Figure 2

Proteomic Analysis for detection of A3G in LNCaP cells The cell lysate from LNCaP cells was loaded onto SDS-polyacrylamide gel (4-12% gradient gel) and resolved through electrophoresis as described in Figure 1. Pure recombinant A3G was also run on a separate gel. Both the gels were stained with Coomassie brilliant blue. One section of the gel was subjected to immunoblotting using anti-ApoC29 to assist in A3G identification in the gel for mass spectrometry. The gel bands corresponding to A3G protein were excised and were subjected to in-gel trypsin digestion based on the manufacturer's protocol (Thermo Scientific). The resulting peptides were analyzed using a Thermo Finnigan LTQ ion trap instrument ESI. Peptides were separated on a packed capillary tip (Polymicro Technologies, 100 μm × 11 cm) with Jupiter C18 resin (5 μm, 300 Å, Phenomenex) using an in-line solid-phase extraction column (100 μm × 6 cm) packed with the same C18 resin. The total ion chromatogram for the digested pure A3G and A3G from LNCaP cells were compared. The common regions of similar retention time (≈2 minutes) were analyzed to search for m/z peaks corresponding to A3G in the LNCaP sample. The representative peaks were matched with the m/z peaks corresponding to pure A3G. This search revealed that more than 85% of the sequence could be identified in the spectra in the A3G band detected in LNCaP cells. (A) Spectra of purified A3G (m/z 800 to 1100); (B) Mass spectra of A3G from LNCaP cell lysate (m/z 800 to 1100); (C) Spectra of purified A3G (m/z 1100 to 1500); and (D) Spectra of A3G from LNCaP cell lysate (m/z 1100 to 1500). The detected peptides from A3G sequence have been shown in red. The X-axis represents m/z and the Y-axis represents intensity.