Glycosylation is the most abundant protein posttranslational modification and is involved in many relevant biological processes and crucial to the understanding of many diseases. In depth analysis of glycosylation sites is difficult, however, as glycopeptides exhibit a significant micro heterogeneity at glycosylation sites. In addition, ion suppression effects require selective methods for glycopeptide enrichment. Mass spectrometric analysis of glycopeptides is challenging because both the peptide as well as the glycan moiety have to be elucidated for a full structural understanding. We used Fetuin, Asialo-Fetuin and Alpha-1-Acidglycoprotein to equally representing sialylated and non-sialylated glycosylic structures. In addition, monoclonal antibodies were analyzed as a dedicated example for pharmaceutical QC. Proteins were digested with trypsin and glycopeptides were enriched using a dedicated ZICHILIC glycocapture beads in combination with an optimized buffer system (EMD Chemicals Inc.). The glycopeptides were analyzed using ESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for in depth characterization of the glycopeptides. For database searches, an integrated software approach was used: protein searches of the glycopeptide MS/MS spectra were performed for obtaining the amino acid sequence of the glycopeptide, and searches in glycan databases based on the same glycopeptide MS/MS spectra were carried out to complete the characterization of N-linked glycopeptides. In the current study, two important features of glycoprotein analysis are shown: (1) The employed integrated software approach allowed the glycan identification in a similar way as peptide identification. The important step of interactive result validation was facilitated by a suite of dedicated data and result viewers. (2) Compared to MS analysis of native glycoprotein digests, the enriched samples allowed to detect more glycopeptides and permitted the acquisition of higher quality MS/MS spectra. For MALDI-TOF/TOF-MS analysis, linear positive ion mode detection of precursor ions proved to be highly suitable for the analysis of even multi-sialylated glycopeptides.

RP-14 Glycosylation is the most abundant protein posttranslational modification and is involved in many relevant biological processes and crucial to the understanding of many diseases. In depth analysis of glycosylation sites is difficult, however, as glycopeptides exhibit a significant micro heterogeneity at glycosylation sites. In addition, ion suppression effects require selective methods for glycopeptide enrichment. Mass spectrometric analysis of the two distinct glycopeptide moieties is challenging because both the peptide as well as the glycan moiety have to be elucidated for a full structural assignment. We used Fetuin, Alpha-1-Acidglycoprotein and Asialo-Fetuin to equally representing sialylated and non-sialylated glycosylic structures. In addition, monoclonal antibodies were analyzed as a dedicated example for pharmaceutical QC. Samples were digested with trypsin and glycopeptides were enriched using a dedicated ZIC glycocapture resin in combination with an optimized buffer system (EMD Chemicals Inc.). Subsequently, the glycopeptides were analyzed using ESI ion trap MS for glycoprofiling and MALDI-TOF/TOF-MS for in depth characterization of the glycopeptides. In MALDI-TOF/TOF instruments, N-linked glycopeptides undergo an indicative cross-ring fragmentation of the GlcNAc that links to the Asn. Indicative fragments allow obtaining the peptide mass safely as well as the glycan composition. Subsequent Mascot searches of the glycopeptide MS/MS spectra provided for the sequence of the glycopeptide and the localization of the glycosylation site. Searches in glycan databases based on the same glycopeptide MS/MS spectra allowed to complete the characterization of N-linked glycopeptides. For the first time, MALDI-TOF/TOF-MS analysis of multi-sialylated glycopeptides was shown after linear positive ion mode detection of the respective precursor ions. In comparison to direct MS analysis of glycoprotein digests, the enriched samples allowed to detect more glycopeptides and permitted the acquisition of MS/MS spectra of higher quality. Additional separation techniques prior to MS analysis such as RP-HPLC may allow even greater insights into the complexity of protein glycosylation.

Dedicated and specific sample preparation and adequate chromatographic resolution prior to MS are necessary for comprehensive and site-specific glycosylation analysis to compensate for high heterogeneity of protein glycosylation, low-abundance of specific glycoforms and ion-suppression effects caused by coelution of other peptides. This article describes a scheme for glycopeptide profiling, which comprises HILIC batch enrichment followed by complementary HILIC and RP-LC in 1-D and 2-D approaches. For reproducible and sensitive nano-LC/ESI-MS analysis, we used ZIC-HILIC and RP18e monolithic silica capillaries and assessed their retention characteristics and complementarity for glycopeptide separations. The experiments revealed that pre-enrichment of glycopeptides in combination with LC employing both phases considerably improves site-specific elucidation of glycosylation heterogeneity. Zwitterionic hydrophilic interaction liquid chromatography showed high capability to separate glycopeptides by their glycan composition, which coeluted on RP18e. By varying solvent conditions, retention can be well tuned, and efficient separations were achieved even in absence of any additives like salt or formic acid. RP18e facilitated glycopeptide separations with high peak capacity based on peptide sequence and degree of sialylation. Implementing both orthogonal and complementary phases in 1-D and 2-D LC setups was shown to significantly increase the number of different identified glycoforms and possesses great potential for comprehensive glycoproteomics approaches.

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.

Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP-1 transcription factor is considered as facilitator of hypoxia-induced VEGF expression through interaction with hypoxia-inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP-1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP-1 subunit junB is a target gene of hypoxia-induced signaling via NF-kappaB. Loss of JunB in various cell types results in severely impaired hypoxia-induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB-deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB(-/-) teratocarcinomas because of severely impaired paracrine-acting VEGF and the subsequent inability to efficiently recruit host-derived vessels.

The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 microm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the low column back pressures. Also, monolithic columns showed a lower clogging rate in long-term usage for biological samples as compared to particulate columns. The applied system set-up was tested to be useful for the routine peptide screening in search of disease biomarkers.