Computational protocol: A method for in vivo identification of bacterial small RNA-binding proteins

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Protocol publication

[…] Peptide digests were analyzed as previously described (Whitney et al. ) by electrospray ionization in the positive ion mode on a hybrid quadrupole-orbitrap mass spectrometer (Q Exactive™; Thermo Fisher, San Jose, CA). The Q Exactive was equipped with a nanoflow HPLC system (NanoAcquity; Waters Corporation, Milford, MA) fitted with a home-built helium-degasser. Peptides were trapped on a homemade 100 μm i.d. × 20 mm long precolumn packed with 200 Å (5 μm, C18AQ; Michrom BioResources Inc., Auburn, CA). Subsequent peptide separation was on an in-house constructed 75 μm i.d. × 180 mm long analytical column pulled using a Sutter Instruments P-2000 CO2 laser puller (Sutter Instrument Company, Novato, CA) and packed with 100 Å (5 μm, C18AQ: Michrom) particle. For each injection, an estimated amount of 1 μg of peptide mixture was loaded onto the precolumn at 4 μL/min in water/acetonitrile (95/5) with 0.1% (v/v) formic acid. Peptides were eluted using an acetonitrile gradient flowing at 250 nL/min using mobile phase consisting of: A, water, 0.1% formic acid; B, acetonitrile, 0.1% formic acid. Peptides were eluted using an acetonitrile gradient flowing at 250 nL/min using mobile phase gradient of 5–35% acetonitrile over 60 min with a total gradient time of 95 min. Ion source conditions were optimized using the tuning and calibration solution recommended by the instrument provider. Data-dependent analyses were acquired using MS survey scans in the Orbitrap followed by data-dependent selection of the 20 most abundant precursors for tandem mass spectrometry. Singly charged ions were excluded from data-dependent analysis. Data redundancy was minimized by excluding previously selected precursor ions for 60 sec following their selection for tandem mass spectrometry. Data were acquired using Xcalibur, version 2.2 (Thermo Fisher).Tandem mass spectra were searched for sequence matches against the UniProt P. aeruginosa PAO1 database using Comet search engine. The following modifications were set as search parameters: peptide mass tolerance at 10 ppm, trypsin digestion cleavage after K or R (except when followed by P), one allowed missed cleavage site, carbamidomethylated cysteine (static modification), and oxidized methionine. Search results were validated by PeptideProphet probability ≥0.9 and ProteinProphet probability ≥0.95 at an error rate less than 1%. […]

Pipeline specifications

Software tools PeptideProphet, ProteinProphet
Application MS-based untargeted proteomics
Organisms Pseudomonas aeruginosa
Diseases Multiple Sclerosis
Chemicals Iron