The continuous gas-phase ion separation and atmospheric pressure focusing properties of high-field asymmetric waveform ion mobility spectrometry (FAIMS) offer significant advantages for the mass spectrometric analysis of tryptic digests of proteins. In this study, tryptic peptides of pig hemoglobin were examined by ESI-FAIMS-MS using a newly designed FAIMS device. The new, hemispherical geometry of the inner electrode served to deliver the ions, via the gas flows, to the center axis of the FAIMS analyzer, improving the sensitivity relative to previous prototypes. Mass spectra collected using this new FAIMS showed significantly less chemical background noise than conventional ESI-MS, while maintaining approximately the same absolute sensitivity as that observed with ESI-MS. As a consequence of the ion separation in FAIMS, the identification of the tryptic fragments was simplified and some peptides, such as the triply protonated WAGVANALAHK3+, that were obscured by the intense background of ESI-MS, were readily detected using ESI-FAIMS-MS. In addition, the FAIMS device was shown to separate isobaric ions at m/z 532.4. Correlations between CV and mass-to-charge ratio, as well as CV and ionic collision cross section, were evaluated for 38 peptide ions identified in the tryptic digest. The correlation between the CV of the peptide and the mass-to-charge ratio is very poor, indicating good orthogonality between the separation by FAIMS and the separation by mass spectrometry.
Recent studies indicate that extracellular vesicles are an important source material for many clinical applications, including minimally-invasive disease diagnosis. However, challenges for rapid and simple extracellular vesicle collection have hindered their application. We have developed and validated a novel class of peptides (which we named venceremin, or Vn) that exhibit nucleotide-independent specific affinity for canonical heat shock proteins. The Vn peptides were validated to specifically and efficiently capture HSP-containing extracellular vesicles from cell culture growth media, plasma, and urine by electron microscopy, atomic force microscopy, sequencing of nucleic acid cargo, proteomic profiling, immunoblotting, and nanoparticle tracking analysis. All of these analyses confirmed the material captured by the Vn peptides was comparable to those purified by the standard ultracentrifugation method. We show that the Vn peptides are a useful tool for the rapid isolation of extracellular vesicles using standard laboratory equipment. Moreover, the Vn peptides are adaptable to diverse platforms and therefore represent an excellent solution to the challenge of extracellular vesicle isolation for research and clinical applications.
High-field asymmetric waveform ion mobility spectrometry (FAIMS) separates gas-phase analyte ions from chemical background, offering substantial improvements in the detection of peptides from complex protein digests. For a digest of enolase 1 (baker's yeast), the focusing and separation offered by FAIMS produced an average intensity gain of 3.5 for the tryptic ions and reductions in background intensity of 5- to 10-fold when compared with ESI-MS. The increased signal-to-background in the ESI-FAIMS-MS experiment resulted in a greater number of identifiable peptides and therefore greater sequence coverage. Compensation voltage (CV) maps for a total of 282 tryptic peptides from thirteen proteins, generated according to charge-state, mass-to-charge ratios, and chain length, show that a majority of tryptic peptides can be detected by operating FAIMS at a few discrete values of CV rather than scanning CV across a wide range. The ability to reduce scanning requirements has potential benefits for coupling FAIMS with LC-MS. In select cases, FAIMS can be used to eliminate isobaric MS overlap between tryptic peptides; however, the primary advantage of FAIMS in an LC-FAIMS-MS analysis is foreseen to be the attenuation of chemical background noise rather than the separation of individual peptides. Using FAIMS to reduce mass spectral noise will offer improved detection of peptides from low abundance proteins in complex biological samples.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.