Scientific (1 mentions)
Agilent 1100 series, by Agilent Technologies (1 mentions)
Q exactive hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Vanquish uhplc system, by Waters Corporation (1 mentions)
Acquity uplc beh c18 column, by Waters Corporation (1 mentions)
Xcalibur qual browser, by Thermo Fisher Scientific (1 mentions)
Statistica 13.3 pl, by StatSoft (1 mentions)
30 protocols using lipidsearch 4
1
Lipidomic analysis of CARM1 knockout cells
2
Lipid Extraction and LC-MS/MS Analysis
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The endogenous bound lipids from SPNS2 proteins were extracted by adding chloroform-methanol (2:1, v/v) as previously described.48 (link) The extracted lipids were dried and resuspended in 80% methanol. For LC-MS/MS analysis, the lipids were separated on a C18 column (Acclaim PepMap 100, C18, 75 μm × 15 cm, Thermo Fisher Scientific) using a Dionex UltiMate 3000 RSLC nano LC System using a previously described mobile phase and gradient.49 (link) The column eluent was loaded to Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific). The Orbitrap Eclipse was operated in negative mode using data-dependent acquisition. The detailed mass spectrometer parameters are as follows: spray voltage, -1.9 kV; capillary temperature, 320°C; mass range, 250-1500; full MS resolution, 120K; MS/MS resolution, 15K; NCE, 25/30/35; cycle time, 1.5s. The data was analysed by LipidSearch 4.2 software (Thermo Fisher Scientific).
3
Lipid Separation and Identification by Reverse-Phase UHPLC-MS
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The lipids were separated by reverse-phase LC using a Thermo Scientific Accucore Vanquish C18+ 2.1 (i.d.) × 150 mm column with 1.5 μm particles. The UHPLC used a binary solvent system at a 0.26 mL min−1 flow rate with a column oven set to 55 °C. Before injection of the sample, the column was equilibrated for 2 min with a solvent mixture of 99% mobile phase A1 (CH3CN/H2O, 50/50, v/v, with 5 mM ammonium formate and 0.1% formic acid) and 1% mobile phase B1 (CH3CHOHCH3/CH3CN/H2O, 88/10/2, v/v/v, with 5 mM ammonium formate and 0.1% formic acid) and after the sample injection (typically 10 μL). The A1/B1 ratio was maintained at 99/1 for 1.0 min followed by a linear gradient to 35% B1 over 2.0 min, then a linear gradient to 60% B1 over 6 min followed by a linear gradient to 100% B1 over 11 min. At this step the run was held at 100% B1 for 5 min, followed by a 2.0 min gradient return to 99/1 (A1/B1). The column was re-equilibrated with 99/1 (A1/B1) for 2.0 min before the next run. Each sample was injected twice for analysis in both positive and negative modes. For the initial full scan MS (range 300 to 200 m/z), the resolution was set to 120 000 with a data-dependent MS2 triggered for any analyte reaching 3e6 or above signal. Data-dependent MS2 was collected at 30 000 resolutions. Data were analyzed using Thermo Scientific's Lipid Search 4.2 software.
4
Untargeted Lipidomics with LC-MS Using C30 Column
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The nonpolar lipids were reconstituted using 200 µL of 2-propanol: acetonitrile: water (v:v:v 30:65:5). Then 5 µL of reconstituted sample was injected into the LC-MS. The untargeted lipidomics method was modified from a published method23 (link) that used a C30 column (Acclaim C30, 3 µm, 2.1 × 150 mm). The LC method used two elution solutions; buffer A (60% acetonitrile and 40% water with 0.1% formic acid and 10 mM ammonium formate) and buffer B (90% 2-propanol and 10% acetonitrile with 0.1% formic acid and 10 mM ammonium formate). The 0.2 mL/min LC gradient was started from 0 to 1.5 min, 32% B, 4 min, 45% B, 5 min, 52% B, 8 min, 58% B, 11 min, 66% B, 14 min, 70% B, 18 min, 75% B, 21–25 min, 97% B, 25–32 min 32% B. The samples were acquired by a Orbitrap Exploris 480 (Thermo Fisher Scientific) using polarity switching approach with DDA mode. All the lipidomics .RAW files were processed on LipidSearch 4.0 (Thermo Fisher Scientific) for the lipid identification.
5
Lipid Profiling of Shrimp Muscle
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Peak areas of lipid molecular species were analyzed using LipidSearch 4.0 software (Thermo Fisher Scientific, CA, USA). Heatmap imaging, partial least squares discriminant analysis (PLS-DA), and variable importance in projection (VIP) analysis were performed in MetaboAnalyst 5.0 (https://www.metaboanalyst.ca accessed on 10 September 2021) [20 (link)]. The mean values and standard deviations were calculated using Microsoft Excel software. One-way ANOVA was carried out using SPSS software (version 25.0) to test the significant differences (p < 0.05) of physical properties and lipid levels during low-temperature storage, and highly significant differences (p < 0.01) between physical properties and lipid species. In addition, Pearson’s correlation analysis of individual lipid variables and physical quality characteristics (including L*, a*, b*, springiness, gumminess, cohesiveness, chewiness, and hardness) of L. vannamei muscles was carried out using SPSS software. All results were determined from 6 samples.
6
Lipid Profiling via Mass Spectrometry
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Acquired data was converted from raw to mzXML file format using Mass Matrix (Cleveland, OH, USA). Samples were analyzed in randomized order with a technical mixture injected interspersed throughout the run to qualify instrument performance. Lipidomics data were analyzed using LipidSearch 4.0 (Thermo Scientific), which provides lipid identification on the basis of accurate intact mass, isotopic pattern, and fragmentation pattern to determine lipid class and acyl chain composition. Graphs, heat maps and statistical analyses (T-Test) and Partial Least Squares-Discriminant Analysis (PLS-DA) were performed using MetaboAnalyst 5.0 [54 (link)].
7
Lipid Identification and Quantitation
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Lipid features were identified using LipidSearch 4.0 software (Thermo Scientific, San Jose, CA, USA). The feature identification and quantitation parameters used are mentioned in Supplementary File S2 .
8
Lipid Molecular Profiling Workflow
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Structures of individual lipid molecules were conducted from the QC sample by searching LipidSearch 4.0 software (Thermo Fisher Scientific, Hemel Hempstead, UK) , false positives were checked manually according to the MS/MS fragmentation pattern. Peak extraction, deconvolution, alignment and gap-filling of each molecule in different egg samples were conducted by utilizing MZmine 2 software. Molecules with intensities that lower than 1％ of base peak (most abundant) in their class were discarded. Peak intensity data of individual lipid molecules was then normalized by dividing the sum of all lipid molecules in same sample.
9
Lipidomics Analysis by LC-MS/MS
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The lipid samples were re-suspended in 30 µL of 1:1 LC/MS grade isopropanol:methanol prior to LC-MS/MS analysis, 5 µL were injected. A Cadenza 150 mm × 2 mm 3 µm C18 column (Imtakt) heated to 40 °C at 260 µL/min was used with a 1100 quaternary pump HPLC with room temperature autosampler (Agilent). Lipids were eluted over a 20 min. gradient from 32% B buffer (90% IPA/10% ACN/10 mM ammonium formate/0.1% formic acid) to 97% B. A buffer consisted of 59.9% ACN/40% water/10 mM ammonium formate/0.1% formic acid. Lipids were analyzed using a high resolution hybrid QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) in DDA mode (Top 8) using positive/negative ion polarity switching. DDA data were acquired from m/z 225-1450 in MS1 mode and the resolution was set to 70,000 for MS1 and 35,000 for MS2. MS1 and MS2 target values were set to 5e5 and 1e6, respectively. Lipidomics data were analyzed using LipidSearch 4.1.9 software (Thermo Fisher Scientific) and Elements for Metabolomics (Proteome Software) NIST database incorporated.
10
Comprehensive Lipid Profiling by LC-MS/MS
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The dried lipid layer was re-suspended in 30 μL of 1:1 LC/MS grade isopropanol:methanol prior to LC-MS/MS analysis, 5 μL were injected. A Cadenza 150 mm x 2 mm 3 μm C18 column (Imtakt) heated to 40°C at 260 μL/min was used with a quaternary pump HPLC with room temperature autosampler (Agilent 1100 series). Lipids were eluted over a 20 min. gradient from 32% B buffer (90% IPA/10% ACN/10 mM ammonium formate/0.1% formic acid) to 97% B. A buffer consisted of 59.9% ACN/40% water/10 mM ammonium formate/0.1% formic acid. Lipids were analyzed using a hybrid QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) in DDA mode using positive/negative ion polarity switching with 1 MS1 scan followed by 8 MS2 HCD scans per cycle (Top 8). DDA data were acquired from m/z 200–1450 in MS1 mode and the resolution was set to 70,000 for MS1 and 35,000 for MS2. MS1 and MS2 target values were set to 5e5 and 1e6, respectively. Lipidomics data were analyzed using LipidSearch 4.1.9 software (Thermo Fisher Scientific) for identification and validation.
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