sepharose and tris(2-carboxyethyl)phosphine

Topics: Buffers; Cations; Cysteine; Osmolar Concentration; Phosphines; Research Design; Sepharose

Reduction of disulfide bonds is - in many cases - a critical pretreatment step for the determination of thiols in real samples. This study reports the first systematic investigation of the potentials of the on-line reduction of disulfide bonds under flow conditions in a sequential injection setup. One of the most promising reducing agents, tris-(2-carboxyethyl)phosphine (TCEP) was selected for this purpose while the Ellman's disulfide (DTNB) was used as model compound. The study involved the investigation of several parameters that affected the kinetics and efficiency of the reaction, including stopped-flow experiments. Both soluble and immobilized TCEP on agarose beads were examined. The results confirmed that both forms of TCEP can be used as an advantageous on-line reducing reagent for disulfide bonds under flow conditions.

Topics: Disulfides; Flow Injection Analysis; Kinetics; Microspheres; Online Systems; Phosphines; Sepharose; Solubility

Dynamic range and the presence of highly abundant proteins limit the number of proteins that may be identified within a complex mixture. Cysteine (Cys) has unique chemical reactivity that may be exploited for chemical tagging/capture with biotin/avidin reagents or affinity chromatography allowing specific isolation and subsequent identification of peptide sequences by mass spectrometry. Organomercurial agarose (Hg-beads) specifically captures Cys-containing peptides and proteins from cell lysates. Tryptic peptides from yeast lysates containing Cys were captured and eluted from Hg-beads after incubation with TCEP and trypsin. From two 1 h nano 1-D LC DDA/MS of the eluate >700 proteins were identified with an estimated false positive rate of approximately 1%. Few peptides were identified with high confidence without Cys within their sequence after capture, and extensive washing, indicating little nonspecific binding. The number of fragmentation spectra was increased using automated 2-D nano-LC/MS and allowed identification of 1496 proteins with an estimated false positive rate of 1.1%. Approximately 4% of the proteins identified were from peptides that did not contain Cys, and these were biased toward higher abundance proteins. Comparison of the 1496 proteins to those reported previously showed that >25% were from yeast proteins not previously observed. Most proteins were identified from a single peptide, and sequence coverage was sacrificed by focusing only on identifying Cys-containing peptides, but large numbers of proteins were rapidly identified by eliminating many of the peptides from the higher abundance proteins.

Topics: Chromatography, Affinity; Cysteine; Organomercury Compounds; Phosphines; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sepharose; Tandem Mass Spectrometry; Trypsin