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Quantification of the compositional information provided by immonium ions on a quadrupole-time-of-flight mass spectrometer.

ABSTRACT: Immonium ions have been largely overlooked during the rapid expansion of mass spectrometry-based proteomics largely due to the dominance of ion trap instruments in the field. However, immonium ions are visible in hybrid quadrupole-time-of-flight (QTOF) mass spectrometers, which are now widely available. We have created the largest database to date of high-confidence sequence assignments to characterize the appearance of immonium ions in CID spectra using a QTOF instrument under "typical" operating conditions. With these data, we are able to demonstrate excellent correlation between immonium ion peak intensity and the likelihood of the appearance of the expected amino acid in the assigned sequence for phenylalanine, tyrosine, tryptophan, proline, histidine, valine, and the indistinguishable leucine and isoleucine residues. In addition, we have clearly demonstrated a positional effect whereby the proximity of the amino acid generating the immonium ion to the amino terminal of the peptide correlates with the strength of the immonium ion peak. This compositional information provided by the immonium ion peaks could substantially improve algorithms used for spectral assignment in mass spectrometry analysis using QTOF platforms.

SUBMITTER: Hohmann LJ

PROVIDER: S-EPMC2638499 | biostudies-literature | 2008 Jul

REPOSITORIES: biostudies-literature

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Quantification of the compositional information provided by immonium ions on a quadrupole-time-of-flight mass spectrometer.

Hohmann Laura J LJ Eng Jimmy K JK Gemmill Andrew A Klimek John J Vitek Olga O Reid Gavin E GE Martin Daniel B DB

Analytical chemistry 20080620 14

Immonium ions have been largely overlooked during the rapid expansion of mass spectrometry-based proteomics largely due to the dominance of ion trap instruments in the field. However, immonium ions are visible in hybrid quadrupole-time-of-flight (QTOF) mass spectrometers, which are now widely available. We have created the largest database to date of high-confidence sequence assignments to characterize the appearance of immonium ions in CID spectra using a QTOF instrument under "typical" operati ...[more]

PMID: 18564857

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Project description:There is an immediate need for improved methods to systematically and precisely quantify large sets of peptides in complex biological samples. To date protein quantification in biological samples has been routinely performed on triple quadrupole instruments operated in selected reaction monitoring mode (SRM), and two major challenges remain. Firstly, the number of peptides to be included in one survey experiment needs to be increased to routinely reach several hundreds, and secondly, the degree of selectivity should be improved so as to reliably discriminate the targeted analytes from background interferences. High resolution and accurate mass (HR/AM) analysis on the recently developed Q-Exactive mass spectrometer can potentially address these issues. This instrument presents a unique configuration: it is constituted of an orbitrap mass analyzer equipped with a quadrupole mass filter as the front-end for precursor ion mass selection. This configuration enables new quantitative methods based on HR/AM measurements, including targeted analysis in MS mode (single ion monitoring) and in MS/MS mode (parallel reaction monitoring). The ability of the quadrupole to select a restricted m/z range allows one to overcome the dynamic range limitations associated with trapping devices, and the MS/MS mode provides an additional stage of selectivity. When applied to targeted protein quantification in urine samples and benchmarked with the reference SRM technique, the quadrupole-orbitrap instrument exhibits similar or better performance in terms of selectivity, dynamic range, and sensitivity. This high performance is further enhanced by leveraging the multiplexing capability of the instrument to design novel acquisition methods and apply them to large targeted proteomic studies for the first time, as demonstrated on 770 tryptic yeast peptides analyzed in one 60-min experiment. The increased quality of quadrupole-orbitrap data has the potential to improve existing protein quantification methods in complex samples and address the pressing demand of systems biology or biomarker evaluation studies.

Project description:RationaleThe wide chemical diversity and complex matrices inherent to metabolomics still pose a challenge to current analytical approaches for metabolite screening. Although dedicated front-end separation techniques combined with high-resolution mass spectrometry set the benchmark from an analytical point of view, the increasing number of samples and sample complexity demand for a compromise in terms of selectivity, sensitivity and high-throughput analyses.MethodsPrior to low-field drift tube ion mobility (IM) separation and quadrupole time-of-flight mass spectrometry (QTOFMS) detection, rapid ultrahigh-performance liquid chromatography separation was used for analysis of different concentration levels of dansylated metabolites present in a yeast cell extract. For identity confirmation of metabolites at the MS2 level, an alternating frame approach was chosen and two different strategies were tested: a data-independent all-ions acquisition and a quadrupole broad band isolation (Q-BBI) directed by IM drift separation.ResultsFor Q-BBI analysis, the broad mass range isolation was successfully optimized in accordance with the distinctive drift time to m/z correlation of the dansyl derivatives. To guarantee comprehensive sampling, a broad mass isolation window of 70 Da was employed. Fragmentation was performed via collision-induced dissociation, applying a collision energy ramp optimized for the dansyl derivatives. Both approaches were studied in terms of linear dynamic range and repeatability employing ethanolic extracts of Pichia pastoris spiked with 1 μM metabolite mixture. Example data obtained for histidine and glycine showed that drift time precision (<0.01 to 0.3% RSD, n = 5) compared very well with the data reported in an earlier IM-TOFMS-based study.ConclusionsChimeric mass spectra, inherent to data-independent analysis approaches, are reduced when using a drift time directed Q-BBI approach. Additionally, an improved linear dynamic working range was observed, representing, together with a rapid front-end separation, a powerful approach for metabolite screening.

Dataset Information

Quantification of the compositional information provided by immonium ions on a quadrupole-time-of-flight mass spectrometer.

Publications

Quantification of the compositional information provided by immonium ions on a quadrupole-time-of-flight mass spectrometer.

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