D R Dupont | PubMed Health

PubMed2000

The alkylated thiohydantoin method for C-terminal sequence analysis.

The alkylated-thiohydantoin method for C-terminal sequencing makes a significant improvement to the thiohydantoin method first described by Schlack and Kumpf. Prior to cleavage from the protein, the C-terminal thiohydantoin is alkylated, making it a better leaving group than the unmodified thiohydantoin. The C-terminal alkylated-thiohydantoin can be cleaved from the protein under conditions that simultaneously form the next thiohydantoin. Combining cleavage and thiohydantoin formation in one step eliminates the need for activating the C-terminal carboxyl group before every sequencing cycle and prevents detection of C-termini formed by random cleavage of peptide bonds in the protein during the sequencing chemistry. The alkylated-thiohydantoin method includes the presequencing modification of cysteine and lysine and the automated modification of aspartic and glutamic acids, serine and threonine. Modifying the reactive side-chain groups improves the ability to sequence through and detect these amino acids. The alkylated-thiohydantoin method can sequence through and detect 19 of the 20 genetically coded amino acids. Sequencing stops at proline residues.

Alkylation↗

PubMed1998

Visualization of proteins by modification of lysines, cysteines, and phosphorylated serines facilitates sample preparation for microsequencing.

A procedure for visualization and sensitive detection of protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent sample preparation for sequence analysis is described. This procedure utilizes either fluorescent or visible tags for certain amino acids in protein molecules, e.g., lysines modified with dansyl/dabsyl chloride and cystines/cysteines or phosphorylated serines modified with iodoacetamidofluorescein (I-15) after proper sample pretreatments. Modifications are performed prior to SDS-PAGE, eliminating the need for fixing, staining, and destaining as required for the conventional procedures. After electrophoresis, the fluorescent or visible bands are excised from the gel, homogenized in microcentrifuge tubes, and soaked in an appropriate buffer to release the separated proteins into solution. Enzymatic digestion can then be carried out in solution for better efficiency of digestion and recovery. The subsequent HPLC mapping and collection of protein digests are performed on PE Applied Biosystems Model 173A MicroBlotter. The separated peptides containing tagged amino acids are visible on the PVDF membrane and can be excised for direct sequence analysis. This approach has been employed for selectively isolating the lysine, cysteine, or phosphorylated serine containing peptides using model proteins. The sequencing results of the peptides generated from premodified proteins demonstrate that this approach facilitates sample preparation for microsequence analysis at low picomole level. Overall recoveries of 20-30% by sequencing initial yields have been achieved using our model proteins.

Amino Acid Sequence↗