Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Abstract

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip-based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C(18) LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C(18) chips. On the other hand, hydrophobic glycopeptides were better retained on C(18) chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four-protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase.

Figure 1

Extracted-ion chromatograms depicting the retention time reproducibility and repeatability of activated graphitized carbon for (a) the glycopeptide SRNLTK-GlcNAc₂Man₅ derived from the tryptic digestion of bovine ribonuclease B, and (b) the glycopeptide NVGLNR-GlcNAc₂(Fuc)Man₃(Xyl) derived from tryptic digestion of horseradish peroxidase. Ribonuclease B tryptic digest (500 fmol) was injected 10 times in one day, while horseradish peroxidase tryptic digest (500 fmol) was injected 10 times over two days (5 injections per day).

Figure 2

Extracted-ion chromatogram of glycopeptides derived from tryptic digestion of bovine ribonuclease B and analyzed using the activated graphitized carbon chip. Inset, CID spectrum of the glycopeptide to which Man₅ is attached.

Figure 3

Extracted-ion chromatogram of the tryptic glycopeptide NVGLNR-GlcNAc₂(Fuc)Man₃(Xyl) derived from horseradish peroxidase and analyzed with an activated graphitized carbon chip. Inset, CID spectrum of the glycopeptide.

Figure 4

Extracted-ion chromatogram for the horseradish peroxidase tryptic glycopeptide LYNFSNTGLPDPTLNTTYLQTLR to which GlcNAc₂(Fuc)Man₃(Xyl) glycan structure is attached. Inset, ETD tandem mass spectrum of the glycopeptide.

Figure 5

Extracted-ion chromatogram of the tryptic glycopeptide LHFHDCFVNGCDASILLDNTTSFR-GlcNAc₂(Fuc)Man₃(Xyl) derived from horseradish peroxidase and analyzed using activated graphitized carbon (a) and C₁₈ (b) chips. This peptide was identified by accurate mass matching.

Figure 6

Extracted-ion chromatogram of the tryptic glycopeptide GLIQSDQELFSSPNATDTIPLVR-GlcNAc₂(Fuc)Man₃(Xyl) derived from horseradish peroxidase and analyzed using C₁₈ chip. Inset, CID spectrum of the glycopeptide.

Figure 7

Base-peak chromatogram of a 4-protein mixture attained using activated graphitized carbon (a) and C₁₈ (b) chips. The concentration of each protein was 100 fmol/μl, while 1 μl was injected in each case.