Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While mass spectrometry (MS)-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry (IMS) separations with liquid chromatography (LC) and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the LC-IMS-MS platform analyzing blood serum samples from 60 post-liver transplant patients with recurrent fibrosis progression and 60 non-transplant patients illustrates its potential utility for disease characterization.
[Show abstract][Hide abstract] ABSTRACT: A new ion mobility spectrometer (IMS) platform was developed to improve upon the sensitivity and reproducibility of our previous platforms, and further enhance IMS-MS utility for broad ‘pan-omics’ measurements. The new platform incorporated an improved electrospray ionization source and interface for enhanced sensitivity, and providing the basis for further benefits based upon implementation of multiplexed IMS. The ion optics included electrodynamic ion funnels at both the entrance and exit of the IMS, an ion funnel trap for ion injection, and a design in which nearly all ion optics (e.g., drift rings, ion funnels) were fabricated using printed circuit board technology. The IMS resolving power achieved was ∼73 for singly-charged ions, very close to the predicted diffusion-limited resolving power (∼75). The platform’s performance evaluation (e.g., for proteomics measurements) include LC-IMS-TOF MS datasets for 30 technical replicates for a trypsin digested human serum, and included platform performance in each dimension (LC, IMS and MS) separately.
International Journal of Mass Spectrometry 07/2014; 377(1). DOI:10.1016/j.ijms.2014.07.034 · 1.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for co-fragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted co-fragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average 9 peptide-spectrum matches (PSMs) per second on a Q-Exactive workbench, while the actual MS/MS acquisition rate was close to 7 spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5,000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.
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