Online coupling of reverse-phase and hydrophilic interaction liquid chromatography for protein and glycoprotein characterization.
ABSTRACT: We have developed a novel system for coupling reverse-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) online in a micro-flow scheme. In this approach, the inherent solvent incompatibility between RP and HILIC is overcome through the use of constant-pressure online solvent mixing, which allows our system to perform efficient separations of both hydrophilic and hydrophobic compounds for mass spectrometry-based proteomics applications. When analyzing the tryptic digests of bovine serum albumin, ribonuclease B, and horseradish peroxidase, we observed near-identical coverage of peptides and glycopeptides when using online RP-HILIC--with only a single sample injection event--as we did from two separate RP and HILIC analyses. The coupled system was also capable of concurrently characterizing the peptide and glycan portions of deglycosylated glycoproteins from one injection event, as confirmed, for example, through our detection of 23 novel glycans from turkey ovalbumin. Finally, we validated the applicability of using RP-HILIC for the analysis of highly complex biological samples (mouse chondrocyte lysate, deglycosylated human serum). The enhanced coverage and efficiency of online RP-HILIC makes it a viable technique for the comprehensive separation of components displaying dramatically different hydrophobicities, such as peptides, glycopeptides, and glycans.
Project description:The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in ?2-3 and ?2-6 linkages, while these glycans remain attached to peptides.
Project description:Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed "Drop-HILIC", which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical Abstract The organic solvent plays a crucial role in ZIC-HILIC glycopeptide enrichment.
Project description:Analysis of the glycosylation of proteins is a challenge that requires orthogonal methods to achieve separation of the diverse glycoforms. A combination of reversed phase chromatography with tandem mass spectrometry (RP-LC-MS/MS) is one of the most powerful tools for glycopeptide analysis. In this work, we developed and compared RP-LC and hydrophilic interaction liquid chromatography (HILIC) in nanoscale on a chip combined with MS/MS in order to separate glycoforms of two peptides obtained from the tryptic digest of hemopexin. We observed reduction of the retention time with decreasing polarity of glycans attached to the same peptide backbone in HILIC. The opposite effect was observed for RP-LC. The presence of sialic acids prolonged the retention of glycopeptides in both chromatographic modes. The nanoHILIC method provided higher selectivity based on the composition of glycan, compared to nanoRP-LC but a lower sensitivity. The nanoHILIC method was able to partially separate linkage isomers of fucose (core and outer arm) on bi-antennary glycoform of SWPAVGDCSSALR glycopeptide, which is beneficial in the elucidation of the structure of the fucosylated glycoforms.
Project description:The system-wide site-specific analysis of intact glycopeptides is crucial for understanding the exact functional relevance of protein glycosylation. A dedicated workflow with the capability to simultaneously characterize and quantify intact glycopeptides in a site-specific and high-throughput manner is essential to reveal specific glycosylation alteration patterns in complex biological systems. In this study, an enhanced, dedicated, large-scale site-specific quantitative N-glycoproteomics workflow has been established, which includes improved specific extraction of membrane-bound glycoproteins using the filter aided sample preparation (FASP) method, enhanced enrichment of N-glycopeptides using sequential hydrophilic interaction liquid chromatography (HILIC) and multi-lectin affinity (MLA) enrichment, site-specific N-glycopeptide characterization enabled by EThcD, relative quantitation utilizing isobaric N,N-dimethyl leucine (DiLeu) tags and automated FDR-based large-scale data analysis by Byonic. For the first time, our study shows that HILIC complements to a very large extent to MLA enrichment with only 20% overlapping in enriching intact N-glycopeptides. When applying the developed workflow to site-specific N-glycoproteome study in PANC1 cells, we were able to identify 1067 intact N-glycopeptides, representing 311 glycosylation sites and 88 glycan compositions from 205 glycoproteins. We further applied this approach to study the glycosylation alterations in PKM2 knockout cells vs. parental breast cancer cells and revealed altered N-glycoprotein/N-glycopeptide patterns and very different glycosylation microheterogeneity for different types of glycans. To obtain a more comprehensive map of glycoprotein alterations, N-glycopeptides after treatment with PNGase F were also analyzed. A total of 484 deglycosylated peptides were quantified, among which 81 deglycosylated peptides from 70 glycoproteins showed significant changes. KEGG pathway analysis revealed that the PI3K/Akt signaling pathway was highly enriched, which provided evidence to support the previous finding that PKM2 knockdown cancer cells rely on activation of Akt for their survival. With glycosylation being one of the most important signaling modulators, our results provide additional evidence that signaling pathways are closely regulated by metabolism.
Project description:Protein glycosylation analysis is challenging due to the structural variety of complex conjugates. However, chromatographically separating glycans attached to tryptic peptides enables their site-specific characterization. For this purpose, we have shown the importance of selecting a suitable hydrophilic interaction liquid chromatography (HILIC) stationary phase in the separation of glycopeptides and their isomers. Three different HILIC stationary phases, i.e., HALO® penta-HILIC, Glycan ethylene bridged hybrid (BEH) Amide, and ZIC-HILIC, were compared in the separation of complex N-glycopeptides of hemopexin and Immunoglobulin G glycoproteins. The retention time increased with the polarity of the glycans attached to the same peptide backbone in all HILIC columns tested in this study, except for the ZIC-HILIC column when adding sialic acid to the glycan moiety, which caused electrostatic repulsion with the negatively charged sulfobetaine functional group, thereby decreasing retention. The HALO® penta-HILIC column provided the best separation results, and the ZIC-HILIC column the worst. Moreover, we showed the potential of these HILIC columns for the isomeric separation of fucosylated and sialylated glycoforms. Therefore, HILIC is a useful tool for the comprehensive characterization of glycoproteins and their isomers.
Project description:Heterogeneity of protein glycosylation poses great challenges for analysis that is key to understand structure and function of glycoproteins. Resolving this conundrum requires efficient and specific enrichment of intact glycopeptides for identification and quantitation. To this end, hydrophilic interaction chromatography (HILIC) has been commonly used to enrich intact N- and O-linked glycopeptides. However, its effectiveness to enrich isobarically labeled glycopeptides remains unclear. Here, we studied three different enrichment methods for enrichment of N- and O-linked glycopeptides. It was found that removal of N-glycans prior to enrichment of O-linked glycopeptides by HILIC improved identification of O-linked glycopeptides by mass spectrometry. We also compared the enrichment of intact N- and O-linked glycopeptides using other chromatography methods and found that using cartridges containing materials for strong anion exchange (SAX) chromatography increased yield and identification of N- and O-linked glycopeptides. The enrichment of O-linked glycopeptides was further improved when a Retain AX cartridge (RAX) was used. In particular, isobaric tag labeled glycopeptides after C18 desalting could be readily enriched by SAX and RAX cartridges but not by HILIC to enable quantitative glycoproteomics. It is anticipated that the use of SAX and RAX cartridges will facilitate broad applications of identifications and quantitation of glycoproteins.
Project description:Liquid chromatography-mass spectrometry-based metabolomics studies require highly selective and efficient chromatographic techniques. Typically employed reversed-phase (RP) methods fail to target polar metabolites, but the introduction of hydrophilic interaction liquid chromatography (HILIC) is slow due to perceived issues of reproducibility and ruggedness and a limited understanding of the complex retention mechanisms. In this study, we present a comparison of the chromatographic performance of a traditional RP-C18 column with zwitterionic, amide-, alkyl diol-, and aminoalkyl-based HILIC and mixed-mode columns. Our metabolite library represents one of the largest analyte sets available and consists of 764 authentic metabolite standards, including amino acids, nucleotides, sugars, and other metabolites, representing all major biological pathways and commonly observed exogenous metabolites (drugs). The coverage, retention patterns, and selectivity of the individual methods are highly diverse even between conceptually related HILIC methods. Furthermore, we show that HILIC sorbents having highly orthogonal selectivity and specificity enhance the coverage of major metabolite groups in (semi-) targeted applications compared to RP. Finally, we discuss issues encountered in the analysis of biological samples based on the results obtained with human plasma extracts. Our results demonstrate that fast and highly reproducible separations on zwitterionic columns are feasible, but knowledge of analyte properties is essential to avoid chromatographic bias and exclusion of key analytes in metabolomics studies. Graphical Abstract The chromatographic parameters of 764 authentic metabolite standards provide the basis for a comparison of coverage, selectivity and orthogonality of 7 reversed-phase (RP), mixed-mode (MM) and hydrophilic interaction liquid chromatography (HILIC) methods.
Project description:In this work, the capability of newly developed hydrophilic interaction liquid chromatography (HILIC) coupled with matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) platform for quantitative analysis of N-glycans has been demonstrated. As a proof-of-principle experiment, heavy and light stable-isotope labeled hydrazide reagents labeled maltodextrin ladder were used to demonstrate the feasibility of the HILIC-MALDI-MSI platform for reliable quantitative analysis of N-glycans. MALDI-MSI analysis by an Orbitrap mass spectrometer enabled high-resolution and high-sensitivity detection of N-glycans eluted from HILIC column, allowing the re-construction of LC chromatograms as well as accurate mass measurements for structural inference. MALDI-MSI analysis of the collected LC traces showed that the chromatographic resolution was preserved. The N-glycans released from human serum was used to demonstrate the utility of this novel platform in quantitative analysis of N-glycans from a complex sample. Benefiting from the minimized ion suppression provided by HILIC separation, comparison between MALDI-MS and the newly developed platform HILIC-MALDI-MSI revealed that HILIC-MALDI-MSI provided higher N-glycan coverage as well as better quantitation accuracy in the quantitative analysis of N-glycans released from human serum. Graphical abstract Reconstructed chromatograms based on HILIC-MALDI-MSI results of heavy and light labeled maltodextrin enabling quantitative glycan analysis.
Project description:Reversed-phase chromatographic separation of glycopeptides tends to be dominated by the peptide composition. In contrast, capillary zone electrophoresis separation of glycopeptides is particularly sensitive to the sialic acid composition of the glycan. In this paper, we combine the two techniques to achieve superior N-glycopeptide analysis. Glycopeptides were first isolated from a tryptic digest using hydrophilic interaction liquid chromatography (HILIC) solid-phase extraction. The glycopeptides were separated using reversed-phase ultra high-performance liquid chromatography (UHPLC) to generate four fractions corresponding to different peptide backbones. Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) was used to analyze the fractions. We applied this method for the analysis of alpha-1-acid glycoprotein (AGP). A total of 268 site-specific N-glycopeptides were detected, representing eight different glycosylation sites from two isomers of AGP. Glycans included tetra-sialic acids with multi N-acetyllactosamine (LacNAc) repeats and unusual pentasialylated terminal sialic acids. Reversed-phase UHPLC coupled with CZE generated ?35% more N-glycopeptides than direct reversed-phase UHPLC-ESI-MS/MS analysis and ?70% more N-glycopeptides than direct CZE-ESI-MS/MS analysis. This approach is a promising tool for global, site-specific glycosylation analysis of highly heterogeneous glycoproteins with mass-limited samples.
Project description:A MS-based methodology has been developed for analysis of core-fucosylated versus antennary-fucosylated glycosites in glycoproteins. This procedure is applied to the glycoprotein alpha-1-antitrypsin (A1AT), which contains both core- and antennary-fucosylated glycosites. The workflow involves digestion of intact glycoproteins into glycopeptides, followed by double digestion with sialidase and galactosidase. The resulting glycopeptides with truncated glycans were separated using an off-line HILIC (hydrophilic interaction liquid chromatography) separation where multiple fractions were collected at various time intervals. The glycopeptides in each fraction were treated with PNGase F and then divided into halves. One half of the sample was applied for peptide identification while the other half was processed for glycan analysis by derivatizing with a meladrazine reagent followed by MS analysis. This procedure provided site-specific identification of glycosylation sites and the ability to distinguish core fucosylation and antennary fucosylation via a double digestion and a mass profile scan. Both core and antennary fucosylation are shown to be present on various glycosites in A1AT.