Multidimensional nano-HPLC coupled with tandem mass spectrometry for analyzing biotinylated proteins.

Multidimensional high-performance liquid chromatography (HPLC) is a key method in shotgun proteomics approaches for analyzing highly complex protein mixtures by complementary chromatographic separation principles. Here, we describe an integrated 3D-nano-HPLC/nano-electrospray ionization quadrupole time-of-flight mass spectrometry system that allows an enzymatic digestion of proteins followed by an enrichment and subsequent separation of the created peptide mixtures. The online 3D-nano-HPLC system is composed of a monolithic trypsin reactor in the first dimension, a monolithic affinity column with immobilized monomeric avidin in the second dimension, and a reversed phase C18 HPLC-Chip in the third dimension that is coupled to a nano-ESI-Q-TOF mass spectrometer. The 3D-LC/MS setup is exemplified for the identification of biotinylated proteins from a simple protein mixture. Additionally, we describe an online 2D-nano-HPLC/nano-ESI-LTQ-Orbitrap-MS/MS setup for the enrichment, separation, and identification of cross-linked, biotinylated species from chemical cross-linking of cytochrome c and a calmodulin/peptide complex using a novel trifunctional cross-linker with two amine-reactive groups and a biotin label.

Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid.

BACKGROUND: Proteins and peptides occurring in human body fluids can be useful biological markers for neurological diseases and can even contribute to the pathogenesis of such diseases. However, proteins and peptides are potential substrates of proteases and other enzymes. Proteolysis and enzymatic modification may lead to their degradation and modification. METHODS: Using mass spectrometry we investigated the degradation and modification of indicator peptides in the presence of cerebrospinal fluid (CSF). We further applied a fluorometric assay to study the activity of the presumed enzyme glutaminyl cyclase. RESULTS: In CSF we observed an aminopeptidase activity that could partially be inhibited by protease inhibitors and EDTA. In addition, the formation of pyroglutamate (pGlu) from N-terminal glutamine (Gln) was regularly observed. The reaction to pGlu was rapid and protected the indicator peptides from further N-terminal degradation. The conversion of Gln to pGlu could be attributed to the activity of the enzyme glutaminyl cyclase (QC). The QC activity was a characteristic feature of all 45 CSF samples collected from multiple sclerosis patients and controls. CONCLUSION: Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid. The presence of QC in CSF can stabilize peptides from degradation by aminopeptidases. This may have impact for neurological disorders that are characterized by both, the presence of QC and the occurrence of appropriate peptide substrates.

Cyclodextrins found in enzyme- and heat-processed starch-containing foods.

Native and branched-type (glucosylated and maltosylated) cyclodextrins have been isolated and identified in different enzyme- and heat-processed starch-containing food products. Amylolytic enzyme-processed foods such as different beer samples, corn syrup of different dextrose equivalents, and thermally-processed food such as bread, contained minute amounts of different types of cyclodextrins. HPLC/MS Analyses of appropriately preconcentrated and purified food samples indicated the presence of parent beta- and gamma-cyclodextrins and all the three, alpha-, beta-, and gamma-branched cyclodextrins with different degrees of glycosylation. The data presented in this account are thought to be of practical importance in terms of both the analysis methods used for the cyclodextrins and the approval status of different cyclodextrin-containing food, cosmetic, and pharmaceutical products.

Workplace monitoring of isocyanates using ion trap liquid chromatography/tandem mass spectrometry.

A sensitive liquid chromatography/ion trap tandem mass spectrometry method was developed for the qualitative and quantitative detection of isocyanates in air. The method is based on derivatization of isocyanates with 1-(2-methoxyphenyl)piperazine during air sampling. The extracts are analyzed using an ion trap LC/MS system equipped with an electrospray (ESI) ion source. The method shows high linearity, specificity, accuracy and precision. The limits of detection are 40x to 55x lower than with UV-based methods.