Christopher A. Carroll

Early N-Terminal Changes and Caspase-6 Cleavage of Tau in Alzheimer's Disease

Alzheimers disease (AD) is a progressive amnestic dementia that involves post-translational hyperphosphorylation, enzymatic cleavage, and conformational alterations of the microtubule-associated protein tau. The truncation state of tau influences many of its pathologic characteristics, including its ability to assume AD-related conformations and to assemble into filaments. Cleavage also appears to be an important marker in AD progression. Although C-terminal truncation of tau at D421 has recently been attributed to the apoptotic enzyme caspase-3, N-terminal processing of the protein remains mostly uncharacterized. Here, we report immunohistochemical staining in a cohort of 35 cases ranging from noncognitively impaired to early AD with a panel of three N-terminal anti-tau antibodies: Tau-12, 5A6, and 9G3-pY18. Of these three, the phosphorylation-independent epitope of 5A6 was the earliest to emerge in the pathological lesions of tau, followed by the appearance of the Tau-12 epitope. The unmasking of the Tau-12 epitope in more mature 5A6-positive tangles was not correlated with tau phosphorylation at tyrosine 18 (9G3-pY18). Still, later in the course of tangle evolution, the extreme N terminus of tau was lost, correlating temporally with the appearance of a C-terminal caspase-truncated epitope lacking residues 422-441. In addition, caspase-6 cleaved the N terminus of tau in vitro, preventing immunoreactivity with both Tau-12 and 5A6. Mass spectrometry confirmed that the in vitro caspase-6 truncation site is D13, a semicanonical and hitherto undescribed caspase cleavage site in tau. Collectively, these results suggest a role for caspase-6 and N-terminal truncation of tau during neurofibrillary tangle evolution and the progression of Alzheimers disease.

Serum Protein Expression Profiling for Cancer Detection: Validation of a SELDI-Based Approach for Prostate Cancer

Multiple studies have reported that analysis of serum and other bodily fluids using surface enhanced laser desorption/ionization time of flight mass spectroscopy (SELDI-TOF-MS) can identify a “fingerprint” or “signature” of spectral peaks that can separate patients with a specific disease from normal control patients. Ultimately, classification by SELDI-TOF-MS relies on spectral differences in position and amplitude of resolved peaks. Since the reproducibility of quantitation, resolution and mass accuracy of the SELDI-TOF-MS, or any high throughput mass spectrometric technique, has never been determined this method has come under some skepticism as to its clinical usefulness. This manuscript describes a detailed design of a three-phase study to validate the clinical usefulness of SELDI-TOF-MS in the identification of patients with prostatic adenocarcinoma (PCA). At the end of this validation study, the usefulness of the general SELDI-TOF-MS approach to identifying patients with PCA will be demonstrated and how it compares with PCA diagnosis by measuring prostate specific antigen.

Anti-apoptotic proteins are oxidized by Aβ25–35 in Alzheimer's fibroblasts

We have examined the effects of the beta-amyloid peptide (Abeta(25-35)) on fibroblasts derived from subjects with Alzheimers disease (AD) and from age-matched controls. The peptide was significantly more cytotoxic to the AD-derived fibroblasts. The level of protein oxidation was also greater in the cells from AD subjects. Two-dimensional electrophoresis (2-DE) coupled with immunostaining for protein carbonylation revealed specific oxidation-sensitive proteins (OSPs) in both the control and AD-derived cells. Two specific OSPs were identified by mass spectrometry as heat shock protein 60 (HSP 60) and vimentin. Exposure of the cells to Abeta(25-35) resulted in a twofold increase in the level of oxidation of these two OSPs in the cells derived from controls, but a ninefold increase in their level of oxidation in the fibroblasts from AD subjects. These observations are of particular interest because of the proposed anti-apoptotic roles of both HSP 60 and vimentin and our recent observation that these same two proteins are particularly susceptible to oxidation in neuronally derived cells.

Quantification of phosphorylation of insulin receptor substrate-1 by HPLC-ESI-MS/MS

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) regulates the function and subsequent insulin signaling of this protein. Human IRS-1 has 1242 amino acid residues, including 182 serines and 60 threonines. The size, complexity, and relatively low abundance of this protein in biological samples make it difficult to map and quantify phosphorylation sites by conventional means. A mass spectrometry peak area based quantification approach has been developed and applied to assess the relative abundance of IRS-1 phosphorylation in the absence or presence of stimuli. In this method, the peak area for a phosphopeptide of interest is normalized against the average of peak areas for six selected representative IRS-1 peptides that serve as endogenous internal standards. Relative quantification of each phosphopeptide is then obtained by comparing the normalized peak area ratios for untreated and treated samples. Two non-IRS-1 peptides were added to each digest for use as HPLC retention time markers and additional standards as well as references to the relative quantity of IRS-1 in different samples. This approach does not require isotopic or chemical labeling and can be applied to various cell lines and tissues. Using this method, we assessed the relative changes in the quantities of two tryptic phosphopeptides isolated from human IRS-1 expressed in L6 cells incubated in the absence or presence of insulin or tumor necrosis factor-α. Substantial increases of phosphorylation were observed for Thr446 upon stimulation. In contrast, no obvious change in the level of phosphorylation was observed for Ser1078. This mass spectrometry based strategy provides a powerful means to quantify changes in the relative phosphorylation of peptides in response to various stimuli in a complex, low-abundance protein.

Tryptophan 334 Oxidation in Bovine Cytochrome c Oxidase Subunit I Involves Free Radical Migration

A single tryptophan (W334(I)) within the mitochondrial‐encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W334(I) is converted to hydroxytryptophan as identified by reversed‐phase HPLC‐electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS–PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W334(I) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear‐encoded subunits, W48(IV) and W19(VIIc), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003–1009). Two aromatic‐rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface‐exposed tryptophans, where they produce hydroxytryptophan.

O-Glycosylated 24 kDa human growth hormone has a mucin-like biantennary disialylated tetrasaccharide attached at Thr-60

MS was used to characterize the 24 kDa human growth hormone (hGH) glycoprotein isoform and determine the locus of O‐linked oligosaccharide attachment, the oligosaccharide branching topology, and the monosaccharide sequence. MALDI‐TOF/MS and ESI‐MS/MS analyses of glycosylated 24 kDa hGH tryptic peptides showed that this hGH isoform is a product of the hGH normal gene. Analysis of the glycoprotein hydrolysate by high‐performance anion‐exchange chromatography with pulsed amperometric detection and HPLC with fluorescent detection for N‐acetyl neuraminic acid (NeuAc) yielded the oligosaccharide composition (NeuAc2, N‐acetyl galactosamine1, Gal1). After β‐elimination to release the oligosaccharide from glycosylated 24 kDa hGH, collision‐induced dissociation of tryptic glycopeptide T6 indicated that there had been an O‐linked oligosaccharide attached to Thr‐60. The sequence and branching structure of the oligosaccharide were determined by ESI‐MS/MS analysis of tryptic glycopeptide T6. The mucin‐like O‐oligosaccharide sequence linked to Thr‐60 begins with N‐acetyl galactosamine and branches in a bifurcated topology with one appendage consisting of galactose followed by NeuAc and the other consisting of a single NeuAc. The oligosaccharide moiety lies in the high‐affinity binding site 1 structural epitope of hGH that interfaces with both the growth hormone and the prolactin receptors and is predicted to sterically affect receptor interactions and alter the biological actions of hGH.

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization–tandem mass spectrometry, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 microg protein) or preparative (>250 microg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.