An automated high performance capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometer for high-throughput proteomics

Abstract

We describe a fully automated high performance liquid chromatography 9.4 tesla Fourier transform ion resonance cyclotron (FTICR) mass spectrometer system designed for proteomics research. A synergistic suite of ion introduction and manipulation technologies were developed and integrated as a high-performance front-end to a commercial Bruker Daltonics FTICR instrument. The developments incorporated included a dual-ESI-emitter ion source; a dualchannel electrodynamic ion funnel; tandem quadrupoles for collisional cooling and focusing, ion selection, and ion accumulation, and served to significantly improve the sensitivity, dynamic range, and mass measurement accuracy of the mass spectrometer. In addition, a novel technique for accumulating ions in the ICR cell was developed that improved both resolution and mass measurement accuracy. A new calibration methodology is also described where calibrant ions are introduced and controlled via a separate channel of the dual-channel ion funnel, allowing calibrant species to be introduced to sample spectra on a real-time basis, if needed. We also report on overall instrument automation developments that facilitate high-throughput and unattended operation. These included an automated version of the previously reported very high resolution, high pressure reversed phase gradient capillary liquid chromatography (LC) system as the separations component. A commercial autosampler was integrated to facilitate 24 h/day operation. Unattended operation of the instrument revealed exceptional overall performance: Reproducibility (1-5% deviation in uncorrected elution times), repeatability (Ͻ20% deviation in detected abundances for more abundant peptides from the same aliquot analyzed a few weeks apart), and robustness (high-throughput operation for 5 months without significant downtime). When combined with modulated-ionenergy gated trapping, the dynamic calibration of FTICR mass spectra provided decreased mass measurement errors for peptide identifications in conjunction with high resolution capillary LC separations over a dynamic range of peptide peak intensities for each spectrum of 10 3 , and Ͼ10 5 for peptide abundances in the overall separation. (J Am Soc Mass Spectrom 2004, 15, 212-232)

Advances in proteomics data analysis and display using an accurate mass and time tag approach

“…We observed that the run-to-run reproducibility of proteome analyses improved dramatically with implementation of a fully automated capillary LC-FTICR-MS platform (Belov, et al, 2004). An example of the excellent reproducibility obtained with this automated platform is illustrated in Figure 19, which shows the variation in peptide intensities obtained from six replicate analyses of the same S. oneidensis proteome sample.…”

“…This method benefits from high dynamic range, mass resolution and mass accuracy , Shen, et al, 2001b. Over the last decade we have developed and refined nanoscale capillary LC (nanoLC)-FTICR-MS approaches that have evolved into an accurate mass and time (AMT) tag approach (described below) that has proved to be a robust method for automated high-throughput proteomics studies (Belov, et al, 2004, Paša-Tolić et al, 2004.…”

A proteomic study of the HUPO Plasma Proteome Project's pilot samples using an accurate mass and time tag strategy

“…The column was re-equilibrated with 5% B for 20 minutes and peptides were eluted with a linear gradient from 5% to 70% B over 80 minutes. The capillary LC column was interfaced to either an ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) or to a PNNL-modified 9.4 T Bruker-FTICR mass spectrometer using electrospray ionization [24]. The peptide loading quantity was either 10 or 5 μg for the ion trap MS or FTICR-MS, respectively.…”

“…A Bruker Daltonics 9.4 tesla FTICR mass spectrometer was modified and configured for highthroughput proteomics use as described by Belov et al [24]. Briefly, the FTICR mass spectrometer was combined with the capillary LC system (described above) and modified for concurrent internal mass calibration and auto-sampling.…”

“…Injected samples contained tryptically digested peptides equivalent to 5 μg protein. These analyses typically result in analyzed peptides with <5 part per million (ppm) mass measurement accuracy (MMA), depending on the dynamic range of the measurements, see example spectrum in Figure 2 [24]. While the total analysis time was 4 days of instrument time, the analyses were performed as time became available on the cLC-FTICR MS.…”