Highly specific capture and direct MALDI MS analysis of phosphopeptides by zirconium phosphonate on self-assembled monolayers.

The dynamic range and low stoichiometry of protein phosphorylation frequently demands the enrichment of phosphorylated peptides from protein digests prior to mass spectrometry. Several techniques have been reported in literature for phosphopeptide enrichment, including metal oxides such as TiO(2) and ion metal affinity chromatography (IMAC). While the metal oxides have been used with reasonable success, IMAC has suffered from reduced selectivity and poor reproducibility.

Comparison between vacuum sublimed matrices and conventional dried droplet preparation in MALDI-TOF mass spectrometry.

The properties of several cinnamic acid compounds used as matrices for matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) were investigated as standard dried droplet (DD) and vacuum sublimed preparations. The differences between both preparation methods were analyzed with regard to matrix grain size, internal ion energy, initial velocity, analyte intensity, and analyte incorporation depth. Some of the used cinnamic acid derivatives exhibit clearly reduced grain sizes as sublimed preparations compared with standard DD approaches.

Improved identification of membrane proteins by MALDI-TOF MS/MS using vacuum sublimated matrix spots on an ultraphobic chip surface.

Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface.

High-throughput expression and purification of 6xHis-tagged proteins in a 96-well format.

By using automation and affinity-tag technologies, analysis of the large number of ORFs generated by genome-sequencing projects is greatly accelerated. Protocols describing culture of E. coli in automation-compatible formats and subsequent micro-to large-scale automated purification of 6xHis-tagged proteins are presented.

A highly specific system for efficient enzymatic removal of tags from recombinant proteins.

The TAGZyme system allows efficient and precise exoproteolytic cleavage of N-terminal affinity tags, such as the 6xHis tag, from recombinant proteins. In combination with Ni-NTA technology, the TAGZyme system provides high-purity proteins free of vector-encoded amino acids for use in applications that demand recombinant reagents, an absence of nonspecific cleavage, and a complete removal of all impurities from the target protein preparation. We present results of recent studies on the use of the TAGZyme system.

Automated high-throughput purification of 6xHis-tagged proteins.

Methods based on immobilized-metal affinity chromatography (IMAC) technology for the isolation of 6xHis-tagged proteins offer a one-step purification process that is both robust and meets the challenge of quantitatively purifying thousands of proteins with differing structures and characteristics. To perform this method in a high-throughput, automated format, protocols have been developed that can be run on lab automation workstations. Ready-to-run protocols covering a wide range of protein purification and assay applications, and which rely on the well-established 6xHis-Ni-NTA IMAC technology, are available.