High-Resolution Multistage MS, MS², and MS³ Matrix-Assisted Laser Desorption/Ionization FT-ICR Mass Spectra of Peptides from a Single Laser Shot

Abstract

By combined and repeated use of sustained off-resonance irradiation (SORI) for ion dissociation, stored waveform inverse Fourier transform (SWIFT) waveforms for ion isolation, and ion axialization and remeasurements techniques, we obtain for the first time MS, MS2, and MS3 FT-ICR mass spectra from peptide ions (enzymatic digest products of horse cytochrome c) produced from a single laser shot. The successive fragmentation of gas-phase ions detected from the same initial batch of ions increases the sensitivity of analysis of trace amounts of biological samples in structural mass spectrometry, and fragment identification is facilitated by resolution of carbon-13 isotopic distributions. The method is illustrated by analyses of subfemtomole amounts of crudely purified samples of tryptic digest solutions of horse cytochrome c and bovine cytochrome c. The high-resolution primary ion mass spectrum, along with the collision-induced dissociation (CID) and MSn capabilities of FT-ICR, help to determine the primary amino acid sequence of the fragment ions beyond what is obtained from enzymatic digestion alone, without prior chromatographic separation and purification.

Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics

“…Tandem mass spectrometry (MS/MS) was accomplished by isolating the precursor ions of interest in the linear trap quadrupole (LTQ) element, which were then collided with N 2 to activate fragmentation. Multistep activation experiments (MS n ) (Solouki et al 1996;Collings et al 2001) were completed by properly isolating first-generation and subsequent fragments prior to activation. The fragmentation of mass-selected ions was activated by using a typical 25 V collision voltage.…”

“…High-resolution determinations enable the unambiguous differentiation of mononucleotides with very similar elemental compositions that produce nearly overlapping isotopic distributions (Quinn et al 2013). At the same time, multistep tandem mass spectrometry (MS n ) (Solouki et al 1996;Collings et al 2001) and ion mobility spectrometry mass spectrometry (IMS-MS) (von Helden et al 1995;Clemmer and Jarrold 1997;Verbeck et al 2002) have been proven capable of tackling mixtures of isobaric mononucleotides that share the same elemental composition, but display different structures (Quinn et al 2013). These capabilities are exemplified by the analysis of the isomeric species uridine and pseudouridine, which include either an N-or C-glycosidic bond between the pyrimidine ring and ribose unit.…”

Activation of large lons in FT‐ICR mass spectrometry

“…This was attributed to a smaller number of ions and inefficient trapping of fragment ions after multiple SORI-CID stages. High-resolution and high-sensitivity multistage (up to MS 3 ) SORI-CID spectra of peptides were obtained for MALDI generated ions (Solouki et al, 1996). In this work, SORI-CID was followed by axialization of fragment ions.…”

“…In this case, fragment ion identification is facilitated by comparison of experimental and calculated isotopic distributions. The number of consecutive MS stages is practically limited by the fragmentation efficiency of SORI-CID and the efficiency of ion remeasurement (Solouki et al, 1996). Greatest efficiency is achieved if only one major fragment ion is formed in each MS stage.…”

Global Epitranscriptomics Profiling of RNA Post-Transcriptional Modifications as an Effective Tool for Investigating the Epitranscriptomics of Stress Response

“…The selected workflow eschews high-resolution chromatography to minimize possible separation bias, reduce losses during sample transfer/manipulation, and expedite the analysis by reducing the number of necessary operations. Alternative combinations of high-resolution and multistep tandem mass spectrometry (MS n ) (36,37), or ion mobility spectrometry-mass spectrometry (IMS-MS) (38 -40) and gas-phase fragmentation techniques can be independently employed to complete the characterization of the mononucleotide mixtures with excellent sensitivity (34,41). Either combination can afford both identity and abundance of any PTM present in the total RNA extract, thus providing comprehensive and unbiased profiles at the entire transcriptome level.…”