Sylvia W. Yuen

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.

Applications of tandem microbore liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/electroblotting in microsequence analysis.

Protein isolation by microbore HPLC is compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/electroblotting methods for several major proteins from rabbit muscle. Although single-mode HPLC or SDS-PAGE/electroblotting provides excellent speed and sensitivity for submicrogram-level protein purification, neither one alone has adequate resolution for separating such a complex protein mixture. Tandem procedures, utilizing two different modes of HPLC in separate steps or a combination of single HPLC separation and SDS-PAGE/electroblotting, offer the necessary versatility. One of the major concerns in this investigation was to evaluate electroblotting techniques for microsequencing. The Aebersold et al. procedure (R.H. Aebersold, D.B. Teplow, L.E. Hood, and S.B.H. Kent (1986) J. Biol. Chem. 261, 4229-4238) was substantially modified and improved; the details of this work will be published elsewhere. These changes significantly improve repetitive yields at the low microgram level without producing high backgrounds. At lower levels the recovery of sequenceable protein currently limits our ability to obtain useful results. Starting with 250-750 micrograms of rabbit muscle crude extract, several proteins (15-70 kDa) were isolated by tandem microbore LC and PAGE/electroblotting for amino-terminal sequence analysis. It appears that the combination of electroblotting and microbore LC represents a powerful approach for microsample preparation.

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.

Progress Toward Polybrene Purification and Utility in Micro-Peptide/Protein Sequencing

Since the introduction of automated Edman degradation(1) there have been continuing attempts to minimize sample loss during the organic extraction steps inherent to the chemistry. These are required to remove excess reagent, buffer, reaction by-products and, of course, the ATZ- amino acid prior to its conversion to the PTH derivative for identification. Various attempts have been made to reduce sample loss: addition of blocked proteins(2), synthetic amino acid polymers(3), and chemically introducing groups which render the sample more hydrophilic(4). Another modification was the covalent attachment of the sample onto an appropriately derivatized matrix, ie. solid phase sequencing(5).