Computational protocol: Freeze-dried plasma proteins are stable at room temperature for at least 1 year

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Protocol publication

[…] Plasma samples of 25 μl were dissolved in 225 μl of ice cold 5% formic acid prior to collection of the peptides over a preparative, ZipTip, C18 column []. The ~ 2 µl elution volume was aspirated and ejected across the C18 resin bed carefully 5 times to avoid permitting air bubbles into the resin bed. Collected peptides were eluted off the ZipTip in 2 µl of acidified 65% acetonitrile and immediately diluted with 18 µl of 5% formic acid and injected for analytical HPLC separation over a 15 cm × 300 µm ID column coupled to an electrospray source for the LTQ XL linear ion trap mass spectrometer (Thermo Electron Corporation). A federated library of human proteins was assembled from NCBI, Ensembl and Swiss-Prot and made non-redundant using Structured Query Language (SQL) [, ]. The experimental MS and MS/MS spectra of peptides recorded were correlated to predicted spectra from the federated library at a charge state of 2+ and 3+ to identify fully tryptic peptides using the X!TANDEM [], OMSSA [], MASCOT [] and SEQUEST algorithms [, ] set within ± 3 m/z in the precursor mass and within ± 0.5 Da in the fragment mass with up to three missed cleavages [, ], as proteins may be only partially digested by proteases. Only the best fit peptide to each MS/MS spectra in terms of charge state or amino acid sequence was accepted. […]

Pipeline specifications

Software tools OMSSA, Comet
Databases UniProt
Application MS-based untargeted proteomics
Chemicals Edetic Acid, Nitrogen