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Iterative accumulation multiplexing Fourier transform ion cyclotron resonance mass spectrometry.

ABSTRACT: A multiplexed tandem mass spectrometry (MS/MS) technique known as iterative accumulation multiplexing (IAM) has been implemented on a hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer (Q-FTICR-MS). The IAM experiment resulted in obtaining MS/MS spectra for six analytes in two MS/MS experiments while characteristic resolving power and mass measurement accuracies were maintained. Parent-product ion correlations were graphically represented in a "ratiogram" where each product ion is encoded with a ratio unique to the parent ion from which it was formed. This is the first example of multiplexed MS on a FTICR instrument where the ions are encoded externally to the ICR cell. By performing the encoding external to the ICR cell, one set of ions can be encoded while the previous set of ions is being analyzed in the cell, maximizing the use of the continuous ion current emanating from the electrospray ionization source.

SUBMITTER: Bushey JM

PROVIDER: S-EPMC3141178 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Iterative accumulation multiplexing Fourier transform ion cyclotron resonance mass spectrometry.

Bushey Jared M JM Danell Ryan M RM Glish Gary L GL

Analytical chemistry 20090701 14

A multiplexed tandem mass spectrometry (MS/MS) technique known as iterative accumulation multiplexing (IAM) has been implemented on a hybrid quadrupole Fourier transform ion cyclotron resonance mass spectrometer (Q-FTICR-MS). The IAM experiment resulted in obtaining MS/MS spectra for six analytes in two MS/MS experiments while characteristic resolving power and mass measurement accuracies were maintained. Parent-product ion correlations were graphically represented in a "ratiogram" where each pr ...[more]

PMID: 19548663

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Project description:Mass spectrometry (MS) based top-down proteomics provides rich information about proteoforms arising from combinatorial amino acid sequence variations and post-translational modifications (PTMs). Fourier transform ion cyclotron resonance (FT-ICR) MS affords ultrahigh resolving power and provides high-accuracy mass measurements, presenting a powerful tool for top-down MS characterization of proteoforms. However, the detection and characterization of large proteins from complex mixtures remain challenging due to the exponential decrease in S: N with increasing molecular weight (MW) and coeluting low-MW proteins; thus, size-based fractionation of complex protein mixtures prior to MS analysis is necessary. Here, we directly combine MS-compatible serial size exclusion chromatography (sSEC) fractionation with 12 T FT-ICR MS for targeted top-down characterization of proteins from complex mixtures extracted from human and swine heart tissue. Benefiting from the ultrahigh resolving power of FT-ICR, we isotopically resolved 31 distinct proteoforms (30-50 kDa) simultaneously in a single mass spectrum within a 100 m/ z window. Notably, within a 5 m/ z window, we obtained baseline isotopic resolution for 6 distinct large proteoforms (30-50 kDa). The ultrahigh resolving power of FT-ICR MS combined with sSEC fractionation enabled targeted top-down analysis of large proteoforms (>30 kDa) from the human heart proteome without extensive chromatographic separation or protein purification. Further separation of proteoforms inside the mass spectrometer (in-MS) allowed for isolation of individual proteoforms and targeted electron capture dissociation (ECD), yielding high sequence coverage. sSEC/FT-ICR ECD facilitated the identification and sequence characterization of important metabolic enzymes. This platform, which facilitates deep interrogation of proteoform primary structure, is highly tunable, allows for adjustment of MS and MS/MS parameters in real time, and can be utilized for a variety of complex protein mixtures.

Dataset Information

Iterative accumulation multiplexing Fourier transform ion cyclotron resonance mass spectrometry.

Publications

Iterative accumulation multiplexing Fourier transform ion cyclotron resonance mass spectrometry.

Similar Datasets

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