Top-down identification and quantification of stable isotope labeled proteins from Aspergillus flavus using online nano-flow reversed-phase liquid chromatography coupled to a LTQ-FTICR mass spectrometer.
Online liquid chromatography-mass spectrometric (LC-MS) analysis of intact proteins (i.e., top-down proteomics) is a growing area of research in the mass spectrometry community. A major advantage of top-down MS characterization of proteins is that the information of the intact protein is retained over the vastly more common bottom-up approach that uses protease-generated peptides to search genomic databases for protein identification. Concurrent to the emergence of top-down MS characterization of proteins has been the development and implementation of the stable isotope labeling of amino acids in cell culture (SILAC) method for relative quantification of proteins by LC-MS. Herein we describe the qualitative and quantitative top-down characterization of proteins derived from SILAC-labeled Aspergillus flavus using nanoflow reversed-phase liquid chromatography directly coupled to a linear ion trap Fourier transform ion cyclotron resonance mass spectrometer (nLC-LTQ-FTICR-MS). A. flavus is a toxic filamentous fungus that significantly impacts the agricultural economy and human health. SILAC labeling improved the confidence of protein identification, and we observed 1318 unique protein masses corresponding to 659 SILAC pairs, of which 22 were confidently identified. However, we have observed some limiting issues with regard to protein quantification using top-down MS/MS analyses of SILAC-labeled proteins. The role of SILAC labeling in the presence of competing endogenously produced amino acid residues and its impact on quantification of intact species are discussed in detail.
Collier
Timothy S
TS
W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA.
Hawkridge
Adam M
AM
Georgianna
D Ryan
DR
Payne
Gary A
GA
Muddiman
David C
DC
D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001231:Aspergillus flavus / Q000737:chemistry* / Q000378:metabolism; D002250:Carbon Radioisotopes / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D005583:Fourier Analysis; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002250:Carbon Radioisotopes; D005656:Fungal Proteins; D001120:Arginine