Strategies for automating top-down protein analysis with Q-FTICR MS

Abstract

Thirty years after its invention, the Fourier transform ion cyclotron resonance (FTICR) mass spectrometer continues its evolution into an enabling technology to deepen our understanding of biological systems. For contemporary protein analysis, a quadrupole FTICR hybrid with electrospray ionization is forwarded here as an engine for high resolution tandem mass spectrometry in a high-throughput setting. Three basic strategies for MS/MS of ions >10 kDa are illustrated by identification and characterization of proteins from stationary-phase yeast cells. From samples containing 1–13 proteins, introduced by a nanospray robot, 2–6 can be isolated automatically and fragmented in 15–45 min. Features used commonly for peptide analyses (e.g., multidimensional separations, data dependent acquisition, and probability-based protein identification) are now available in an off-line top-down platform. In one set of 9 samples, 20 proteins (6–17 kDa) were processed through the platform yielding a mean P score=0.002 (99.8% identification confidence) upon database retrieval with characterization of N-terminal post-translational modifications. On occasion of his 60th birthday, we offer this work in celebration of the steadily advancing technology that Alan Marshall helped to invent (now in over 400+ labs worldwide).