1. Top‐down protein identification of proteasome proteins with nanoLC‐FT‐ICR‐MS employing data‐independent fragmentation methods
- Author
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Lakshmanan, Rajeswari, Wolff, Jeremy J, Alvarado, Rudy, and Loo, Joseph A
- Subjects
Biological Sciences ,Biomedical and Clinical Sciences ,Chromatography ,Liquid ,Humans ,Peptide Fragments ,Proteasome Endopeptidase Complex ,Proteomics ,Spectrometry ,Mass ,Electrospray Ionization ,Tandem Mass Spectrometry ,Electrospray ionization ,Fourier transform-ion cyclotron resonance ,Proteasome ,Protein LC-MS ,Technology ,Top-down mass spectrometry ,Information and Computing Sciences ,Medical and Health Sciences ,Biochemistry & Molecular Biology ,Biological sciences ,Biomedical and clinical sciences - Abstract
A comparison of different data-independent fragmentation methods combined with LC coupled to high-resolution FT-ICR-MS/MS is presented for top-down MS of protein mixtures. Proteins composing the 20S and 19S proteasome complexes and their PTMs were identified using a 15 T FT-ICR mass spectrometer. The data-independent fragmentation modes with LC timescales allowed for higher duty-cycle measurements that better suit online LC-FT-ICR-MS. Protein top-down dissociation was effected by funnel-skimmer collisionally activated dissociation (FS-CAD) and CASI (continuous accumulation of selected ions)-CAD. The N-termini for 9 of the 14 20S proteasome proteins were found to be modified, and the α3 protein was found to be phosphorylated; these results are consistent with previous reports. Mass-measurement accuracy with the LC-FT-ICR system for the 20- to 30-kDa 20S proteasome proteins was 1 ppm. The intact mass of the 100-kDa Rpn1 subunit from the 19S proteasome complex regulatory particle was measured with a deviation of 17 ppm. The CASI-CAD technique is a complementary tool for intact-protein fragmentation and is an effective addition to the growing inventory of dissociation methods that are compatible with online protein separation coupled to FT-ICR-MS.
- Published
- 2014