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DEIMoS: an open-source tool for processing high-dimensional mass spectrometry data

ABSTRACT: LC-IMS-MS/MS data from a large study of human plasma samples consisting of 40 quality control samples from the NIST Standard Reference Material 1950 and 112 study samples. An internal standard mixture consisting of D4-malonic acid, D4-succinic acid, D5-glycine, D4-citric acid, 13C6-fructose, D5-L-tryptophan, D4-lysine, D7-alanine, D35-stearic acid, D5-benzoic acid, and D15-octanoic acid was added prior to extraction. Each sample was spiked with 50 uL of a solution of the internal standards at 0.166 mg/mL in water. Metabolites and lipids were extracted with concomitant protein precipitation using the Matyash protocol. The metabolite layer was removed and dried in vacuo. Lipid and protein layers were not analyzed. An Agilent 1260 Infinity II high flow liquid chromatography system was used to inject and chromatographically separate samples prior to introduction to the ion mobility spectrometry-mass spectrometry instrument. A steady flowrate of 0.300 mL/min was delivered through a Millipore-Sigma SeQuant Zic-pHILIC column (15 cm length, 2.1 mm inner diameter, packed with 5 um particles). Ion mobility spectrometry-tandem mass spectrometry analysis was performed using an Agilent 6560 Ion Mobility LC/Q-TOF system. Spectra were acquired separately in both positive and negative ionization modes. Fixed collision energies were employed at 10, 20, and 40 eV on alternating frames.

INSTRUMENT(S): 6560 Q-TOF LC/MS

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Thomas O. Metz

PROVIDER: MSV000088849 | MassIVE | Wed Feb 16 18:12:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:Purpose: to reveal the small RNA profile of postnatal retina in Wistar WU albino rat by Ion-Torrent PGM sequencing technology. Methods : Wistar WU rats ranging in age from P1 to P21 were anesthetized by inhalation using Forane prior to sacrifice at the same hour to avoid circadian variation. Total RNA was extracted from the retinal tissues of various developmental stages using NucleoSpin miRNA kit (Macherey–Nagel, Düren, Germany) following manufacturer's instructions. The miRNA concentration was assessed using Qubit microRNA Assay Kit (ThermoFisher Scientific, MA, USA). The RNA integrity number (RIN), an algorithm for judging the integrity of RNA samples, were evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies, USA) following the manufacturing instruction of the RNA 6000 Nano kit and RIN>7 was considered acceptable. MicroRNAs was also determined using the Agilent Small RNA Kit to have deeper view in the 10 to 40-nucleotide size range. Small RNA library construction from pooled retinal samples (N=3, in each age-group) were carried out according to the Ion Total RNA-Seq Kit v2 protocol (Revision E) with slight modification. Enzymatic reaction was performed in half reaction volume, while cleaning procedures were executed with the accurate final volume according to the protocol. The Ion 316 or 318 ™ Chip v2 was used for sequencing on the Ion Torrent PGM™ instrument according to the protocol of Ion PGM™ Hi-Q View Sequencing Kit. To assess reliability of miRNA workflow solution in 316 v2 chip, two independent P7 samples were assayed (biological replicates) and P21 sample were sequenced as technical replicates in every sequencing procedure. Some samples were also sequenced in 318 v2 chip (P3, P5, P10, P15 and P21) as biological replicate. Ion Torrent Suite Platform was used to trim the raw sequence data and remove any residual sequencing adapter fragments that remained on the 5′ or 3′ ends. Reads were mapped to the non-coding RNAs from ENSEMBL [Rnor_6.0 (GCA_000001895.4)] using TMAP algorithm. These aligned BAM (Binary Alignment Map) files were further processed in Galaxy Web-based platform (Afgan, 2018) via Cufflinks, Cuffmerge and Cuffdiff (Version 2.2.1.3) application (Trapnell, 2010). Further analysis and visualising of the datasets was carried out in R Studio Software environment. qRT–PCR validation was performed using TaqMan assays.

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DEIMoS: an open-source tool for processing high-dimensional mass spectrometry data

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