Applications of hydrophilic interaction chromatography to amino acids, peptides, and proteins.

This review summarizes the recent advances in the analysis of amino acids, peptides, and proteins using hydrophilic interaction chromatography. Various reports demonstrate the successful analysis of amino acids under such conditions. However, a baseline resolution of the 20 natural amino acids has not yet been published and for this reason, there is often a need to use mass spectrometry for detection to further improve selectivity.

Comparative profiling of human saliva by intact protein LC/ESI-TOF mass spectrometry.

Human saliva is finding increasing interest for proteomic and biomarker-discovery studies, due to the ease of collection and potential for simpler processing workflows compared to serum or plasma. However, it is known that salivary protein composition can vary with physiological and environmental factors. In this work, we have examined intra- and inter-person variability of saliva protein composition using an LC/MS methodology to profile low molecular weight human salivary proteins.

Phosphopeptide analysis by directly coupling two-dimensional planar electrochromatography/thin-layer chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A novel strategy is presented for the fractionation of complex peptide mixtures using two-dimensional planar electrochromatography/thin-layer chromatography (2D PEC/TLC). Phosphopeptides migrate more slowly in the first dimension, based upon their anionic phosphate residues, and certain predominantly acidic phosphopeptides even migrate in the opposite direction, relative to the bulk of the peptides. Phosphopeptides are further distinguished based upon hydrophilicity in the second dimension.

Integration of multidimensional chromatographic protein separations with a combined "top-down" and "bottom-up" proteomic strategy.

In this paper, we present a combined top-down/bottom-up proteomic analysis workflow for the characterization of proteomic samples. This workflow combines protein fractionation (multidimensional chromatographic separation) with parallel online ESI-TOF-MS intact protein analysis, and fraction collection. Collected fractions were digested and protein identifications were produced using MALDI Q-TOF-MS analysis.

Evaluation of multidimensional (ion-exchange/reversed-phase) protein separations using linear and step gradients in the first dimension.

The performance characteristics of multidimensional liquid chromatographic protein separations were evaluated using on-line electrospray mass detection, and a novel workflow for automated LC/MS data processing. Two-dimensional ion exchange/reversed-phase LC separations of Escherichia coli cytosol were conducted using either a continuous linear or discontinuous step gradient in the first dimension. Chromatographic profiles of the top 100 most abundant components were characterized to assess overall separation reproducibility within each mode, and to characterize differences in component distribution between the two modes of operation.

Rapid quantitative capillary zone electrophoresis method for monitoring the micro-heterogeneity of an intact recombinant glycoprotein.

A simple high-resolution capillary zone electrophoresis (CZE) method capable of rapidly assessing the micro-heterogeneity of a 24 kDa molecular weight glycoprotein, has been developed. Separation is carried out using a bare silica capillary at a pH of 2.5 in a commercially available electrophoresis buffer system composed of triethanolamine and phosphoric acid.

Capillary isoelectric focusing and affinity capillary electrophoresis approaches for the determination of binding constants for antibodies to the prion protein.

For the development of specific immunological assays, the binding of a specific antibody (Ab) to the target antigen (Ag) has to be relatively strong. In this study, we have utilized affinity capillary electrophoresis (ACE), a form of capillary zone electrophoresis (CZE) to determine the binding constant (Kb) of specific Abs against bovine serum albumin (BSA) and the healthy prion protein (PrPc), in buffer solutions at fixed pHs, approximating in vivo conditions. We have also utilized capillary isoelectric focusing (cIEF) to determine the complexity and recognition of the various isoforms of PrPc Abs towards their Ag, PrPc.

Separation of quaternary ammonium diastereomeric oligomers by capillary electrophoresis.

The separation of novel diastereomeric trimers (3M) and pentamers (5M), derived from quaternary ammonium salts, was studied in conventional, uncoated and coated capillaries using capillary zone electrophoresis (CZE) with a variety of buffers and additives. Resolution of 5M diastereomers was best achieved using gamma-cyclodextrin (gamma-CD) as a chiral selector, while no diastereomeric resolution was realized for the 3M material.

Derivatization of phospholipids.

Phospholipids are major components of biological membranes. Without chemical derivatization, it is difficult to identify and quantitate phospholipids in biological samples. Chemical derivatization can improve both the selectivity and sensitivity of the analytes.

Complexation with iron at sub-ppm levels in HPLC analyses of phospholipids.

Gradient RP-HPLC analysis of a phospholipid, E5564, utilizing water, methanol and phosphoric acid occasionally results in the appearance of a broad unknown peak in the chromatogram before a well-resolved E5564 peak. This unknown peak does not elute in a reproducible fashion with regards to peak shape and retention time, and is not present in chromatograms resulting from injections of the diluent alone. Investigation of this phenomenon revealed that iron ions in sub-ppm levels in the HPLC mobile phase chelated E5564 and the resultant complex(es) comprised the broad peak.

Application of affinity capillary electrophoresis for the determination of binding and thermodynamic constants of enediynes with bovine serum albumin.

The binding constants and thermodynamic properties of a series of novel enediyne compounds with bovine serum albumin (BSA) were determined. The enediynes were synthesized, characterized, and then studied by affinity capillary electrophoresis (ACE) methods to derive these recognition parameters. Change in electrophoretic mobility of BSA as a function of enediyne concentration was determined at 25 degrees C providing binding constants of 1.