The filamentous fungus Aspergillus flavus is one of the chief fungal contaminants in world agriculture, wreaking damages and crop losses numbering hundreds of millions of dollars annually.  Inhalation of the fungus causes aspergillosis in humans with compromised immune systems and its generated aflatoxins are the most carcinogenic natural compounds known, causing liver cancer and other hematological malignancies.

Top-down proteomics is a powerful method using accurate intact mass and MS/MS data, covering a large percentage of the protein's sequence, facilitating identification and PTM localization.  The throughput of the method is limited, however, by front end separations necessary to reduce the dynamic range of complex mixtures and by the estimative task of fragmenting large biomolecules in the gas-phase for MS/MS.  Using Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC) generates an internal standard facilitating amino acid counting and relative protein quantification which, when searched against a database along with intact molecular weight, can identify a large portion of the proteome, often eliminating the need for tandem mass spectrometry.

Here, we characterize experimental configurations for reverse phase HPLC-FTMS.  Upon determining the conditions best suited to the experiment using commercial protein standards, they were utilized to fractionate a 1:1 mixture of ¹³C₆-labeled and unlabeled cell lysates from A. flavus which were analyzed downstream on an LTQ-FT to identify isotopic pairs for subsequent quantification and identification against a predicted proteome database.