Lynn R. Zieske

Hydrophilic-interaction chromatography of complex carbohydrates

Complex carbohydrates can frequently be separated using hydrophilic-interaction chromatography (HILIC). The mechanism was investigated using small oligosaccharides and a new column, PolyGLYCOPLEX. Some carbohydrates exhibited anomer separation, which made it possible to determine the orientation of the reducing end relative to the stationary phase. Amide sugars were consistently good contact regions. Relative to amide sugars, sialic acids and neutral hexoses were better contact regions at lower levels of organic solvents than at higher levels. HILIC readily resolved carbohydrates differing in residue composition and position of linkage. Complex carbohydrate mixtures could be resolved using volatile mobile phases. This was evaluated with native glycans and with glycans derivatized with 2-aminopyridine or a nitrobenzene derivative. Both asialo- and sialylated glycans could be resolved using the same set of conditions. With derivatized carbohydrates, detection was possible at the picomole level by UV detection or on-line electrospray mass spectrometry. Selectivity compared favorably with that of other modes of HPLC. HILIC is promising for a variety of analytical and preparative applications.

A general approach for characterizing glycosylation sites of glycoproteins

Recent advances in glycobiology have greatly stimulated carbohydrate research; however, improving techniques for identification and isolation of specific glycosylation sites in protein structure analysis remains a challenge. We report here a practical approach utilizing a membrane staining technique on Problott, a PVDF-type membrane, to screen glycoproteins and glycopeptides derived from enzymatic digests of glycoproteins. To improve the detection sensitivity, an amplified staining technique using biotinylated lectins, avidin, and biotinylated peroxidase was employed. In addition, we describe a micro-batch affinity binding procedure to isolate glycopeptides from complex glycoprotein enzymatic digests. These protocols allow us to start with a subnanomole quantity of glycoprotein and locate the glycosylation sites; isolate glycopeptides in a homogeneous form; and perform amino acid composition, amino acid sequence, and mass analyses on the isolated glycopeptides. The characterization of glycosylation site of a model glycoprotein, carboxypeptidase P, of which the structure is still largely unknown, has been investigated.

Multi-dimensional mapping of pyridylamine-labeled N-linked oligosaccharides by capillary electrophoresis

A simple, sensitive and reproducible multi-dimensional capillary electrophoresis (CE) oligosaccharide mapping method is reported. The structures of 20 identified N-linked oligosaccharides have been assigned mapping positions from which co-migrating unknown oligosaccharides can be characterized. The separation protocols developed have been demonstrated to separate both charged and neutral oligosaccharides. One dimension involves electroendosmotic flow-assisted CE in a sodium acetate buffer, pH 4.0. A second dimension involves separation based on borate complexation electrophoresis in a polyethylene glycol-containing buffer. A third dimension developed specifically for neutral oligosaccharides, using a sodium phosphate buffer, pH 2.5, has been shown to resolve neutral species not able to be separated by the other two dimensions. Thus, a three-dimensional map was generated to facilitate structural characterization of these oligosaccharides.

Structural determination of the essential serine and glycosylation sites of carboxypeptidase P.

Through a series of kinetic studies involving the inactivation effects of diisopropylfluorophosphate, an affinity label that modifies the active site serine residue involved in the mechanism of action, it has been firmly established that carboxypeptidase P (CPP) requires a serine residue for catalytic activity. The essential kinetic parameters were determined to be 1.33 mM for the apparent dissociation constant with a limiting half-life of inactivation of 20.1 min. Structural elucidation of the primary amino acid sequence surrounding the essential serine, and comparing that with the reactive site of carboxypeptidase Y (CPY), revealed a significant degree of homology at the active site between these two enzymes. These regions, however, were quite divergent from other known serine proteases, leading to the speculation that these serine exopeptidases may comprise a unique family in the overall classification of serine proteases. It was established that CPY could be inactivated with either of the classic histidine affinity labels tosylphenylalanylchloromethyl ketone (TPCK) or carbobenzoxyphenylalanylchloromethyl ketone (ZPCK) with Kis of 1.2 and 12.8 microM, respectively. This is in marked contrast to CPP, which was unaffected by saturating levels of the known histidine affinity labels, TPCK, tosyllysylchloromethyl ketone, or ZPCK. This point may be a significant element in differentiating specificity among these two serine proteases. Further investigation into the structural nature of CPP revealed that it is a glycoprotein with a single site of carbohydrate attachment. In addition, the carbohydrate moiety itself appears to contribute 1217 Da to the overall molecular weight and it is characterized as an asparagine linked high mannose type. This is significantly different from CPY with its four sites of carbohydrate attachment contributing approximately 17% to its molecular weight.

A Facile Method for the Release, Labeling and ce Analysis of Glycoprotein Oligosaccharides

Publisher Summary This chapter describes a facile method for the release, labeling, and capillary electrophoresis (CE) analysis of glycoprotein oligosaccharides. It describes a finger printing method in which N-linked oligosaccharides of glycoproteins are rapidly and effectively released and labeled, facilitating their analytical separation by high-performance liquid chromatography (HPLC) or CE. In this method, oligosaccharides are enzymatically released using peptide- N -glycosidase F, followed by chemical derivatization with the chromophore l-phenyl-3-methyl-5-pyrazolone. PNGase F releases accessible Asn-linked oligosaccharides by cleaving β-aspartylglucosylamine bonds, resulting in the release of intact oligosaccharides. During the process, the asparagine residue to which the carbohydrate was attached is converted to aspartic acid. The protocol developed requires no purification of the released oligosaccharides prior to labeling, and the oligosaccharides once labeled are ready for analysis by CE and/or HPLC immediately following a simple liquid–liquid extraction used for their clean-up. Increasing the PNGase F concentration up to 10-fold did not increase the extent of deglycosylation past the end point that was rapidly achieved with much less enzyme.

A strategy of obtain internal sequence information from blotted proteins after initial N-terminal sequencing

Publisher Summary To generate internal peptide fragments for the identification of sequences from N-terminally blocked proteins, or for the maximization of sequence, information from larger proteins require purification of additional protein sample. With the advent of high sensitivity sample preparation systems employing capillary HPLC, it has become feasible to explore the generation and purification of internal peptide fragments from modest amounts of protein (60 picomole) immobilized onto polyvinylidene difluoride (PVDF) membrane that have previously been subjected to Edman degradation. Initial investigations reveal that after proteins have subjected to Edman chemistry, they are refractory to digestion by the enzymes trypsin, Lys-C, and Glu-C. It is possible to generate internal fragments using chymotrypsin, but the extensive auto-digestion products contaminate the subsequent peptide maps. Chemical cleavage methods were employed resulting in great success. Two proteins—that is, carbonic anhydrase and transferrin, are chosen as models for this study. The experiments discussed in this chapter demonstrates the generation, extraction, and the subsequent purification strategy of internal fragments using both cyanogen bromide to cleave proteins at methionine, and incubation in formic acid at elevated temperature to cut between the aspartic acid and proline. There are two advantages to performing cyanogen bromide digestions in 70% formic acid at an elevated temperature: first, the methionine specific cleavage occurred faster, and second, the cleavage between aspartic acid and proline pairs were catalyzed. This resulted in the generation of more peptide fragments for all samples tested in a relatively short time.

A Unified Approach to Glycoprotein Primary Structure Analysis: Identification, Isolation, and Characterization of both Peptide and Pendant Carbohydrate of Glycopeptides

Publisher Summary This chapter outlines the general strategy for the analysis of glycoproteins, utilizing lectin staining, available peptide separation and sequencing techniques, a simple micro-batch affinity method, PDMS, and PMP labeling of carbohydrates. A classical approach of carbohydrate chemists to glycoprotein analysis is to completely deglycosylate the glycoprotein (using hydrazinolysis), isolate and purify the resulting oligosaccharides, and then do structure determinations. Disadvantages to this scheme are that site information is lost and the peptide bonds are usually completely destroyed as well. Advantages are that most protein laboratories have the necessary instrumentation, and both peptide and carbohydrate structural information can be acquired at levels commensurate with the demands of modern protein chemistry.