Notes: The asparagine-glycine-arginine (NGR) sequence can be converted to isoaspartate-glycine-arginine (isoDGR) by asparagine deamidation, and isoDRG can interact with αvβ3, an integrin involved in angiogenesis. The authors tested the hypothesis that a cyclic isoDGR peptide (CisoDGRC) can be used to target delivery of drugs or nanoparticles to tumor neovasculature. The authors produced tumor necrosis factor α tagged with NGR (NGR-TNF) and subjected it to conditions favoring asparagine deamidation to produce CisoDGRC-TNF. The isoaspartate content was determined using the IsoQuant® Isoaspartate Detection Kit. (3900)

Notes: The authors examined the effect of ethanol ingestion on the accumulation of proteins containing atypical isoaspartate residues and determined whether betaine prevented the accumulation of proteins with isoaspartate residues. The authors used the ISOQUANT^® Isoaspartate Detection Kit and [³H] methanol to measure the levels of isoaspartate residues in 20–50µg of liver proteins extracted from rats fed control, ethanol- or betaine-supplemented diets. (3972)

Notes: The authors used H₂¹⁸O to increase the mass shift of recombinant antibody fragments upon asparagine deamidation and aspartic acid isomerization during a month-long incubation at 50°C, as detected by HPLC-mass spectrometry. The increase shift in mass makes it easier to detect these amino acid modifications. The levels of isoaspartate residues in the antibody fragments were quantified using the IsoQuant^® Isoaspartate Detection Kit and HPLC. (3973)

Notes: The authors used the ISOQUANT Isoaspartate Detection Kit to methylate isoaspartyl residues of proteins in mouse brain tissue extracts. The use of the radiolabeled methyl donor S-adenosyl-[³H-methyl]methionine allowed the quantitation of methylation and the identification of L-isoaspartate O-methyltransferase substrates using one dimensional and two-dimensional electrophoresis. Extracts were methylated with recombinant bovine protein isoaspartyl methyltransferase using 20µM S-adenosyl-[³H-methyl]methionine in 100 mM sodium phosphate, pH 6.8, containing 1mM EGTA, 0.16% Triton^® X-100, and 0.004% sodium azide for 30 minutes at 30°C. (3559)

Notes: Aspartyl modifications of the amyloid peptide Aβ are related to protein conformation changes associated with amyloid deposits found in Alzheimer disease patients. The authors investigated region 1–16 of Aβ, the minimal zinc-binding domain, to determine if in vitro aging of Aβ-(1–16) results in aspartyl modifications. The level of isoaspartic acid was quantitated using the ISOQUANT^® Isoaspartate Detection Kit. Reactions were performed with 10µl of a 7.5µM peptide solution at 30°C for 30 minutes, and reaction products were quantitated by reverse phase HPLC analysis. (3665)

Notes: The authors used tetanus toxin C fragment (TTCF) antigen as a model to show that asparagine deamidation interferes with processing by asparagine endopeptidase (AEP) and can influence antigen presentation in T cells. The level of iso-aspartic acid, a product of Asn deamidation, in aged TTCF was quantitated using the IsoQuant^® Isoaspartate Detection Kit. Asn deamidation was confirmed by tryptic digestion using Sequencing Grade Modified Trypsin, followed by MALDI-TOF mass spectrometry. The observed sizes of two tryptic peptides were one mass unit larger than expected, consistent with the fact that Asn deamidation leads to a gain of one mass unit. (3664)

Notes: These authors used mice deficient in the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT) to examine immune tolerance. Spleen, lymph node, and thymus cells were removed from 4-6-week-old wildtype and PCMT-/- mice. These cells were sonicated and the total protein content determined prior to measuring  isoaspartyl content using a PCMT vapor diffusion assay, the radioactive protocol for the ISOQUANT^® Isoaspartate Detection Kit.  PBS was used as a negative control and the isoaspartyl delta sleep-inducing peptide was used as a positive control. (3119)

Notes: In this paper, methyltransferase assays were performed using the ISOQUANT^® Isoaspartate Detection Kit. (2523)

Notes: These authors examined the possible link between autoimmune diseases and isoaspartate residues in self-peptides. Splenic B or T lymphocytes were purified from autoimmune MRL lpr/lpr or non-autoimmune prone B10.A mice. The isolated lymphocytes were treated with lipopolysaccharide or concanavalin A. Cell lysates prepared from 10⁴ cells were assayed for total isoaspartyl content using the radioactive method in the ISOQUANT® Isoaspartate Detection Kit. In addition, native snRNP complexes and native mouse cytochrome c were tested for the presence of isoaspartylated forms of protein by the same method. (3118)

Notes: These authors determined the amount of deamidation in α/ß-type small, acid-soluble spore protein (SASP).  The protein was digested with trypsin then analyzed by reverse-phase high-performance liquid chromatography (HPLC) on a C-18 column using the ISOQUANT^® Isoaspartate Detection Kit.  Modifications included different mobile phases  (0.06% aqueous trifluoroacetic acid and 0.052% trifluoroacetic acid in 80% acetonitrile) and the proteins (100 to 200 pmol) and peptides (50 pmol) analyzed were methylated at 30°C for 1 or 2 hours, respectively. (3122)

Notes: The rat bcl-xL gene was cloned and expressed in E. coli. After isolation, the expressed protein was passed over an anion exchange column to separate the  Bcl-x_L(A) and Bcl-x_L(B) forms.  These proteins were analyzed for isoaspartate content using the ISOQUANT^® Isoaspartate Detection Kit.  To determine which sites in the rat Bcl-x_L protein were deaminated, the protein was digested with lysylendopeptidase and the fragments separated by HPLC.  The isolated peptides were then analyzed by the ISOQUANT^® Isoaspartate Detection Kit; format not specified. (3120)