Top-down protein identification of proteasome proteins with nanoLC-FT-ICR-MS employing data-independent fragmentation methods.
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.
Department of Chemistry and Biochemistry, Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA, USA.
Rajeswari
R
Lakshmanan
Jeremy J
JJ
Wolff
Rudy
R
Alvarado
Joseph A
JA
Loo
D002853:Chromatography, Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry*; D046988:Proteasome Endopeptidase Complex / Q000032:analysis / Q000737:chemistry*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000295:instrumentation / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000295:instrumentation / Q000379:methods*
D010446:Peptide Fragments; D046988:Proteasome Endopeptidase Complex
Electrospray ionization; Fourier transform-ion cyclotron resonance; Proteasome; Protein LC-MS; Technology; Top-down mass spectrometry