Brad J. Williams

Hepatic phenotype of liver fatty acid binding protein gene-ablated mice

Although the function of liver fatty acid binding protein in hepatic fatty acid metabolism has been extensively studied, its potential role in hepatic cholesterol homeostasis is less clear. Although hepatic cholesterol accumulation was initially reported in L-FABP-null female mice, that study was performed with early N2 backcross generation mice. To resolve whether the hepatic cholesterol phenotype in these L-FABP(-/-) mice was attributable to genetic inhomogeneity, these L-FABP(-/-) mice were further backcrossed to C57Bl/6 mice up to the N10 (99.9% homogeneity) generation. Hepatic total cholesterol accumulation was observed in female, but not male, L-FABP(-/-) mice at all (N2, N4, N6, N10) backcross generations examined. The greater total cholesterol was due to increased hepatic levels of both unesterified (free) cholesterol and esterified cholesterol. Altered hepatic cholesterol accumulation correlated directly with L-FABPs ability to bind cholesterol with high affinity as shown by direct L-FABP binding of fluorescent cholesterol analogs (NBD-cholesterol, dansyl-cholesterol), a photoactivatable cholesterol analog [free cholesterol benzophenone (FCBP)], and free cholesterol (circular dichroism, isothermal titration microcalorimetry). One mole of fluorescent sterol was bound per mole of L-FABP. This was confirmed by photo-cross-linking studies with the photoactivatable cholesterol analog FCBP and by isothermal titration calorimetry with free cholesterol, which showed that L-FABP bound only one sterol molecule per L-FABP molecule. In contrast, the hepatic phenotype of male, but not female, L-FABP(-/-) mice was characterized by decreased hepatic triacylglycerol levels at all backcross generations examined. Taken together, these data support the hypothesis that L-FABP plays a role in physiological regulation of not only hepatic fatty acid metabolism, but also that of hepatic cholesterol.

Structure and Function of the Virulence-Associated High-Temperature Requirement A of Mycobacterium tuberculosis

The high-temperature requirement A (HtrA) family of serine proteases has been shown to play an important role in the environmental and cellular stress damage control system in Escherichia coli. Mycobacterium tuberculosis ( Mtb) has three putative HtrA-like proteases, HtrA1, HtrA2, and HtrA3. The deletion of htrA2 gives attenuated virulence in a mouse model of TB. Biochemical analysis reveals that HtrA2 can function both as a protease and as a chaperone. The three-dimensional structure of HtrA2 determined at 2.0 A resolution shows that the protease domains form the central core of the trimer and the PDZ domains extend to the periphery. Unlike E. coli DegS and DegP, the protease is naturally active due to the formation of the serine protease-like catalytic triad and its uniquely designed oxyanion hole. Both protease and PDZ binding pockets of each HtrA2 molecule are occupied by autoproteolytic peptide products and reveal clues for a novel autoregulatory mechanism that might have significant importance in HtrA-associated virulence of Mtb.

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

Although sterol carrier protein-2 (SCP-2) is encoded as a precursor protein (proSCP-2), little is known regarding the structure and function of the 20-amino acid N-terminal presequence. As shown herein, the presequence contains significant secondary structure and alters SCP-2: (i) secondary structure (CD), (ii) tertiary structure (aqueous exposure of Trp shown by UV absorbance, fluorescence, and fluorescence quenching), (iii) ligand binding site [Trp response to ligands, peptide cross-linked by photoactivatable free cholesterol (FCBP)], (iv) selectivity for interaction with anionic phospholipid-rich membranes, (v) interaction with a peroxisomal import protein [FRET studies of Pex5p(C) binding], the N-terminal presequence increased SCP-2s affinity for Pex5p(C) by 10-fold, and (vi) intracellular targeting in living and fixed cells (confocal microscopy). Nearly 5-fold more SCP-2 than proSCP-2 colocalized with plasma membrane lipid rafts and caveolae (AF488-CTB); 2.8-fold more SCP-2 than proSCP-2 colocalized with a mitochondrial marker (Mitotracker), but nearly 2-fold less SCP-2 than proSCP-2 colocalized with peroxisomes (AF488 antibody to PMP70). These data indicate the importance of the N-terminal presequence in regulating SCP-2 structure, cholesterol localization within the ligand binding site, membrane association, and, potentially, intracellular targeting.

Enhanced Detection of Low-Abundance Host Cell Protein Impurities in High-Purity Monoclonal Antibodies Down to 1 ppm Using Ion Mobility Mass Spectrometry Coupled with Multidimensional Liquid Chromatography

The enormous dynamic range of proteinaceous species present in protein biotherapeutics poses a significant challenge for current mass spectrometry (MS)-based methods to detect low-abundance HCP impurities. Previously, an HCP assay based on two-dimensional chromatographic separation (high pH/low pH) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry and developed in the authors laboratory has been shown to achieve a detection limit of about 50 ppm (parts per milion) for the identification and quantification of HCPs present in monoclonal antibodies following Protein A purification.1 To improve the HCP detection limit we have explored the utility of several new analytical techniques for HCP analysis and thereby developed an improved liquid chromatography-mass spectrometry (LC-MS) methodology for enhanced detection of HCPs. The new method includes (1) the use of a new charge-surface-modified (CSH) C18 stationary phase to mitigate the challenges of column saturation, peak tailing, and distortion that are commonly observed in the HCP analysis; (2) the incorporation of traveling-wave ion mobility (TWIM) separation of coeluting peptide precursors, and (3) the improvement of fragmentation efficiency of low-abundance HCP peptides by correlating the collision energy used for precursor fragmentation with their mobility drift time. As a result of these improvements, the detection limit of the new methodology was greatly improved, and HCPs present at a concentration as low as 1 ppm (1 ng HCP/mg mAb) were successfully identified and quantified. The newly developed method was applied to analyze two high-purity mAbs (NIST mAb and Infliximab) expressed in a murine cell line. For both samples, low-abundance HCPs (down to 1 ppm) were confidently identified, and the identities of the HCPs were further confirmed by targeted MS/MS experiments. In addition, the performance of the assay was evaluated by an interlaboratory study in which three independent laboratories performed the same HCP assay on the mAb sample. The reproducibility of this assay is also discussed.

Autoinducer AI-2 is involved in regulating a variety of cellular processes in Salmonella Typhimurium.

Salmonella Typhimurium is known to exhibit LuxS/AI-2-mediated cell signaling. We investigated the role of LuxS/AI-2 system on Salmonella Typhimurium protein expression using a proteomics approach based on two-dimensional gel electrophoresis (2DGE)-MALDI-MS. The global protein expression profiles of the wild-type, a luxS mutant, and a luxS mutant strain supplemented with AI-2 were compared. Seven proteins were differentially expressed when comparing the wild-type and luxS mutant strains, whereas 13 proteins were differentially expressed when comparisons were made between luxS mutant strains with and without AI-2 supplementation. The seven proteins that were differentially expressed between the wild-type and the luxS mutant strain were also differentially expressed in the luxS mutant strain supplemented with AI-2. The level of PhoP, a virulence determinant, was higher in the presence of AI-2. Proteins associated with the carbohydrate metabolism (pfkA, gpmI, and talB) and ATP synthesis (Pta gene product) were up-regulated by the presence of AI-2 molecules. These results provide experimental evidence that AI-2 molecules regulate a variety of cellular processes in Salmonella Typhimurium.

High-throughput method for on-target performic acid oxidation of MALDI-deposited samples

An information-rich on-target performic acid oxidation method, which is compatible with alkylation for differentiation of free cysteine versus disulfide-containing peptides, is described. On-target oxidation is achieved using performic acid vapor to oxidize disulfide-containing peptides and/or small proteins on the matrix-assisted laser desorption/ionization (MALDI) sample deposits. The on-target oxidation method is preferred over solution-phase oxidation methods because (1) less sample handling is required, (2) oxidation throughput is drastically increased and (3) ion suppression effects are reduced because performic acid is not added directly to the MALDI spot. The utility of this method is demonstrated by simultaneous oxidation of multiple MALDI sample deposits containing model disulfide-linked peptides, intact bovine insulin and a bovine ribonuclease A proteolytic digest.

Negative Ion Fragmentation of Cysteic Acid Containing Peptides: Cysteic Acid as a Fixed Negative Charge

We present here a study of the collision induced dissociation (CID) of deprotonated cysteic acid containing peptides produced by MALDI. The effect of cysteic acid (Cox) position is interrogated by considering the positional isomers, CoxLVINVLSQG, LVINVLSQGCox, and LVINVCoxLSQG. Although considerable variation between the CID spectra is observed, the mechanistic picture that emerges involves charge retention at the deprotonated cysteic acid side chain. Fragmentation occurs in the proximity of the cysteic acid group by charge directed mechanisms as well as remote from this group to form ions, which may be rationalized by charge remote mechanisms. Additionally, the formation of the SO3–• ion is observed in all cases. Fragmentation of CoxLVINVLSQCox provides both N- and C-terminal, y and b ions, respectively indicating that the negative charge may be retained at either of the cysteic acids; however, there is some evidence that charge retention at the C-terminal cysteic acid may be preferred. Fragmentation of tryptic type peptides containing a C-terminal arginine or lysine residue is considered through comparison of three peptides CoxLVINKLSQG, CoxLVINVLSQK, and CoxLVINVLSQR. Lastly, we rationalize the formation of bn–1 + H2O and an–1 ions through a mechanism involving rearrangement of the C-terminal residue to form a mixed anhydride intermediate.

Effect of Cysteic Acid Position on the Negative Ion Fragmentation of Proteolytic Derived Peptides

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M – H]− ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a β-γ carbon–carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (CoxLRRASLG) [M – H + O3]− ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The di fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of ai ions followed by the elimination of the side chain to form di ions).