Coupling methanol denaturation, immobilized trypsin digestion, and accurate mass and time tagging for liquid-chromatography-based shotgun proteomics of low nanogram amounts of RAW 264.7 cell lysate.

ABSTRACT: We report the shotgun proteomic analysis of mammalian cell lysates that contain low nanogram amounts of protein. Proteins were denatured using methanol, digested using immobilized trypsin, and analyzed by UPLC-ESI-MS/MS. The approach generated more peptides and higher sequence coverage for a mixture of three standard proteins than the use of free trypsin solution digestion of heat- or urea-denatured proteins. We prepared triplicate RAW 264.7 cell lysates that contained 6, 30, 120, and 300 ng of protein. An average of 2 ± 1, 23 ± 2, 134 ± 11, and 218 ± 26 proteins were detected for each sample size, respectively. The numbers of both protein and peptide IDs scaled linearly with the amount of sample taken for analysis. Our approach also outperformed traditional methods (free trypsin digestion

SUBMITTER: Sun L 

PROVIDER: S-EPMC3477608 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature