Non GelBased LCBased Resolution of Integral Membrane Proteins

RP-HPLC-MS/MS. Heazlewood et al. [14] performed complex analysis of Ara-bidopsis whole mitochondrial protein extracts. Batches of 500 | g of mitochondrial protein were acetone precipitated and digested in 100 mM Tris-HCl, pH 8.5 overnight at 37°C with trypsin 1:10 (w/w). Soluble peptides from 15-20 |g of this material were analyzed on a QStar Pulsar MS/MS system (Applied Biosystems, Foster City, CA) using an in-line Agilent 1100 capillary LC system incorporating a 0.150-mm Zorbax C18 reverse-phase column (Agilent, Palo Alto, CA). Peptides were analyzed by MS over a 10-h elution period with increasing acetonitrile concentrations from 2% to 80% (v/v) in water and 0.1% (v/v) formic acid at 4 |L/min. Ions were selected for CID from an initial time-of-flight scan (1 s) if they occurred between 400 and 1500 atomic mass units (m/z ratio), had a charge series of MH2, MH3, or MH4, and had an ion count >10 cps. CID spectra were accumulated for 5 s. The resulting MS/MS-derived spectra were analyzed using Pro ID (Applied Biosystems) at error tolerances of +0.15 for MS and +0.05 for MS/MS, and only the top CID match for each spectrum was accepted. The matching procedure initially used the calculated parent mass of an ion to screen the database for theoretical peptides that fell within the MS error tolerance range. Using this procedure, five independent mitochondrial isolations were digested and analyzed by LC-MS/MS. Proteins qualified for inclusion in the experimental set if the protein matched with a Pro ID confidence score of 98% or greater and if the protein was identified in two or more of the five experiments.

Brugiere et al. [34] performed complex analysis of Arabidopsis mitochondrial hydrophobic protein extracts, but these proteins samples were derived from running each sample on an SDS-PAG in order to embed the proteins in a tight band of a polyacrylamide matrix to facilitate trypsination by a standard in-gel digestion protocol. Following digestion, peptides were extracted from gel pieces with 5% (v/v) formic acid solution and acetonitrile. After drying, tryptic peptides were resuspended in 0.5% aqueous TFA. The samples were injected into an LC-Packings (Dionex) nanoLC system eluted from a C18 column (75 |m x 150 mm) using a gradient from solution A (5% acetonitrile: 95% water: 0.1% formic acid) to solution B (95% ace-tonitrile: 5% water: 0.1% formic acid) over 60 min at a flow rate of 200 nL/min. The LC system was directly coupled to a Q-TOF, and MS and MS/MS data were acquired and processed automatically. Samples from each treatment (C/M extraction, NaOH, and NaCl treatments) were individually analyzed by LC-MS/MS, while a separate sample retrieved from NaCl treatment was analyzed by a 2D chromatography approach using ion-exchange step elutions by 0, 100, 200, and 400 mM KCl upstream to reverse-phase C18 separation. Database searching was carried out using MASCOT. Proteins that were identified with at least two peptides showing a score higher than 40 were validated without any manual validation. For proteins identified by only one peptide having a score higher than 40, the peptide sequence was checked manually. Peptides with scores higher than 20 and lower than 40 were systematically checked and/or interpreted manually to confirm or cancel the MASCOT claim.

The main differences between these studies was the purification of a specific hydrophobic protein samples in the Brugiere et al. [34] study and the longer time used in analysis and the biological replication in the Heazlewood et al. [34] study (1 h versus 10 h and 1 replicate versus 5). These approaches identified 69 proteins in common, with 44 other proteins unique to the study by Brugiere et al. [34] and 347 unique to the study of Heazlewood et al. [14]. Many of the common set are bonafide membrane proteins from Arabidopsis mitochondria.

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