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Implementation of dipolar resonant excitation for collision induced dissociation with ion mobility/time-of-flight MS.

ABSTRACT: An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44% and 84%, respectively. The Mathieu q(x,y) parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

SUBMITTER: Webb IK

PROVIDER: S-EPMC4113219 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Implementation of dipolar resonant excitation for collision induced dissociation with ion mobility/time-of-flight MS.

Webb Ian K IK Chen Tsung-Chi TC Danielson William F WF Ibrahim Yehia M YM Tang Keqi K Anderson Gordon A GA Smith Richard D RD

Journal of the American Society for Mass Spectrometry 20140128 4

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly proto ...[more]

PMID: 24470195

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Project description:Glycopeptide-level mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses are commonly performed to establish site-specific protein glycosylation profiles that are of central importance to gaining structure-function insights on glycoproteins. Confoundingly, the complete characterization of glycopeptide connectivity usually requires the acquisition of multiple MS/MS fragmentation spectra. Complementary ion fragmentation techniques such as collision-induced dissociation (CID) and electron transfer dissociation (ETD) are often applied in concert to address this need. While structurally informative, the requirement for acquisition of two MS/MS spectra per analyte places considerable limitations upon the breadth and depth of large-scale glycoproteomic inquiry. Here, a previously developed method of multiplexing CID and ETD is applied to the study of glycopeptides for the first time. Integration of the two dissociation methods was accomplished through addition of an ion mobility (IM) dimension that disperses the two stages of MS/MS in time. This allows the two MS/MS spectra to be acquired within a few milliseconds of one another, and to be deconvoluted in post-processing. Furthermore, the method allows both fragmentation readouts to be obtained from the same precursor ion packet, thus reducing the inefficiencies imposed by separate CID and ETD acquisitions and the relatively poor precursor ion to fragment ion conversion typical of ETD. N-Linked glycopeptide ions ranging in molecular weight from 1.8 to 6.5 kDa were generated from four model glycoproteins that collectively encompassed paucimannosidic, high mannose, and complex types of N-glycosylation. In each case, IM-resolved CID and ETD events provided complete coverage of the glycan topology and peptide sequence coverages ranging from 48.4% (over 32 amino acid residues) to 85.7% (over eight amino acid residues). The potential of this method for large-scale glycoproteomic analysis is discussed.

Project description:Ion mobility-mass spectrometry (IM-MS) has become a powerful tool for glycan structural characterization due to its ability to separate isomers and provide collision cross section (CCS) values that facilitate structural assignment. However, IM-based isomer analysis may be complicated by the presence of multiple gas-phase conformations of a single structure that not only increases difficulty in isomer separation but can also introduce the possibility for misinterpretation of conformers as isomers. Here, the ion mobility behavior of several sets of isomeric glycans, analyzed as their permethylated derivatives, in both nonreduced and reduced forms, was investigated by gated-trapped ion mobility spectrometry (G-TIMS). Notably, reducing-end reduction, commonly performed to remove anomerism-induced chromatographic peak splitting, did not eliminate the conformational heterogeneity of permethylated glycans in the gas phase. At a mobility resolving power of ∼100, 14 out of 22 structures showed more than one conformation. These results highlight the need to use IMS devices with high mobility resolving power for better separation of isomers and to acquire additional structural information that can differentiate isomers from conformers. Online electronic excitation dissociation (EED) MS/MS analysis of isomeric glycan mixtures following G-TIMS separation showed that EED can generate isomer-specific fragments while producing nearly identical tandem mass spectra for conformers, thus allowing confident identification of isomers with minimal evidence of any ambiguity resulting from the presence of conformers. G-TIMS EED MS/MS analysis of N-linked glycans released from ovalbumin revealed that several mobility features previously thought to arise from isomeric structures were conformers of a single structure. Finally, analysis of ovalbumin N-glycans from different sources showed that the G-TIMS EED MS/MS approach can accurately determine the batch-to-batch variations in glycosylation profiles at the isomer level, with confident assignment of each isomeric structure.

Dataset Information

Implementation of dipolar resonant excitation for collision induced dissociation with ion mobility/time-of-flight MS.

Publications

Implementation of dipolar resonant excitation for collision induced dissociation with ion mobility/time-of-flight MS.

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