Improvement of Automatic In-Gel Digestion by in Situ Alkylation of Proteins

We have recently improved the automation of an in-gel digestion system, DigestPro 96, using in situ alkylation of proteins with acrylamide, conducted during one-dimensional (1D) SDS-PAGE. The improved method included the processes of destaining, dehydration, trypsin digestion, and extraction but excluded the reduction and alkylation steps following staining of proteins with CBB. The extracted peptide mixtures were directly loaded onto a micro C18 LC column of the mass spectrometer. The resultant spectra were processed with “Mascot” search engine to estimate the sequence coverage of the bovine serum albumin (BSA). The original method, designed for Laemmli 1D SDS gel applications, consisted of reduction and post-alkylation with iodoacetamide, which produced carboxyamidemethyl (CAM; –S–CH₂CONH₂) derivatives. The original method also included a desalting step essential for mass spectrometry, especially matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We compared the original and improved methods using BSA (3 pmol loaded to the gel, one third of digested peptide mixture injected into LC-MS). The original method yielded both CAM and propionicamide (PAM; –S–CH₂CH₂CONH₂) derivatives. The source of PAM derivatives is the unpolymerized acrylamide formed during electrophoresis. The sequence coverage of CAM derivatives of BSA by the original method was 10% with desalting and 19% without desalting. The sequence coverage of PAM derivative by the improved method was 32%. Our results clearly show the advantage of our improved automated in-gel digestion method for in situ PAM alkylated protein with respect to peptide recovery, compared with the original method with CAM post-alkylation.     

Asymmetric synthesis of β-amino alcohol by reductive cross-coupling of planar chiral ferrocenecarboxaldehyde with N-tosyl ferrocenylideneamine, and its application to asymmetric reaction

Samarium iodine-mediated cross-coupling of N-tosyl ferrocenylideneamine with planar chiral ferrocenecarboxaldehyde gave diastereoselectively anti-β-amino alcohol derivative in good yield. The obtained anti-β-amino alcohol with ferrocene ring at 1,2-positions was utilized as chiral auxiliary for asymmetric alkylation and acylation reactions.     

An efficient synthesis of anhydrides of 2-monosubstituted succinic acid monoesters and their application to the Dakin–West reaction: Isolation of some 5-succinyloxyoxazole intermediates

A rapid and versatile synthesis of 2-monosubstituted monosuccinates and their bimolecular anhydrides is described. The reaction of these anhydrides with N-Fmoc-protected dipeptides under the modified Dakin–West reaction affords mainly the corresponding 5-succinyloxyoxazoles.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S--2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 m 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.Reference to a company or product name does not imply approval or recommendation of the product by the U.S. Department of Agriculture to the exclusion of others that may be suitable.Abbreviations used are Mops: 3-(N-morpholino)propanesulfonic acid; STI: soybean trypsin inhibitor (Kunitz); 2-PE-STI:S--2-pyridylethylated STI; 2-QEC:S--(2-quinolylethyl)-l-cysteine; 2-QE-STI:S--2-quinolylethylated STI; TosPheCH₂-trypsin: bovine trypsin treated withp-toluenesulfonyl phenylalanine chloromethyl ketone.     

Incorporation of 3H-N-ethylmaleimide into sheep red cell membrane thiol groups following protection by diamide-induced oxidation

The thiol oxidant diazene dicarboxylic acid bis [N,N-dimethylamide] (diamide) is known to reversibly activate K-Cl cotransport in sheep red blood cells [1]. Although the detailed mechanism of activation is unknown, functional thiols at the membrane or at the cytoplasmic level are recognized as important. To search for membrane bound thiols involved in the regulation of K-Cl cotransport, sheep red cells were first exposed to diamide at concentrations activating K-Cl cotransport, and then to the alkylating agent N-ethylmaleimide (NEM) in order to block non-oxidized thiols. White ghosts, prepared by osmotic lysis from these cells, were again treated with NEM followed by reduction of the diamide-induced dithiols with dithio-threitol (DTT) concentrations known to reverse the diamide-induced K-Cl flux [1]. Maximum ³H-NEM incorporation into the DTT-reduced thiols occurred at 50 M DTT. Saturation labelling by ³H-NEM of about 2 × 10⁴ diamide-protected thiols/cell occurred at 25 M NEM. Diamide protected about 0.1% of all membrane thiols chemically determined earlier [2]. Membranes from high K (HK) and low K (LK) sheep red cells did not differ significantly in the number of diamide-protected thiols, and polyacrylamide gels revealed a similar protein distribution of 3H-NEM-labelled thiols. Since diamide is known to stimulate K-Cl flux in LK cells ten times more than in HK cells this finding is consistent with the hypothesis of a cytoplasmic control effecting different K-Cl flux activities in the membranes of the two cation genotypic red blood cells.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

The alkaline hydrolysis of phosphotriesters in alkylated mammalian DNA

Isolated DNA was alkylated with $\text{N-[}{}^{14}\text{C]methyl}\text{-N-}\text{nitrosourea}$ or $\text{N-[}{}^{14}\text{C]ethyl}\text{-N-}\text{nitrosourea}$. Sedimentation analysis of the alkylated DNA before and after alkaline hydrolysis was used to determine the number of single-strand breaks introduced by hydrolysis of the triesters. Vacuum distillation from alkylated DNA solutions before and after alkaline hydrolysis was used to determine the numbers of triesters hydrolysing to the alcohol.     

Inhibition of human DNA polymerase β activity by the anticancer prodrug Cloretazine

The antineoplastic prodrug Cloretazine exerts its cytotoxicity via a synergism between 2-chloroethylating and carbamoylating activities that are cogenerated upon activation in situ. Cloretazine is reported here to inhibit the nucleotidyl-transferase activity of purified human DNA polymerase β (Pol β), a principal enzyme of DNA base excision repair (BER). The 2-chloroethylating activity of Cloretazine alkylates DNA at the O⁶ position of guanine bases resulting in 2-chloroethoxyguanine monoadducts, which further react to form cytotoxic interstrand DNA crosslinks. Alkylated DNA is often repaired via BER in vivo. Inhibition of the polymerase activity of Pol β may account for some of the synergism between Cloretazine’s two reactive subspecies in cytotoxicity assays. This inhibition was only observed using agents with carbamoylating activity. Furthermore, while therapeutically relevant concentrations of Cloretazine inhibited the polymerase activity of Pol β, the enzyme’s lyase activity, which may also participate in BER, was not significantly inhibited.     

Stereospecificity of sterol biosynthesis in Calendula officinalis flowers

Flowers of Calendula officinalis were incubated with mevalonic acid doubly labelled with ¹⁴C in position 2 and ³H in positions 2R, 2S, 4R or 5R,S and the [³H/¹⁴C] ratios determined in squalene β-sitosterol, stigmasterol, Δ⁷-sterols and stigmastan-3 β-ol. The results indicated that in the biosynthesis of these sterols: formation of the Δ⁷ double bond is associated with elimination of hydrogen from the 7β position, formation of the Δ⁵ double bond with elimination of hydrogens from the 5 and 6α positions, and formation of the Δ²² double bond with elimination of the 22-pro-S and 23 hydrogens. Demethylation in position 4 is associated with elimination of hydrogen from the 3α position whereas demethylation in position 14 occurs without hydrogen loss from position 15. Alkylation in position 24 is associated with hydrogen elimination from this position.