Fig 1.
Coomassie Brilliant Blue stained SDS-electrophoresis gel of immunopurified Gas1-GFP.
Gas1-GFP was constitutively expressed in wild type yeast cells, released by PI-PLC treatment from the cellular membrane fraction, immunopurified from the supernatant with GFP-Trap_A (ChromoTek) and electrophoresed by SDS-PAGE. The staining of the SDS-PAGE gel with Coomassie Brilliant Blue revealed only a band of 160 kDa, which corresponds to the plasma membrane form of Gas1-GFP. This stained band was excised from the gel and the Gas1-GFP protein in the gel slice was extracted for trypsin digestion and subsequent MS analysis of the GPI glycan.
Fig 2.
MS analysis of remodeled GPI-glycan structure of Gas1-GFP.
(A) The base peak chromatogram (BPC) and the extracted ion chromatograms (EICs) of GPI glycan found in Gas1-GFP immunoprecipitated from wild-type. The bottom panel is EIC of a GPI-specific fragment ion treated with PI-PLC (m/z 422.1, GlcN-Ino-P) in MS/MS. (B) The averaged mass spectra (Ave. MS) for 1.60 to 1.88 min on BPC. (C) The proposed structures and the relative abundances (%) of the GPI glycan found in Gas1-GFP immunoprecipitated from wild-type. The relative abundance (%) of each GPI glycan was calculated from the peak intensity of monoisotopic masses of each GPI glycan in EIC using Xcalibur software ver. 2.2. (Thermo Fisher Scientific). These structures contain GPI peptides (KN) bearing hexosamine (glucosamine) attached to Man1 as reported previously [6].
Fig 3.
MS/MS analysis of remodeled GPI-glycan structure of Gas1-GFP.
(A) MS/MS spectra of GPI glycan [3] found in Gas1-GFP immunoprecipitated from wild-type. Each fragment ions are shown with the deduced structures and the observed mass. (B) The theoretical fragmentation pattern of GPI glycan [3] found in Gas1-GFP immunoprecipitated from wild-type. The b-series fragments are indicated by green arrows, and the terminal and internal fragments of the GPI structure are indicated by red and black arrows, respectively. The orange arrows indicate m/z 422 and 243, a GPI-specific fragment ion treated with PI-PLC [6]. (C) Scheme of GPI glycan remodeling of Gas1-GFP. The remodeling of the GPI glycan during transport of Gas1-GFP from the ER to the plasma membrane (PM) involves the addition of a 5th Man on the 4th Man and the removal of the side-branch EtNP of Man1 and Man2.