A Molecular Code for Identity in the Vomeronasal System.

In social interactions among mammals, individuals are recognized by olfactory cues, but identifying the key signals among thousands of compounds remains a major challenge. To address this need, we developed a new technique, component-activity matching (CAM), to select candidate ligands that "explain" patterns of bioactivity across diverse complex mixtures. Using mouse urine from eight different sexes and strains, we identified 23 components to explain firing rates in seven of eight functional classes of vomeronasal sensory neurons. Focusing on a class of neurons selective for females, we identified a novel family of vomeronasal ligands, steroid carboxylic acids. These ligands accounted for much of the neuronal activity of urine from some female strains, were necessary for normal levels of male investigatory behavior of female scents, and were sufficient to trigger mounting behavior. CAM represents the first step toward an exhaustive characterization of the molecular cues for natural behavior in a mammalian olfactory system.

Screening of Polysorbate-80 Composition by High Resolution Mass Spectrometry with Rapid H/D Exchange.

Polysorbate (PS) is a widely used polymeric excipient in biotherapeutic formulations to stabilize and protect protein drugs. Commercial PS is a highly heterogeneous mixture of structurally related components. PS composition can impact the stabilizer performance of PS in formulated protein drugs. Characterization of PS heterogeneity is, however, analytically challenging. In this work, a high-throughput screening protocol is presented for the profiling of the PS-80 polysorbate form using high resolution mass spectrometry (HRMS) coupled with a rapid hydrogen/deuterium (H/D) exchange in deuterated methanol. The protocol takes advantage of accurate mass measurements from HRMS analysis and utilizes H/D exchange-induced mass shifts that are characteristic to structures (particularly the number of terminal hydroxyl groups) of PS molecules to definitively identify species. In particular, mass shifts caused by deuterium uptake were used (1) to confirm molecular identities assigned by accurate mass measurements (which adds an extra level of identification confidence) and (2) to differentiate isomers that have an identical mass (thus, undistinguishable by high mass accuracy), but differ in the number of terminal hydroxyls. These data were input to an automated searching algorithm against a molecular mass database covering over 17000 potential PS-80 molecular species. The identified species were then visualized with Kendrick Mass Defect plots. The analysis protocol identified and profiled over 180 species from PS-80 samples in a high-throughput fashion without requiring chromatographic separation to reduce complexity of mixtures or tandem mass spectrometric analysis to conduct structural elucidation.

MS-based cross-linking analysis reveals the location of the PsbQ protein in cyanobacterial photosystem II.

PsbQ is a luminal extrinsic protein component that regulates the water splitting activity of photosystem II (PSII) in plants, algae, and cyanobacteria. However, PsbQ is not observed in the currently available crystal structures of PSII from thermophilic cyanobacteria. The structural location of PsbQ within the PSII complex has therefore remained unknown. Here, we report chemical cross-linking followed by immunodetection and liquid chromatography/tandem MS analysis of a dimeric PSII complex isolated from the model cyanobacterium, Synechocystis sp. PCC 6803, to determine the binding site of PsbQ within PSII. Our results demonstrate that PsbQ is closely associated with the PsbO and CP47 proteins, as revealed by cross-links detected between (120)K of PsbQ and (180)K and (59)K of PsbO, and between (102)K of PsbQ and (440)D of CP47. We further show that genetic deletion of the psbO gene results in the complete absence of PsbQ in PSII complexes as well as the loss of the dimeric form of PSII. Overall, our data provide a molecular-level description of the enigmatic binding site of PsbQ in PSII in a cyanobacterium. These results also help us understand the sequential incorporation of the PsbQ protein during the PSII assembly process, as well as its stabilizing effect on the oxygen evolution activity of PSII.

Quantitative proteomics with siRNA screening identifies novel mechanisms of trastuzumab resistance in HER2 amplified breast cancers.

HER2 is a receptor tyrosine kinase that is overexpressed in 20% to 30% of human breast cancers and which affects patient prognosis and survival. Treatment of HER2-positive breast cancer with the monoclonal antibody trastuzumab (Herceptin) has improved patient survival, but the development of trastuzumab resistance is a major medical problem. Many of the known mechanisms of trastuzumab resistance cause changes in protein phosphorylation patterns, and therefore quantitative proteomics was used to examine phosphotyrosine signaling networks in trastuzumab-resistant cells. The model system used in this study was two pairs of trastuzumab-sensitive and -resistant breast cancer cell lines. Using stable isotope labeling, phosphotyrosine immunoprecipitations, and online TiO(2) chromatography utilizing a dual trap configuration, ~1700 proteins were quantified. Comparing quantified proteins between the two cell line pairs showed only a small number of common protein ratio changes, demonstrating heterogeneity in phosphotyrosine signaling networks across different trastuzumab-resistant cancers. Proteins showing significant increases in resistant versus sensitive cells were subjected to a focused siRNA screen to evaluate their functional relevance to trastuzumab resistance. The screen revealed proteins related to the Src kinase pathway, such as CDCP1/Trask, embryonal Fyn substrate, and Paxillin. We also identify several novel proteins that increased trastuzumab sensitivity in resistant cells when targeted by siRNAs, including FAM83A and MAPK1. These proteins may present targets for the development of clinical diagnostics or therapeutic strategies to guide the treatment of HER2+ breast cancer patients who develop trastuzumab resistance.

A multi-analyte assay of opioids in API and drug products using HPLC and HRMS.

Surveillance testing is an essential component to ensuring safe, effective, and high-quality drug products are available in the commercially marketed US supply chain. Surveillance allows the agency to assess product quality and monitor for potential adulteration of drug products being used by consumers. Opioid drug products can be adulterated to enhance the effect of the intended active ingredient. Numerous accounts have been reported where fentanyl has been used as an adulterant in illicit street drugs such as heroin, cocaine, or methamphetamine. To efficiently surveil the legitimate opioid supply chain, an analytical method with the ability to simultaneously detect, identify and quantify opioid molecules is desired. In this study, a multi-opioid protocol (MOP) using liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) technology was developed and validated for the detection and quantification of 27 opioid drugs. The MOP analytical procedure was applied to the analysis of drug substance and finished dosage forms. MOP was used to identify and quantify active pharmaceutical ingredients (API) listed on the label claim, and in the case of suspected economically motivated adulteration could identify and quantify undeclared opioid APIs. The analytical method analysis time was 16 minutes and the LOD and LOQ in full MS mode were (average) 0.3 and 0.8 ng/mL, respectively. The validation criteria parameters were satisfactory based on international guidelines (ICH). The MOP was successfully applied to the analysis of over 160 drug substances and finished products. For all samples tested in the study, their identities were confirmed, and assays met specifications. Overall, there was no evidence of illegal substitution or adulteration in any of the ingredients and products tested from the legitimate commercial marketed US supply chain.

Carboxyl-group footprinting maps the dimerization interface and phosphorylation-induced conformational changes of a membrane-associated tyrosine kinase.

Her4 is a transmembrane receptor tyrosine kinase belonging to the ErbB-EGFR family. It plays a vital role in the cardiovascular and nervous systems, and mutations in Her4 have been found in melanoma and lung cancer. The kinase domain of Her4 forms a dimer complex, called the asymmetric dimer, which results in kinase activation. Although a crystal structure of the Her4 asymmetric dimer is known, the dimer affinity and the effect of the subsequent phosphorylation steps on kinase domain conformation are unknown. We report here the use of carboxyl-group footprinting MS on a recombinant expressed, Her4 kinase-domain construct to address these questions. Carboxyl-group footprinting uses a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of glycine ethyl ester, to modify accessible carboxyl groups on glutamate and aspartate residues. Comparisons of Her4 kinase-domain monomers versus dimers and of unphosphorylated versus phosphorylated dimers were made to map the dimerization interface and to determine phosphorylation induced-conformational changes. We detected 37 glutamate and aspartate residues that were modified, and we quantified their extents of modification by liquid chromatography MS. Five residues showed changes in carboxyl-group modification. Three of these residues are at the predicted dimer interface, as shown by the crystal structure, and the remaining two residues are on loops that likely have altered conformation in the kinase dimer. Incubating the Her4 kinase dimers with ATP resulted in dramatic increase in Tyr-850 phosphorylation, located on the activation loop, and this resulted in a conformational change in this loop, as evidenced by reduction in carboxyl-group modification. The kinase monomer-dimer equilibrium was measured using a titration format in which the extent of carboxyl-group footprinting was mathematically modeled to give the dimer association constant (1.5-6.8 × 10(12) dm(2)/mol). This suggests that the kinase-domain makes a significant contribution to the overall dimerization affinity of the full-length Her4 protein.

Fast photochemical oxidation of proteins for comparing structures of protein-ligand complexes: the calmodulin-peptide model system.

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based protein footprinting method that modifies proteins on the microsecond time scale. Highly reactive (•)OH, produced by laser photolysis of hydrogen peroxide, oxidatively modifies the side chains of approximately one-half the common amino acids on this time scale. Because of the short labeling exposure, only solvent-accessible residues are sampled. Quantification of the modification extent for the apo and holo states of a protein-ligand complex provides structurally sensitive information at the amino-acid level to compare the structures of unknown protein complexes with known ones. We report here the use of FPOP to monitor the structural changes of calmodulin in its established binding to M13 of the skeletal muscle myosin light chain kinase. We use the outcome to establish the unknown structures resulting from binding with melittin and mastoparan. The structural comparison follows a comprehensive examination of the extent of FPOP modifications as measured by proteolysis and LC-MS/MS for each protein-ligand equilibrium. The results not only show that the three calmodulin-peptide complexes have similar structures but also reveal those regions of the protein that became more or less solvent-accessible upon binding. This approach has the potential for relatively high throughput, information-dense characterization of a series of protein-ligand complexes in biochemistry and drug discovery when the structure of one reference complex is known, as is the case for calmodulin and M13 of the skeletal muscle myosin light chain kinase, and the structures of related complexes are not.

Online, high-pressure digestion system for protein characterization by hydrogen/deuterium exchange and mass spectrometry.

The rapid and complete digestion of proteins is important when protein characterization by hydrogen-deuterium exchange (HDX) is coupled with mass spectrometry. We developed a single-pump, online, high-pressure digestion system that relies on UPLC technology to aid in the digestion of proteins. Two model proteins, amyloid beta-peptide 1-42 (Abeta 1-42) and an HIV-1 capsid mutant protein (NBSA), were used to demonstrate the efficacy of the high-pressure system. Both model proteins readily aggregate and are difficult to digest under normal conditions. Our high-pressure system successfully digests these proteins into small, overlapping peptides. The extra information afforded by overlapping peptides allows us to pinpoint HDX protection to protein segments smaller than the digested peptide. The calculated average segment length (ASL) for both model proteins decreased by 2-fold for high-pressure digestion compared to digestion at ambient pressure.

Marketplace Analysis of Conjugated Estrogens: Determining the Consistently Present Steroidal Content with LC-MS.

Conjugated estrogens purified from pregnant mares urine has been used as estrogen hormone replacement therapy since 1942. Previously, methods were proposed to identify and quantify the components of this complex mixture but ultimately were withdrawn due to incomplete characterization of the product and difficulties in transferring the method between laboratories. The aim of the current study is to develop a LC method that can reliably detect multiple steroidal components in conjugated estrogen tablets and measure their relative amount. The method developed was optimized for UHPLC columns, and the elution profile was analyzed using high-resolution mass spectrometry. A total of 60 steroidal components were identified using their exact m/z, product ion spectra of known, and predicted conjugated estrogen structures. These components were consistently present in 23 lots of Premarin tablets spanning two production years. The ten conjugated estrogens identified in the USP monograph and other additional estrogens reported elsewhere are among the 60 steroidal components reported here. The LC-MS method was tested in different laboratories using multiple samples, and the obtained results were reproducible among laboratories.

Comparing similar spectra: from similarity index to spectral contrast angle.

We investigated a spectral-contrast-angle (theta) method to determine whether mass spectra of structural isomers are the same or significantly different. This method represents collisionally activated dissociation (CAD) spectra as vectors in space. Mass spectra of different isomers are represented as different vectors, having characteristic lengths and direction. The derived spectral contrast angle, which is a measure of the angle between two vectors corresponding to two closely related spectra, is a measure of whether the mass spectra are the same or significantly different. We compare this method with the similarity index (SI) method and show that the spectral contrast angle method is superior and can differentiate between very similar spectra in cases where the SI cannot. Both methods can be implemented simply in situations where the analyst is called on to decide, on the basis of mass or product-ion spectra, whether reference and unknown compounds are the same or to evaluate the reproducibility of spectra comprised of many peaks.

Characterization of N-terminal processing of group VIA phospholipase A2 and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests.

Dysregulation of proteolytic processing of the amyloid precursor protein (APP) contributes to the pathogenesis of Alzheimer's Disease, and the Group VIA phospholipase A(2) (iPLA(2)beta) is the dominant PLA(2) enzyme in the central nervous system and is subject to regulatory proteolytic processing. We have identified novel N-terminal variants of iPLA(2)beta and previously unrecognized proteolysis sites in APP constructs with a C-terminal 6-myc tag by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests. We have developed a Signature-Discovery (SD) program to characterize protein isoforms by identifying signature peptides that arise from proteolytic processing in vivo. This program analyzes MS/MS data from LC analyses of proteolytic digests of protein mixtures that can include incompletely resolved components in biological samples. This reduces requirements for purification and thereby minimizes artifactual modifications during sample processing. A new algorithm to generate the theoretical signature peptide set and to calculate similarity scores between predicted and observed mass spectra has been tested and optimized with model proteins. The program has been applied to the identification of variants of proteins of biological interest, including APP cleavage products and iPLA(2)beta, and such applications demonstrate the utility of this approach.

Azithromycin attenuates airway inflammation in a noninfectious mouse model of allergic asthma.

BACKGROUND: Definitive conclusions regarding the antiinflammatory effects of macrolide antibiotics for treatment of asthma are difficult to formulate since their beneficial effects may be related to their antimicrobial action. We hypothesized that azithromycin possesses distinct antiinflammatory properties and tested this assumption in a noninfectious mouse model of allergic asthma. METHODS: To induce allergic airway inflammation, 7-week-old BALB/cJ mice underwent intraperitoneal ovalbumin sensitization on days 0 and 7 followed by an intranasal challenge on day 14. Mice were treated with azithromycin or phosphate-buffered saline (PBS) solution on days 13 through 16. On day 17, airway inflammation was assessed by quantifying leukocytes in the airway, expression of multiple inflammatory mediators in the BAL fluid, and mucous cell metaplasia. In a separate set of experiments, azithromycin or PBS solution treatment were initiated after the ovalbumin challenge. Each experiment was repeated 3 times (a total of 9 to 11 mice in each group). RESULTS: Compared to treatment with PBS solution, azithromycin attenuated the ovalbumin-dependent airway inflammation. We observed a decrease in total leukocytes in the lung tissue and BAL fluid. In addition, azithromycin attenuated the expression of cytokines (eg, interleukin [IL]-13 and IL-5) and chemokines (eg, CCL2, CCL3, and CCL4) in the BAL fluid and abrogated the extent of mucous cell metaplasia. Similar antiinflammatory effects were observed when azithromycin treatment was initiated after the ovalbumin challenge. CONCLUSION: In this noninfectious mouse model of allergic asthma, azithromycin attenuated allergic airway inflammation. These findings demonstrate an antiinflammatory effect of azithromycin and suggest azithromycin may have beneficial effects in treating noninfectious airway inflammatory diseases, including asthma.

Chemical identification of a low abundance lysozyme peptide family bound to I-Ak histocompatibility molecules.

The processing by antigen-presenting cells (APC) of the protein hen egg-white lysozyme (HEL) results in the selection of a number of peptide families by the class II major histocompatibility complex (MHC) molecule, I-A(k). Some of these families are expressed in very small amounts, in the order of a few picomoles/10(9) APC. We detected these peptides from an extract of class II MHC molecules by using monoclonal anti-peptide antibodies to capture the MHC-bound peptides prior to their examination by HPLC tandem mass spectrometry. Here, we have identified several members of a family of peptides encompassing residues 20-35, which represent less than 1% of the total HEL peptides. Binding analysis indicated that the core segment of the family was represented by residues 24-32 (SLGNWVCAA). Asn-27 (shown in boldface) is the main MHC-binding residue, mapped as interacting with the P4 pocket of the I-A(k) molecule. Analysis of several T cell hybridomas indicated that three residues contacted the T cell receptor: Tyr-23 (P-1), Leu-25 (P3), and Trp-28 (P5). The HEL peptides isolated from the APC extract were sulfated on Tyr-23, but further analysis showed that this modification did not occur physiologically but took place during the peptide isolation.

Autoantibodies and CD4 T cells target a beta cell retroviral envelope protein in non-obese diabetic mice.

We determined that, over a biologic time interval, from 4 to 8 weeks of age, female non-obese diabetic (NOD) mice develop antibodies against pancreatic beta-cell-surface antigens depending upon the presence of both the MHC class II susceptibility allele, I-A(g7), and other NOD background genes. We generated a mAb from a pre-diabetic NOD mouse that binds to the surface of insulinoma cells and isolated mouse beta cells, and identified the target as a retroviral envelope glycoprotein expressed on pancreatic beta cells. The cloned and expressed sequence for this protein was recognized by the mAb. The antibody as well as sera from pre-diabetic NOD mice recognized the recombinant protein. Spontaneous T cell reactivity against a peptide from the cloned protein was found in NOD mice. In conclusion, a beta cell retroviral envelope protein is a target antigen that is selected by the NOD mouse immune system early in the pathogenesis of autoimmune diabetes.

In APCs, the autologous peptides selected by the diabetogenic I-Ag7 molecule are unique and determined by the amino acid changes in the P9 pocket.

We demonstrate in this study the great degree of specificity in peptides selected by a class II MHC molecule during processing. In this specific case of the diabetogenic I-A(g7) molecule, the P9 pocket of I-A(g7) plays a critical role in determining the final outcome of epitope selection, a conclusion that is important in interpreting the role of this molecule in autoimmunity. Specifically, we examined the display of naturally processed peptides from APCs expressing either I-A(g7) molecules or a mutant I-A(g7) molecule in which the beta57Ser residue was changed to an Asp residue. Using mass spectrometry analysis, we identified over 50 naturally processed peptides selected by I-A(g7)-expressing APCs. Many peptides were selected as families with a core sequence and variable flanks. Peptides selected by I-A(g7) were unusually rich in the presence of acidic residues toward their C termini. Many peptides contained short sequences of two to three acidic residues. In binding analysis, we determined the core sequences of many peptides and the interaction of the acidic residues with the P9 pocket. However, different sets of peptides were isolated from APCs bearing a modified I-A(g7) molecule. These peptides did not favor acidic residues toward the carboxyl terminus.