Computational protocol: Serum Neuroinflammatory Disease-Induced Central Nervous System Proteins Predict Clinical Onset of Experimental Autoimmune Encephalomyelitis

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Protocol publication

[…] Single-cell suspensions were obtained from brain tissues by mechanical isolation. Extracellular myelin was removed from all brain and spinal cord suspensions using myelin removal beads (Miltenyi Biotec), according to the manufacturer’s instructions. For surface staining, cell suspensions were blocked with 1% anti-mouse CD16/CD32 (eBioscience; 93) for 20 min on ice and then stained with fluorescently labeled antibodies for murine CD4 (BD Biosciences; RM4-5), CD11c (eBioscience; N418), CD11b (eBioscience; M1/70), or GR1 (BD Biosciences; RB6-8C5), for 45 min. Following staining, cells were washed with PBS, lysed for red blood cell (Beckman Coulter), and fixed using a fixative agent (eBioscience). All samples were analyzed or sorted on a BD FACSAria II (BD Bioscience). Data were analyzed using the FlowJo 10 Data Analysis software (FlowJo LLC) and BD FACSDiva software (BD Biosciences). [...] Capillary LC/FT/MS/MS was performed with a split less nanoLC-2D pump (Eksigent, Livermore, CA, USA), a 50 μm-i.d. column packed with 7 cm of 3 µm-o.d. C18 particles, and a hybrid linear ion trap-Fourier-transform tandem mass spectrometer (LTQ-ELITE; ThermoFisher, San Jose, CA, USA) operated with a lock mass for calibration. The reverse-phase gradient was 2–62% of 0.1% formic acid in acetonitrile over 60 min at 350 nL/min. For unbiased analysis, the top six most abundant eluting ions were fragmented by data-dependent HCD with a mass resolution of 120,000 for MS and 15,000 for MS/MS. For isobaric TMT labeling, probability-based protein database searching of MS/MS spectra against the TrEMBL protein database (release December 29, 2012; 59,862 sequences) was performed with a 10-node MASCOT cluster (v. 2.3.02; Matrix Science, London, UK) with the following search criteria: peak picking with Mascot Distiller; 10 ppm precursor ion mass tolerance, 0.8 Da product ion mass tolerance, three missed cleavages, trypsin, carbamidomethyl cysteines as a static modification, oxidized methionine and deamidated asparagine as variable modifications, an ion score threshold of 20, and TMT-6-plex for quantification. [...] Statistical analysis for relative expression change of proteins revealed by M2 proteomics was performed as described previously (). M2 proteomics results for each technical replicate estimate peptide expression for individual mice, encoded in sample mixtures, relative to pooled reference material from all mice, and spanning all time points. Relative peptide expression levels were transformed to log base 2 for quantile normalization. We tested for changes in relative peptide expression with post-immunization time using a linear mixed-effect model in which time was treated as a multilevel factor. We tested all the pairwise differences in relative peptide expression between all disease time points, using an unpaired, unequal variance t-test on the replicate averages. Statistical analysis was performed with R v3.0.2 (R-Project, Vienna, Austria).Additional statistical analysis, including Student’s t-test, ANOVA, and receiver operating characteristic (ROC), were performed using GraphPad Prism 7 (GraphPad Software) and SigmaPlot 12 (Systat Software Inc.) software. Statistical significance was determined as indicated in the text and/or figure legends. Unless otherwise indicated, normal distribution was assumed for all samples. P values: *≤0.05; **≤0.01; ***≤0.001; ****≤0.0001. […]

Pipeline specifications

Software tools Mascot Distiller, SigmaPlot
Applications Miscellaneous, MS-based untargeted proteomics
Organisms Homo sapiens, Mus musculus
Diseases Central Nervous System Diseases, Encephalomyelitis, Encephalomyelitis, Autoimmune, Experimental, Multiple Sclerosis
Chemicals Glutamic Acid