ch C₁₈, 0.15 x 100 mm
HPLC gradient: 2% acetonitrile, 0.1% formic acid for 3 minutes, ramp to 60% acetonitrile in 90 minutes, ramp to 80% acetonitrile in 5 minutes, hold at 80% for 20 minutes. Discussion Recombinant human tissue plasminogen activator (rt-PA) was expressed in Chinese hamster ovary cells and purified. This glycoprotein has an approximate molecular weight of 64 kDa and a heterogeneous distribution of N-linked glycoforms. The purified protein was reduced and alkylated at two sites, then digested with trypsin. A 2-g aliquot of the digest mixture was analyzed. Automated Data Dependent LC-MS/MS was used to generate the chromatogram shown in Figure 1. Figure 1. Base peak chromatogram from Data Dependent LC-MS/MS of a rt-PA tryptic digest.
The product ion spectra from the tryptic digest mixture were used by TurboSEQUEST software to search the human protein database. Figure 2 shows that the protein was identified as tPA. Sequence coverage is shown. The peptides highlighted in red were immediately identified using TurboSEQUEST, representing 79% coverage of the protein. The peptides labeled in blue were not initially identified, however each of these unknown peptides contains an Asn-X-Ser/Thr motif (with the Asn labeled in green), strongly suggesting that these peptides might be glycosylated.

N-linked oligosaccharides in human proteins are generally predictable but cell lines may incorporate uncommon structures making direct confirmation of complete structure with MSⁿ important. All have the same core structure with extensions and branches composed prim
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