Computational protocol: The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery

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Protocol publication

[…] For PMF, in-gel tryptic peptides of each spot of interest were mixed with an acidic solid matrix such as α-cyano-4-hydroxy cinnamic acid (CHCA, Bruker Daltonics) matrix 10 mg/ml, which provides high sensitivity and negligible matrix adduction during the laser absorption and subjected to laser radiation. The matrix was made in 70% acetonitrile and 0.03% TFA. 0.5 μl of the peptide extracts mixed with the 0.5 μl of the matrix were manually spotted onto a 600 μm/384 well AnchorChipTM sample target (Bruker Daltonics) and dried at ambient temperature. Peptide mass spectra were recorded in the reflectron mode using an Ultraflex III TOF/TOF mass spectrometer (Bruker Daltonics) equipped with a 384-sample scout source. The ion acceleration voltage after pulsed extraction was 29000 V. A peptide calibration standard (Bruker Daltonics) was used for external calibration as previously described. MS and MS/MS data were recorded automatically on the MALDI-TOF/TOF instrument using the three most abundant peptide signals of the corresponding peptide mass fingerprint (PMF) spectrum. The monoisotopic peak list was generated in Post Processing s/w and True peptide mass list was generated by Bruker Flex Analysis software version 3.0 and Biotools ver 3.1 without using the smoothing function and the peak filter was applied to exclude the masses lower than 700 Da and the signal to noise ratio of 20. The generated peptide mass list was searched with MASCOT (http://www.matrixscience.com) using entire Uniprot/Swiss-Prot protein database to find and match the protein identity. Databases searches were performed by using following search parameters; Rattus norvegicus as taxonomy, carbamidomethyl modification of cysteines and possible oxidation of methionine, one missed cleavage, a mass accuracy of ≤100 ppm was requested for PMF and for MS/MS searches, a mass accuracy of ≤70 ppm was allowed for peptide masses and their fragments, respectively. For each identified Protein, at least one Peptide was selected for MS/MS (TOF/TOF) to validate the Protein Identity. Instrument was used in the Lift mode (TOF/TOF) to obtain the MS/MS spectra. Again the Flex Analysis 3.0 and Biotools 3.1 s/w were used to generate the fragments mass list and the sequence tag of peptide. The mass list was sent to database in same way as was done in case of above PMF approach. The mass tolerance error of 0.5 Da to 1.0 Da was used for MS/MS ion search. The MS/MS ion search confirmed the protein identity and provided the amino acid sequence of particular peptide. Gene ontology (GO) annotations (functional distribution) for identified proteins were assigned using Blast2GO research tool.All chemicals were of reagent or electrophoresis grade and, unless specified above, were purchased from GE Health Care and Sigma Chemical Company (St Louis, MO, USA). […]

Pipeline specifications

Software tools Biotools, Blast2GO
Application Population genetic analysis
Organisms Rattus norvegicus, Homo sapiens
Diseases Lung Diseases, Pulmonary Edema
Chemicals Oxygen