Large volume injection of aqueous peptide samples on a monolithic silica based zwitterionic-hydrophilic interaction liquid chromatography system for characterization of posttranslational modifications.

Zwitterionic-hydrophilic interaction liquid chromatography (HILIC) has been found very appropriate for separation of polar compounds and peptides with post-translational modifications (PTMs) such as phosphorylation and glycosylation. In this study, a column switching system based on zwitterionic-HILIC silica based monolith columns was used for enrichment and separation of peptides and characterization of N-linked glycosylation by higher-energy collisional dissociation (HCD) Orbitrap mass spectrometry (MS). Peptides were found to be retained on a zwitterionic-HILIC precolumn, even in an aqueous buffer due to electrostatic interactions.

On-line solid phase extraction-liquid chromatography, with emphasis on modern bioanalysis and miniaturized systems.

On-line solid phase extraction (SPE)-liquid chromatography (LC) allows for automated, sensitive, precise and selective bioanalysis. It is a common feature in miniaturized- or nano LC systems, which are well suited for applications requiring high sensitivity and/or treatment of limited samples (laser micro-dissection samples, rare cancer stem cells, etc.).

Comprehensive profiling of N-linked glycosylation sites in HeLa cells using hydrazide enrichment.

The adenocarcinoma cell line HeLa serves as a model system for cancer research in general and cervical cancer in particular. In this study, hydrazide enrichment in combination with state-of-the art nanoLC-MS/MS analysis was used to profile N-linked glycosites in HeLa cells. N-Linked glycoproteins were selectively enriched in HeLa cells by the hydrazide capture method, which isolates all glycoproteins independent of their glycans.

High efficiency, high temperature separations on silica based monolithic columns.

The effect of temperature on separation using reversed-phase monolithic columns has been investigated using a nano-LC pumping system for gradient separation of tryptic peptides with MS detection. A goal of this study was to find optimal conditions for high-speed separations. The chromatographic performance of the columns was evaluated by peak capacity and peak capacity per time unit.

Critical assessment of accelerating trypsination methods.

In LC-MS based proteomics, several accelerating trypsination methods have been introduced in order to speed up the protein digestion, which is often considered a bottleneck. Traditionally and most commonly, due to sample heterogeneity, overnight digestion at 37 °C is performed in order to digest both easily and more resistant proteins. High efficiency protein identification is important in proteomics, hours with LC-MS/MS analysis is needless if the majority of the proteins are not digested.

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter.

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution.

2-D hydrophilic interaction liquid chromatography-RP separation in urinary proteomics--minimizing variability through improved downstream workflow compatibility.

Optimization of every step in a bottom-up urinary proteomics approach was studied with respect to maximize the protein recovery and making the downstream steps in the workflow fully compatible without compromising on the amount of information obtained. Sample enrichment and desalting using centrifugal filtration (5 kDa cut-off) yielded protein recoveries up to 97% when 8 M urea was used. Although yielding lower recoveries (88%), addition of Tris-HCl/NaCl was considered a better choice due to good down-stream compatibility.

Improving the resolution of neuropeptides in rat brain with on-line HILIC-RP compared to on-line SCX-RP.

Our two already established on-line 2-D LC systems, a strong cation exchange-RP chromatography (SCX-RP) system and a hydrophilic interaction LC (HILIC)-RP 2-D LC system, were compared to explore which system is best suited for our further studies of differences in cerebral neuropeptide expression as a function of hypoxia-caused stress. The same mass spectrometer and database search parameters were applied in both systems. In total, 19 first dimension fractions were collected with the novel on-line HILIC-RP system, including a Hypercarb SPE column that was applied to trap the compounds not retained on a Kromasil C18 enrichment column.

Identification of major metal complexing compounds in Blepharis aspera.

Verbascoside and isoverbascoside, present at 0.7% and 0.2% (w/w dryweight), were identified to be major compounds that could contribute to the metal complexation in Blepharis aspera collected in Botswana, Africa.

On-line SPE-Nano-LC-Nanospray-MS for rapid and sensitive determination of perfluorooctanoic acid and perfluorooctane sulfonate in river water.

An instrumental set up including on-line solid-phase extraction, nano-liquid chromatography, and nanospray mass spectrometry is constructed to improve the sensitivity for quantitation of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) in surface water. Sample volumes of 1000 microL are loaded onto a microbore 1.0-mm i.

An alternative multiple-trapping LC-SPE-NMR system.

In this paper, we describe approaches that make RP LC-SPE-NMR simpler, and in our opinion, result in more reliable methods for trapping and subsequent transfer of separated trace-level compounds to the NMR. An SPE unit based on a commercially available, low dead-volume 10 port high-pressure column selector gives the possibility of trapping compounds on nine individual SPEs that have standard fittings. This allows the operator to employ specific stationary phases that are not available as SPEs in commercially available LC-SPE-NMR systems.

Short communication controlling LC-SPE-NMR systems.

There are several stages of the LC-SPE-NMR process that should be monitored closely to ensure an efficient isolation and concentration of the target analyte, for instance analyte break-through and compound transfer from the LC-SPE to the NMR probe. In this study, analyte break-through monitoring was performed with a UV detector and a mass spectrometer placed after the SPE unit. Easy break-through was a problem when attempting multiple trapping of various compounds using C18 SPE cartridges with the original commercial system.