Glycoproteins can present special challenges to those trying to identify them by examining peptides of them using MALDI-MS of gel spots or LC-ESI-MS/MS of purified proteins. In a paper by Valmu et al, they were working with recombinant human transferrin (TRF) purified from mammalian cell hosts in which it was expressed. TRF circulates in serum and its function is to transport the essential element iron (Fe). It is normally N-glycosylated at two sites; N-glycosylation occurs at the Asn-X-Ser/Thr (where X is any amino acid but Pro), and they had a recombinant in which Asn-413 and -611 were changed to Asp (TRF-NG or nonglycosylated form). Human TRF exists as a bilobed globular protein, with a C- and N-side lobes connected by a linking sequence. The group treated TRF and TRF-NG with chymotrypsin, which like trypsin, is often used to generate peptides for doing peptide mass fingerprinting with the MALDI to identify proteins. Chymotrypsin prefers to cut on the carboxyl side of aromatic amino acid residues (Phe, Tyr, and Trp) except when the C-side amino acid is Pro; trypsin, also a member of the serine protease family, works in the same way, but prefers basic amino acids (Lys and Arg). They found that the TRF-NG generated fewer peptides than TRF, and that is because the glycans attached to TRF protected the protein from digestion. Some target sites for chymotrypsin are located near the glycosylation sites of TRF. They observed changes in the HPLC chromatogram (using OD214) as well as MALDI. In using the MALDI, a higher energy setting on the laser was necessary to generate a spectrum for the TRF but not TRF-NG peptides, which they attribute to the difficulty of generating a good yield of peptides. They found some lower intensity peaks in the TRF-NG spectrum and tried to confirm their identity (sequence) using LC-MS/MS. They particularly looked at the chymotryptic peptides that contained the Asn->Asp substitutions, peptides 413-427 (containing Asn/Asp-413, which is C-side of a chymotrypsin target), and peptides 609-622, containing Asn/Asp-611. These peptides were seen in MALDI with about a 3-6 signal-to-nose ratio (and they were using purified proteins NOT from a gel spot cut!!!). They had a difficult time getting fragmentation spectra on the 609-622 peptide; the fragmentation spectrum is used to get the sequence. But they had much better luck getting fragmentation spectra on the 413-472 peptide. They say it was because the peptide contained a Lys, adding more positive charge to the peptide during the electrospray, increasing its ionization efficiency...the additional charges also help fragment the peptides more easily. The mass of the 413-472 peptide is seen at m/z 1690.472 in the MALDI (the [M+H]+ ion), but in ESI, it is seen at m/z 845.46 (the [M+2H]2+ ion). When the collision energy is turned up (to 32 V), you generate the fragmentation. So keep in mind that glycans (oligosaccharide modifications) on proteins can affect their digestion and the peptide masses that you might expect! Many of you might see this in the mass spectral data on your projects, and you need to think about possibly different masses. L Valmu, N Kalkkinen, A Husa, PD Rye (2005) Biochemistry 44: 16007-16013. Differential Susceptibility of Transferrin Glycoforms to Chymotrypsin: A Proteomics Approach To The Detection of Carbohydrate-Deficient Transferrin --------------------------------- Want to start your own business? Learn how on Yahoo! Small Business.