Computational protocol: Tubulointerstitial nephritis antigen-like 1 protein is downregulated in the placenta of pre-eclamptic women

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Protocol publication

[…] Frozen placental tissue was pulverized, and dissolved in lysis buffer (100 mg/0.5 ml) (7 M urea, 2 M thiourea, 4% 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, 2% Dithiothreitol). The homogenate was centrifuged and the supernatant used as a source of placental proteins. Two technical replicates of three pools of normotensive (n = 18) were compared with three pools of PE (n = 18) protein samples using two-dimensional electrophoresis (2DE). For the first dimension, immobilized pH gradient (IPG) strips 4–7 and 12% Tris–Glycine SDS-PAGE gels for the second dimension electrophoresis were used. The stained gels images were scanned on GS-800 Densitometer and analyzed for spot intensity data by PDQuest software (Bio-Rad Laboratories, Hercules, CA, USA). Protein spots excised from 2DE were de-stained, followed by reduction and alkylation. Digested peptides were analyzed in LC-MSE using a NanoAcquity ultra-performance liquid chromatography (UPLC) system coupled to an SYNAPT high definition mass spectrometer (Waters, Milford, MA, USA). Nano C18 reversed phase column (1.7 μm particle size, i.d. 75 μm and length 250 mm) (Waters) and binary solvent system (99.9% water and 0.1% formic acid (mobile phase A) and 99.9% acetonitrile and 0.1% formic acid (mobile phase B)) was used for peptide separation. Data were processed and searched using ProteinLynx Global Server 2.4 (PLGS) software (Waters). UniProtKB H. sapiens proteome (UP000005640), including only reviewed sequences (47,869) from UniprotKB/SwissProt (updated version of H. sapiens proteome, 2008) was used to search against the MS data. A fixed modification included carbamidomethyl C and variable modifications such as oxidation M, deamidation N, and deamidation Q. Automatic setting of PLGS was used for mass accuracy of precursor and fragment ions. Identification was done with minimum two peptides and FDR of 1%. For each protein identified, a number of unique peptides for a given protein was calculated using PepServe bioinformatics tool. The dataset (PASS00711) was uploaded to publicly accessible PeptideAtlas database (www.peptideatlas.org, Institute for Systems Biology, Seattle, WA). […]

Pipeline specifications

Software tools PDQuest 2-D, PLGS, PepServe
Databases PeptideAtlas
Applications MS-based untargeted proteomics, Protein sequence analysis
Organisms Homo sapiens, Mus musculus
Diseases Nephritis, Interstitial