Ying-Qing Yu

Characterization of glycoprotein digests with hydrophilic interaction chromatography and mass spectrometry

A new hydrophilic interaction chromatography (HILIC) column packed with amide 1.7 μm sorbent was applied to the characterization of glycoprotein digests. Due to the impact of the hydrophilic carbohydrate moiety, glycopeptides were more strongly retained on the column and separated from the remaining nonglycosylated peptides present in the digest. The glycoforms of the same parent peptide were also chromatographically resolved and analyzed using ultraviolet and mass spectrometry detectors. The HILIC method was applied to glyco-profiling of a therapeutic monoclonal antibody and proteins with several N-linked and O-linked glycosylation sites. For characterization of complex proteins with multiple glycosylation sites we utilized 2D LC, where RP separation dimension was used for isolation of glycopeptides and HILIC for resolution of peptide glycoforms. The analysis of site-specific glycan microheterogeneity was illustrated for the CD44 fusion protein.

Mixed-mode chromatography for fractionation of peptides, phosphopeptides, and sialylated glycopeptides

A mixed-mode chromatographic (MMC) sorbent was prepared by functionalizing the silica sorbent with a pentafluorophenyl (PFP) ligand. The resulting stationary phase provided a reversed-phase (RP) retention mode along with a relatively mild strong cation-exchange (SCX) retention interaction. While the mechanism of interaction is not entirely clear, it is believed that the silanols in the vicinity of the perfluorinated ligand act as strongly acidic sites. The 2.1 mm x 150 mm column packed with such sorbent was applied to the separation of peptides. Linear RP gradients in combination with salt steps were used for pseudo two-dimensional (2D) separation and fractionation of tryptic peptides. An alternative approach of using linear cation-exchange gradients combined with RP step gradients was also investigated. Besides the attractive forces, the ionic repulsion contributed to the retention mechanism. The analytes with strong negatively charged sites (phosphorylated peptides, sialylated glycopeptides) eluted in significantly different patterns than generic tryptic peptides. This retention mechanism was employed for the isolation of phosphopeptides or sialylated glycopeptides from non-functionalized peptide mixtures. The mixed-mode column was utilized in conjunction with a phosphopeptide enrichment solid phase extraction (SPE) device packed with metal oxide affinity chromatography (MOAC) sorbent. The combination of MOAC and mixed-mode chromatography (MMC) provided for an enhanced extraction selectivity of phosphopeptides and sialylated glycopeptides peptides from complex samples, such as yeast and human serum tryptic digests.

Phosphopeptide enrichment using microscale titanium dioxide solid phase extraction

Identification of phosphopeptides by MS is challenging due to their relatively low abundance in proteomic samples and their limited ionization efficiency. Various affinity enrichment methods have been used in the literature. Titanium dioxide SPE devices have been recently proposed as an alternative to immobilized metal affinity chromatography for phosphopeptide enrichment. This study evaluates the TiO(2 )method using sorbent packed in a 96 well microscale extraction plate operated using a vacuum manifold. The phosphopeptide recovery and enrichment selectivity were investigated at various loading conditions. The effectiveness of organic additives such as dihydroxybenzoic acid derivatives and other nonaliphatic carboxylic acids on enrichment selectivity was examined. The performance of TiO(2) was compared to IMAC sorbent. The results suggest that various additives improve the enrichment selectivity by effectively interfering with the acidic peptides binding to TiO(2) sorbent. Interaction of phosphopeptides with sorbent is also affected, which leads to overall reduction in phosphopeptide recovery. The new SPE device was successfully utilized for the extraction of phosphopeptides from yeast lysate digest using 2,5-dihydroxybenzoic acid to minimize the interference from nonphosphorylated peptides.

Ultra‐performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) and UPLC/MSE analysis of RNA oligonucleotides

Fast and efficient ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) analysis of short interfering RNA oligonucleotides was used for identity confirmation of the target sequence-related impurities. Multiple truncated oligonucleotides and metabolites were identified based on the accurate mass, and their presumed sequence was confirmed by MS/MS and MS(E) (alternating low and elevated collision energy scanning modes) methods. Based on the resulting fragmentation of native and chemically modified oligonucleotides, it was found that the MS(E) technique is as efficient as the traditional MS/MS method, yet MS(E) is more general, faster, and capable of producing higher signal intensities of fragment ions. Fragmentation patterns of modified oligonucleotides were investigated using RNA 2-ribose substitutions, phosphorothioate RNA, and LNA modifications. The developed sequence confirmation method that uses the MS(E) approach was applied to the analysis of in vitro hydrolyzed RNA oligonucleotide. The target RNA and metabolites, including the structural isomers, were resolved by UPLC, and their identity was confirmed by MS(E). Simultaneous RNA truncations from both termini were observed. The UPLC quadrupole time-of-flight (QTOF) MS/MS and MS(E) methods were shown to be an effective tool for the analysis and sequence confirmation of complex oligonucleotide mixtures.

Comprehensive characterization of the N-glycosylation status of CD44s by use of multiple mass spectrometry-based techniques

The CD44 family are type-1 transmembrane glycoproteins which are important in mediating the response of cells to their microenvironment, including regulation of growth, survival, differentiation, and motility. All these important functions have been reported to be regulated by N-glycosylation; however, little is known about this process. In the CD44 family, the most prolific isoform is CD44 standard type (CD44s). In this work, an integrated strategy combining stable isotope labeling, chemical derivatization, hydrophilic-interaction liquid chromatographic (HILIC) separation, and mass spectrometric (MS) identification was used to perform a comprehensive qualitative and quantitative survey of the N-glycosylation of recombinant CD44s. Specifically, the occupation ratios of the N-glycosites were first determined by MS with 18O labeling; the results revealed five glycosites with different occupation ratios. Next, N-glycans were profiled by chemical derivatization and exoglycosidase digestion, followed by MALDI–TOF-MS and HILIC–ESI–MS–MS analysis. Interestingly, the quantitative analysis showed that non-sialylated, fucosylated complex-type glycans dominated the N-glycans of CD44s. Furthermore, the site-specific N-glycan distributions profiled by LC–ESI–MSE indicated that most glycosites bore complex-type glycans, except for glycosite N100, which was occupied by high-mannose-type N-glycans. This is the first comprehensive report of the N-glycosylation of CD44s.FigureStrategies for characterization of the N-glycosylation status of CD44s