Physical properties, including amyloid morphology, FTIR and CD spectra, enhancement of Congo red absorbance, polymerization rate, critical monomer concentration, free energy of stabilization, hydrophobicity, and the partition coefficient between soluble and amyloid states, were measured for the tau-related peptide Ac-VQIVYK amide (AcPHF6) and its single site mutants Ac-VQIVXK amide (X not equal Cys). Transmission electron microscopy showed that 15 out of the 19 peptides formed amyloid in buffer, with morphologies ranging from straight and twisted filaments to sheets and rolled sheets. Using principal component analysis (PCA), measured properties were treated in a comprehensive manner, and scores along the most significant principal components were used to define individual amino acid amyloidogenic propensities. Quantitative structure-activity modeling (QSAM) showed that residues with greater size and hydrophobicity made the largest contributions to the propensity of peptides to form amyloid. Using individual amino acid propensities, sequences within tau with high amyloid-forming potential were estimated and found to include 226VAVVR230 in the proline-rich region, 275VQIINK280 (PHF6) and 306VQIVYK311 (PHF6) within the microtubule binding region, and 392IVYK395 in the C-tail region of the protein. The results suggest that regions outside the microtubule-binding region may play important roles in tau aggregation kinetics or paired helical filament structure.

Development of a quantification method based on isotopic variants of O-methyl isourea (OMIU) in conjunction with reversed-phase (RP) liquid chromatography (LC) electrospray mass spectrometry is described for determining the relative quantification of -related peptides Ac-VQIVXK-NH 2 . Extracted ion chromatograms of the mass spectrometric data derived from online microcapillary LC separation identifies the retention times of the isotopically derivatized peptides together with their ion abundances. Data-dependent MSMS analysis of both derivatized variants of the same peptide provides a complementary method for identification and resolution between isobaric species. In addition, with respect to offline LC MALDI a larger number of analogues are detected and formation of amyloid is also observed for the aspartic acid and histidine-containing peptides. (J Am Soc Mass Spectrom 2007, 18, 201-207) © 2007 American Society for Mass Spectrometry D efining cellular dynamics in terms of changes in protein expression levels means it is no longer sufficient to simply qualitatively identify proteins; instead, methods for protein quantification are required. Relative protein quantification relies largely on the use of differential stable isotope labeling [1,2]. This strategy can be achieved in vivo, by metabolic labeling of proteins by cell growth in the presence of isotopically labeled amino acids [3], or in vitro by chemical derivatization of the proteins or their corresponding proteolysis products. In both cases the proteins or peptides that are to be compared are labeled separately with isotopic variants of a specific compound. The mixtures containing isotopically derivatized peptides are then recombined before mass spectrometric (MS) analysis. Relative quantification is determined by the ratio of the ion abundances of the two derivatized peptide ions under comparison. In vivo metabolic labeling involves the growth of a given cell population in a medium containing the natural form of an essential amino acid, whereas the other cell population is grown in a medium containing the heavy isotopic variant of this amino acid, such as leucine versus deuterated leucine (Leu-d 3 ) [4,5] or arginine versus deuterated arginine ( 13 C 6 -Arg) [6,7]. A number of methods have been developed for the selective derivatization of functional groups within proteins/peptides using isotopic variants of a given chemical reagent. The first major application of this strategy involved the derivatization of cysteine residues with isotopic variants of the ICAT, a reagent composed of an affinity tag (biotin) separated by a polyether linker from the reactive moiety [8,9]. Alternative postdigestion isotopic labeling techniques include selective derivatization of carboxylic acids [10] and primary amines [11][12][13]. However, these chemical reactions suffer from the large number of reaction sites present in the macromolecule. A far easier reaction to control is the guanidination of lysine residues based on the use of O-methyl isourea (OMIU) [14 -17]. Develop...