Aliphatic dipeptide tags for multi-2-plex protein quantification.

Mass-balanced (1)H/(2)H-isotope dipeptide tag (MBIT) is diversified as aliphatic tags for multiplexed protein quantification. Aliphatic MBITs are based on the N-acetyl-Xxx-Ala dipeptide, where Xxx is an artificial amino acid with a linear alkyl side chain from C(2)H(5) to C(8)H(17) (C(2)-C(8) tags). (1)H/(2)H isotopes are encoded in the methyl groups of N-acetyl and Ala to yield a pair of isobaric tags with 2-plex quantitation signals separated by 3 Da. C(2)-C(5) tags are prepared by solid-phase synthesis, while C(6)-C(8) tags are synthesized by olefin metathesis in solution. These aliphatic tags are made reactive toward the primary amines of peptides, and the relative abundances of quantitation signals are characterized using both matrix-assisted laser desorption ionization and electrospray ionization tandem mass spectrometry. MBIT-linked peptides co-migrate in reverse-phase liquid chromatography (LC), and their tandem mass spectra exhibit 2-plex quantitation signals as well as sequence ions in similar abundances. As the length of alkyl side chain increases, C(2)-C(8) tags show a stepwise increase in both the LC retention time and the relative abundance of quantitation signals. In addition, the quantitation linearity is well-maintained in a 15-250 fmol range. The multiplexing capability of aliphatic MBITs is demonstrated by applying three different tags (C(6)-C(8) tags) to the quantification of yeast heat shock proteins expressed under four different physiological conditions.

Iron (II/III) perchlorate electrolytes for electrochemically harvesting low-grade thermal energy.

Remarkable advances have recently been made in the thermocell array with series or parallel interconnection, however, the output power from the thermocell array is mainly limited by the electrolyte performance of an n-type element. In this work, we investigate iron (II/III) perchlorate electrolytes as a new n-type electrolyte and compared with the ferric/ferrous cyanide electrolyte at its introduction with platinum as the electrodes, which has been the benchmark for thermocells. In comparison, the perchlorate electrolyte (Fe2+/Fe3+) exhibits a high temperature coefficient of redox potential of +1.76 mV/K, which is complementary to the cyanide electrolyte (Fe(CN)63-/Fe(CN)64-) with the temperature coefficient of -1.42 mV/K. The power factor and figure of merit for the electrolyte are higher by 28% and 40%, respectively, than those for the cyanide electrolyte. In terms of device performance, the thermocell using the perchlorate electrolyte provides a power density of 687 mW/m2 that is 45% higher compared to the same device but with the cyanide electrolyte for a small temperature difference of 20 °C. The advent of this high performance n-type electrolyte could open up new ways to achieve substantial advances in p-n thermocells as in p-n thermoelectrics, which has steered the way to the possibility of practical use of thermoelectrics.

Mass-balanced 1H/2H isotope dipeptide tag for simultaneous protein quantitation and identification.

Mass-balanced (1)H/(2)H isotope dipeptide tags (MBITs) are presented for simultaneous protein quantitation and identification. MBIT is derived from N-acetyl-Ala-Ala dipeptide and conjugated to primary amines of target peptides. (1)H/(2)H isotopes are encoded in the methyl groups of N-acetylated dipeptide: one tag deuterated on the N-acetyl group and another on the C-terminal alanine. MBIT-linked peptides comigrate in reversed-phase liquid chromatography without significant (1)H/(2)H isotope effects and provide 2-plex quantitation signals at 114 and 117 Th as well as peptide sequence information upon MS/MS analysis with MALDI TOF/TOF. MBIT shows good quantitation linearity in a concentration range of 20-250 fmol. The performance of MBIT on protein quantitation and identification is further tested with yeast heat-shock protein (Hsp82p) obtained from three different physiological states. MBIT using nanogram-scale samples produces the relative abundance ratios comparable to those obtained from optical imaging of microgram-scale samples visualized with SYPRO Ruby stain. The MBIT strategy is a simple and low-cost alternative for 2-plex quantitation of proteins and offers possibilities of tuning the 2-plex signal mass window by replacing the N-terminal alanine with other amino acid residues.

Structure-activity relationship studies of the chromosome segregation inhibitor, Incentrom A.

A series of Incentrom A analogs that inhibit the chromosome segregation process in yeast were synthesized and tested for their effects on chromosome stability and cell proliferation. Pharmacophore and structure-activity relationship of Incentrom A for the anti-yeast activity were established.

N-acylated dipeptide tags enable precise measurement of ion temperature in peptide fragmentation.

Peptide fragmentations into b- and y-type ions are useful for the identification of proteins. The b ion, having the structure of a N-protonated oxazolone, dissociates to the a-type ion with loss of CO. This CO-loss process affords the possibility of characterizing the temperature of the b ion. Herein, we used N-acylated dipeptide tags, isobaric tags originally developed for protein quantification, as internal standards for the measurement of the ion temperature in peptide fragmentation. Amine-reactive dipeptide tags were attached to the N-termini of sample peptides. Collision-induced dissociation (CID) of the tagged peptides yielded a b-type quantitation signal (b(S)) from the tag, which subsequently dissociated into the a(S) ion with CO-loss. As the length of alkyl side chain on the dipeptide tag was extended from C(1) to C(8), the yield of a(S) ion gradually increased for the 4-alkyl-substituted oxazolone ion but decreased for the 2-alkyl-substituted one. To gain insights into the unimolecular dissociation kinetics, we obtained the potential energy surface from ab initio calculations. Theoretical study suggested that the 4-alkyl substitution on N-protonated oxazolone decreased the enthalpy of activation by stabilizing the productlike transition state, whereas the 2-alkyl substitution increased it by stabilizing the reactant. Resulting potential energy surfaces were used to calculate the microcanonical and canonical rate constants as well as the a(S)-ion yield. Arrhenius plots of canonical rate constants provided activation energies and pre-exponential factors for the CO-loss processes in the 600-800 K range. Comparison of experimental a(S)-ion yields with theoretical values led to precise determination of the temperature of b(S) ion. Thus, the b(S)-ion temperature of tagged peptide can be measured simply by combining kinetic parameters provided here and a(S)-ion yields obtained experimentally. Although the b-type fragment patterns varied with the chain length and position of alkyl substituent on the N-protonated oxazolone, the y-type fragment patterns were almost identical under these conditions. Furthermore, b(S)-ion temperatures were nearly the same with only a few degrees K difference. Our results demonstrate a novel use of N-acylated dipeptide tags as internal temperature standards, which enables the reproducible acquisition of peptide fragment spectra.