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J Biomol Tech. 2005 September; 16(3): 294–295.
PMCID: PMC2291724

ABRF/ASBMB EDUCATIONAL SYMPOSIUM

An educational symposium entitled “Ion Mobility Selection as an Emerging Technology in Proteomics” was jointly sponsored by ABRF and ASBMB (American Society for Biochemistry and Molecular Biology) and was held at the ASBMB annual meeting in San Diego, April 3, 2005. This symposium was the latest in a series of ABRF/ASBMB jointly sponsored educational symposia held nearly every year at annual ASBMB meetings since 1991. The ASBMB membership includes ABRF members, but also includes users of the core facilities and technologies led by ABRF members.

Mass spectrometry continues to play an increasing role in biomolecular facilities for sensitive and in-depth structural analyses of trace-level proteins, posttranslational modifications, and expression analyses based on either stable isotope incorporation or unlabeled proteolytic peptide ions. The past few years have also been marked by interesting analytical developments in mass spectrometry, including the integration ion mobility selection to improve the sensitivity, specificity, and selectivity of this technique. The purpose of this symposium was thus to inform ASBMB members on the analytical potentials of this emerging technology in the context of proteomics research. The symposium was arranged by ABRF members Ron Niece, who served as the liaison between ABRF and ASBMB, and Pierre Thibault, who coordinated the scientific program. The symposium (one of nine parallel sessions) drew an audience of approximately 100 attendees.

The symposium started with a keynote address by Richard Smith (Pacific Northwest National Lab) who described the separation principles of ion mobility (IM) and high-field asymmetric waveform ion mobility spectrometry ( J Am Soc Mass Spectrom 2005, 16, 2–12 [PubMed] ). In FAIMS, gas-phase ions traveling through a nonreactive gas flowing in the interspace of two concentric electrodes are separated according to differences in ion mobility between time-dependent, high electric field, Kh (~104 V/cm), and low electric field, Kl (< 103 V/cm). This characteristic distinguishes FAIMS from IM mass spectrometry where separation is based on the speed of ion drift through a time-independent and nearly uniformed low electric field. Although these two techniques operate with different separation principles they can both exploit difference in gas-phase mobilities to probe subtle structural changes in protein and peptide ions. Computational modeling of ion dynamics in FAIMS were presented for small biological ions of primary interest in bottom-up proteomics and for small protein ions such as ubiquitin exhibiting different protein conformations in solutions. The ability to probe protein conformation by FAIMS and IM offers new avenues to access protein polymorphism and misfolding, an advantage of obvious biomedical significance as recently demonstrated for conformers of the amyloidogenic protein β2-microglobulin ( Rapid Commun Mass Spectrom 2004, 18, 2229–2234 [PubMed] )

The second talk by Steve Valentine (Indiana University) presented an overview of the analytical capabilities of capillary liquid chromatography combined with IM mass spectrometry for the analysis of plasma digest samples and for the characterization of the fruit fly proteome. The multiply dispersive LC-IM-MS approach provides increased peak capacities and effectively increases the instrumental dynamic range by removing species from spectral regions containing interfering chemical noise ( J Am Soc Mass Spectrom 2001, 12, 1020–1035 ). Advantages of the technique were demonstrated for an analysis of a human plasma digest and provided more than 10-fold increase in the number of resolved components compared with conventional LC-MS approaches (Briefings of Funct. Genomics & Proteomics 2004, 3, 177–186 ). Such measurements were described for 30-min LC separations of plasma digest mixtures. A key advance of the LC-IM-MS method was the incorporation of a split-field drift tube enabling parent ions to be separated based on differences in their low-field mobilities through the first field region before entering the second mobility region where precursors can be transmitted to the mass spectrometer or collisionally activated to provide MS/MS information ( Anal. Chem. 2003, 75, 6202–6208 [PubMed] ; J. Proteome Res. 2005, 4, 25–35 [PubMed] ). Application of this approach was demonstrated in the analysis of tryptic digests of human plasma and for the identification of key proteins associated with the aging of D. melanogaster.

The presentation of Roger Guevremont (Ionalytics Corporation) highlighted the application of FAIMS to separate protein conformers in solution. Indeed, subtle structural changes reflected by three-dimensional conformation and/or charge distribution within the protein ions can be monitored efficiently by FAIMS ( Rapid Commun Mass Spectrom 2001, 15, 1453–1456 [PubMed] ). For example, FAIMS in combination with stop curve measurements enabled the determination of the cross sections of 19 uniquely separated species corresponding to ions of +5 to +13 charge states of bovine ubiquitin ( J Am Soc Mass Spectrom 2000, 11, 738–745 [PubMed] ). FAIMS also provides the ability to profile the pattern of protein conformers from hemoglobin and cytochrome c to identify structural and relative abundance of protein variants and anomalous amino acid sequences from different mammalian species. Conformers of equine cytochrome c were also investigated using FAIMS and hydrogen/deuterium (H/D) exchange experiments and revealed the existence of various conformers with concurrent m/z shift showing extensive H/D exchange (48–63 H/D exchange for the 17+ ion). The complementary nature of this combined approach provides structural insights far superior than analyses conducted using FAIMS or H/D exchange experiments independently.

The last speaker of this session was Pierre Thibault (University of Montreal) who described the analytical merits of FAIMS for the identification of trace-level peptides present in complex protein digests. Capillary LC-MS analyses using FAIMS are characterized by high transmission of multiply-charged peptides ions and by reduction of chemical noise associated with the electrospray process and/or column bleed. The inherent sensitivity and selectivity of FAIMS typically provide gains in signal-to-noise of 10-fold compared with conventional nanoLC-MS experiments with high attomole to low femtomole detection limits on complex protein digests such as blood biomarkers ( PharmaGenomics 2004, May, 30–40 ). An increase of 20% in the number of detected peptides compared with conventional nanoelectrospray was achieved by transmitting ions of different mobilities at high electric field vs. low field while simultaneously recording each ion population in separate acquisition channels. This approach provided excellent reproducibility across replicate nanoLC-FAIMS-MS runs with more than 90% of all detected peptide ions showing less than 30% variation in intensity. The application of this technique in the context of proteomics research was demonstrated for the identification of trace-level proteins such as the transferrin and the galectin-binding receptors showing differential expression in U937 monocyte cell extracts upon exposure to phorbol ester ( Anal Chem 2005, 77, 2176–2186 [PubMed] ).


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