How to dig deeper? Improved enrichment methods for mucin core-1 type glycopeptides

Abstract

Two different workflows were tested in order to develop methods that provide deeper insight into the secreted O-glycoproteome. Bovine serum samples were subjected to lectin affinity-chromatography both at the protein- and peptide-level in order to selectively isolate glycopeptides with the most common, mucin core-1 sugar. This enrichment step was implemented with either protein-level mixed-bed ion-exchange chromatography or with peptide-level electrostatic repulsion hydrophilic interaction chromatography. Both methods led to at least 65% of the identified products being glycopeptides, in comparison to ≈ 25% without the additional chromatography steps [Darula, Z., and Medzihradszky, K. F. (2009) Affinity enrichment and characterization of mucin core-1 type glycopeptides from bovine serum. Mol. Cell. Proteomics 8, 2515-2526]. In order to improve not only the isolation but also the characterization of the glycopeptides exoglycosidases were used to eliminate carbohydrate extensions from the directly peptide-bound GalNAc units. Consequent tandem MS analysis of the mixtures using higher-energy collision-dissociation and electron-transfer dissociation led to the identification of 124 glycosylation sites in 51 proteins. While the electron-transfer dissociation data provided the bulk of the information for both modified sequence and modification site assignment, the higher-energy collision-dissociation data frequently yielded confirmation of the peptide identity, and revealed the presence of some core-2 or core-3 oligosaccharides. More than two-thirds of the sites as well as the proteins have never been reported modified.

Fig. 1.

Enrichment strategy for mucin-type core-1 O-glycopeptides.

Fig. 2.

Distribution of glycopeptides from nine abundant proteins in the mixed-bed ion exchange separation fractions. Different site assignments count as unique. The x-axis in represents the elution time: 2 min fractions were collected. (The color version of this Fig. can be seen in supplemental Table S3.) In order to illustrate the complexity of the system more liberal acceptance criteria were applied for the data used in this Figure: discriminative score ≥ 0; peptide score ≥15; E ≤ 0.1; mass error ≤ 10 ppm.

Fig. 3.

HCD (upper panel) and ETD (lower panel) spectra of the E1BCW0 glycopeptide, IQPPPT(HexNAc₂)EALLTLPGPT(HexNAc)AAGPAGR. The precursor ion was at m/z 945.4967 (3+). In the HCD spectrum the Gs indicate the number of sugar units on the peptide fragment. In the ETD spectrum the fragments are fully glycosylated. However, sugar loss from the precursor ion was detected as indicated. labels the original and the charge-reduced precursor ions.

Fig. 4.

Distribution of O-glycoproteins (A) and O-glycosylation sites (B) identified by the Jacalin-mixed-bed ion-exchange and the Jacalin-ERLIC experiment.

Fig. 5.

Amino acid distribution around the O-glycosylation sites determined (supplemental Table S7, color version also is presented there). Ser- and Thr-specific distributions are presented in supplemental Table S8. This Figure was generated by http://weblogo.berkeley.edu/. “The height of symbols within the stack indicates the relative frequency of each amino at that position.”