An integrated top-down and bottom-up strategy for broadly characterizing protein isoforms and modifications.

ABSTRACT: We present an integrated top-down and bottom-up approach that is facilitated by concurrent liquid chromatography-mass spectrometry (LC-MS) analysis and fraction collection for comprehensive high-throughput intact protein profiling. The approach employs high-resolution, reversed-phase (RP) LC separations coupled on-line with a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer to profile and tentatively identify modified proteins, using detected intact protein masses in conjunction with bare protein identifications from the bottom-up analysis of the corresponding LC fractions. Selected identifications are incorporated into a target ion list for subsequent off-line gas-phase fragmentation that uses an aliquot of the original fraction used for bottom-up analysis. In a proof-of-principle demonstration, this comprehensive strategy was applied to identify protein isoforms arising from various amino acid modifications (e.g., acetylation, phosphorylation) and genetic variants (e.g., single nucleotide polymorphisms, SNPs). This strategy overcomes major limitations of traditional bottom-up (e.g., inability to characterize multiple unexpected protein isoforms and genetic variants) and top-down (e.g., low throughput) approaches.

SUBMITTER: Wu S 

PROVIDER: S-EPMC2693490 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature