Computational protocol: Altered Retinoic Acid Metabolism in Diabetic Mouse Kidney Identified by 18O Isotopic Labeling and 2D Mass Spectrometry

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Protocol publication

[…] The acquired MS/MS spectra were searched with SEQUEST algorithm against a composite target-decoy mouse protein database consisting of the protein sequences (target) downloaded from SWISSPROT mouse protein database (downloaded July 2006) and reversed versions of these sequences (decoy) as described . All SEQUEST searches were performed on the Bioworks 3.2 platform (ThermoFinnigan, San Jose, CA) using the following parameters: fully tryptic peptide (both tryptic terminus for all peptides), a mass tolerance of ±2.0 Da for precursor ion and ±1.0 Da for fragment ion. The output data from these searches were filtered and sorted by the DTASelect software as previously described . Only the top one peptide sequence match to each acquired MS/MS spectrum was considered. The criteria used in DTASelect were as follows: First, relatively conservative criteria (Sp≥300; ΔCn≥0.12; Xcorr of 1.9, 2.0 and 3.0 for data from a singly, doubly or triply charged precursor ions, respectively) were applied. Second, proteins that passed these thresholds were separated into two groups: proteins identified with two or more peptides and proteins identified with one peptide. Third, since the majority of the false positive identifications in our dataset were within the group of proteins with one identified peptide, much stricter criteria (Xcorr 2.2, 3.2, or 3.75 for precursor charge states of 1+, 2+, or 3+, respectively) were applied to the peptide hits in this group to increase identification certainty. Fourth, if multiple spectra were identified to match precisely the same sequence and charge state, only the spectrum with the highest Xcorr was retained. Finally, proteins that shared all matched peptides with other (homologous) proteins were removed. The false discovery rate (FDR) of identification was calculated to be less than 1% as described . [...] The abundance ratios of 18O/16O-labeled peptide pairs were calculated with in-house software using the following equation :where I0, I2 and I4 are the measured relative peak intensities for the monoisotope peak for an unlabeled peptide, the peak with masses 2 Da higher, and the peak with 4 Da higher masses, respectively; M0, M2, and M4 are the theoretical relative intensities for monoisotopic peak of the unlabeled peptide, the peaks with masses 2 Da and 4 Da higher than the monoisotopic peak, respectively. The “M values” were calculated based on the elemental composition of the peptide by using MS-isotope pattern calculator ( If a peptide was identified more than once, a mean and standard deviation were calculated. The peptide ratios were averaged for all peptides for each protein to give a ratio per protein. […]

Pipeline specifications

Software tools Comet, DTASelect, MS-Isotope
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Diabetes Mellitus, Diabetic Nephropathies, Hyperglycemia, Kidney Diseases
Chemicals Retinaldehyde, Tretinoin, Vitamin A