Fig. 2
From: Rapid characterization of secreted recombinant proteins by native mass spectrometry
Multiple forms of TfR1 are produced in Sf9 cells. a Direct-MS analysis of secreted TfR1 from insect cell growth medium reveals four different populations of the protein. The major population corresponds in mass to the protein, modified with GlcNac2Man3Fuc1 (TfR1a), leading to a 1039 Da shift in mass. In addition, three other populations of TfR1 can be detected, corresponding in mass to the intact protein (TfR1), the protein labeled with two GlcNac2Man3Fuc1 hexasaccharides (TfR1b), as well as a population labeled with the pentasaccharide GlcNac2Man3 (TfR1c). The charge state series labeled with green circles represents GFP, which is accumulating intercellularly, but is present due to cell death, and spillage of the cellular contents into the growth medium. b In order to strip the glycosylations from TfR1, we treated the growth medium with PNGase F. Direct-MS analysis indicated that after 15 min of incubation, only charge state series corresponding to the unmodified form of the protein remained, while the glycosylated TfR1 populations disappeared. The series labeled with brown circles represent PNGase F. c SDS-PAGE and (d) Western blot analysis reveal the different glycoforms of TfR1 prior to and following incubation with PNGase F. The blots in d were reacted with a His Probe HRP-Conjugate to detect TfR1 and an anti-GFP antibody. The uncropped gel and blot images can be seen in Supplementary Fig. 11B-C. e Direct-MS spectra, focused on the low m/z scale, were acquired, following the addition of PNGase F to the growth medium. Two major glycans were detected, corresponding in mass to protonated GlcNac2Man3Fuc1 and GlcNac2Man3, respectively, with a free reducing end. These oligosaccharides were not detected in the medium prior to PNGase F treatment (f). A background peptide from the growth medium is labeled with a red asterisk. g, h MS/MS analysis of the two glycans validated their assignment. Each glycan was selected within the quadrupole mass filter, and subjected to MS/MS fragmentation. The selected glycan is enclosed within a box, and the various ion fragments generated are annotated
