Computational protocol: Na+/K+-ATPase α1 Identified as an Abundant Protein in the Blood-Labyrinth Barrier That Plays an Essential Role in the Barrier Integrity

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Protocol publication

[…] The tryptic peptides were analyzed by 2D-LC-MS/MS. In brief, a 100×2.1 mm strong cation-exchange (SCX) column (Polysulfoethyl A, 5 µm particle size, 300 Å pore diameter) was prepared for ion-exchange chromatography as instructed by the manufacturer (The Nest Group, Inc.). Tryptic peptides were injected onto the SCX column at a 200 µl/min flow rate. The operating conditions for the analytical column were mobile phase A containing 10 mmol/L sodium phosphate (pH 3.0) and 25% acetonitrile, along with mobile phase B containing 10 mmol/L sodium phosphate (pH 3.0), 25% acetonitrile and 350 mmol/L potassium chloride. Following 5 minutes of loading and washing in mobile phase A, peptides were eluted using a linear gradient of 0% to 50% B over 45 min, followed by a linear gradient of 50% to 100% B over 20 min. One-minute fractions were collected and dried by vacuum centrifugation and then redissolved by shaking in 100 µl of 5% formic acid. Portions of each fraction (40 µl) were analyzed by LC-MS/MS using an Agilent 1100 series capillary LC system and an LTQ linear-ion trap mass spectrometer (Thermo Inc.) using a standard electrospray source fitted with a 34-gauge metal needle (Thermo Inc.). Chromatography was achieved using an Eksigent nanoLC to generate a gradient using the following chromatographic conditions: mobile phase A: water, acetonitrile, formic acid, trifluoroacetic acid (95, 4.89, 0.1, 0.01, v/v/v/v); mobile phase B: acetonitrile, isopropanol, water, formic acid, trifluoroacetic acid (80, 10, 9.89, 0.1, 0.01, v/v/v/v/v). Mobile phase B was ramped from 2% to 45% over 40 minutes, increased to 80% in 5 minutes, and held for 5 minutes before being returned to starting conditions. The flow was regulated at 200 nl/min and directed through a 75 µm ×150 mm column packed in house with Astrosil (5 µm particle size, 100 Å pore size, C18 reverse phase chemistry; Stellar Phases) coupled to a 5 µm tapered emitter (New Objectives). Prior to analytical chromatography, 5 µl of tryptic digest was injected onto a 150 µm ×20 mm sample trap packed with Poros R10; the trap was washed with mobile phase A to remove salts and contaminants and then was switched in line with the analytical column. Tandem mass spectrometry data was collected using a QSTAR XL hybrid time-of-flight mass spectrometer (Applied Biosystems) under the following conditions: spray voltage 1800 to 1900 V; TOF-MS scan m/z 400 to 1600, 0.5 sec; TOF-MS/MS scan m/z 50 to 2000, 2.0 sec, 9 sec exclusion.LC-MS/MS data were converted to DTA files using extract_msn software (Thermo Inc.) with the following parameters: MW range 550 to 4000, group scan 1, minimum group count 1, minimum ion count 25, and an absolute threshold of 500. The 175,961 DTA files were analyzed using SEQUEST (version27, Thermo Inc.) and a suite of in-house software . A mouse species subset of the Swiss-Prot protein database (version57.2; 16,115 entries) was prepared with concatenated reversed entries and searched with SEQUEST. A parent ion mass tolerance of 2.5 Da was used with average parent ion mass and average fragment ion masses. Cys had a static modification mass of +57 Da (alkylation with iodoacetimide). No enzyme specificity was used. Discriminant function thresholds were set independently by peptide charge, and the number of tryptic termini was set to allow 34,982 identifications, where 445 matches were to reversed entries for an estimated peptide false discovery rate of 1.3%. Protein identification lists were prepared using DTASelect (Version 1.9, the Scripps Research Institute) with post processing of results to improve spectral-count accuracy and allow more strict protein identification criteria. Proteins were considered present in a given sample, if they had two or more peptides with distinct sequences, having a unique count greater than or equal to one in the respective sample. The number of proteins (including contaminants) was 652, with 9 matches to reversed entries for an estimated protein false discovery rate of 1.3%. Each protein identification was assigned a cellular function by PIPE (http://pipe.systemsbiology.net/pipe/#Summary) based on Genome Ontology (GO) terms. […]

Pipeline specifications

Software tools Comet, DTASelect
Databases UniProt
Application MS-based untargeted proteomics
Organisms Mus musculus
Diseases Labyrinth Diseases, Wounds and Injuries, Protein C Deficiency