Kari B. Green-Church

Targeted delivery of methotrexate to epidermal growth factor receptor–positive brain tumors by means of cetuximab (IMC-C225) dendrimer bioconjugates

We have constructed a drug delivery vehicle that targets the epidermal growth factor receptor (EGFR) and its mutant isoform EGFRvIII. The monoclonal antibody, cetuximab, previously known as C225, which binds to both EGFR and EGFRvIII, was covalently linked via its Fc region to a fifth-generation (G5) polyamidoamine dendrimer containing the cytotoxic drug methotrexate. As measured by mass spectrometry and UV/vis spectroscopy, the resulting bioconjugate, designated C225-G5-MTX, contained 12.6 molecules of methotrexate per unit of dendrimer. Specific binding and cytotoxicity of the bioconjugate was evaluated against the EGFR-expressing rat glioma cell line F98EGFR. Using a competitive binding assay, it was shown that the bioconjugate retained its affinity for F98EGFR cells, with a 0.8 log unit reduction in its EC50. Only cetuximab completely inhibited binding of the bioconjugate, which was unaffected by methotrexate or dendrimer. Cetuximab alone was not cytotoxic to F98EGFR cells at the concentration tested, whereas the IC50 of the bioconjugate was 220 nmol/L, which was a 2.7 log unit decrease in toxicity over that of free methotrexate. The biodistribution of C225-G5-MTX in rats bearing i.c. implants of either F98EGFR or F98WT gliomas was determined 24 hours following convection enhanced delivery of 125I-labeled bioconjugate. At this time, 62.9 ± 14.7% ID/g tumor was localized in rats bearing F98EGFR gliomas versus 11.3 ± 3.6% ID/g tumor in animals bearing F98WT gliomas, thereby showing specific molecular targeting of the tumor. The corresponding radioactivity of normal brain from the F98EGFR tumor-bearing right and non-tumor-bearing left cerebral hemisphere were 5.8 ± 3.4% and 0.8 ± 0.6% ID/g, respectively. Based on these results, therapy studies were initiated in F98EGFR glioma-bearing rats. Animals that received C225-G5-MTX, cetuximab, or free methotrexate had median survival times of 15, 17, and 19.5 days, respectively, which were not statistically different from each other or untreated control animals. Our results, which are both positive and negative, show that specific molecular targeting is but one of several requirements that must be fulfilled if an antibody-drug bioconjugate will be therapeutically useful. [Mol Cancer Ther 2006;5(1):52–9]

Mononucleotide gas-phase proton affinities as determined by the kinetic method

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5′- and 3′-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5′- and 3′-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates ≫ deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5′- vs. 3′-phosphates): the 3′-monophosphates have lower proton affinities than the 5′-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.

Mass spectrometry-based proteomic analyses in contact lens-related dry eye.

Purpose: To identify potential protein biomarkers associated with dry eye in contact lens wearers. Methods: Upon enrollment, current galyfilcon A contact lens wearers completed a previously described questionnaire used to classify dry eye status. Approximately 5 μL of aqueous tears were carefully sampled from the inferior-lateral tear prism of each eye using glass microcapillaries. A variety of proteomic approaches were used to compare samples including quantification by Bradford analyses, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (alone in varying percentages and with MultiPlex analyses for posttranslational modifications), nano-liquid chromatography tandem mass spectometry (nano-LC-MS/MS), and differential gel electrophoresis. Results: Twenty-one subjects were enrolled in the study (age 31.3 ± 11.6 years). Eleven of the subjects were classified with contact lens-related dry eye, while the remaining 10 were normal contact lens wearers. Across all proteomic approaches, several proteins (including several glycoproteins) were identified as potential biomarkers associated with dry eye disease state. In summary across the approaches used, extracellular proteins identified to be altered included β-2 microglobulin, proline rich 4, lacritin, and secretoglobin 1D1, which were found to be decreased in the dry eye state. Secretoglobin 2A2, serum albumin, glycoprotein 340, and prolactin-inducible protein were all found to be increased in the dry eye state. Conclusions: Dry eye in contact lens wearers is related to several changes in the tear film protein. While functional studies for these candidate proteins are ongoing, initial insights into the functions of these proteins suggest roles in altered tear secretion, in addition to possible increased susceptibility to infection.

Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fourier-transform ion cyclotron resonance mass spectrometry.

The gas-phase structures of protonated (deoxy)nucleoside-5′- and 3′-monophosphates (mononucleotides) have been examined by the use of gas-phase hydrogen/deuterium (H/D) exchange and high-field Fourier-transform ion cyclotron resonance mass spectrometry. These nucleotides were reacted with three different deuterating reagents: ND3, D2O, and D2S, of which ND3 was the most effective. All mononucleotides fully exchanged their labile hydrogen for deuterium with ND3 with the exception of deoxycytidine-3′-monophosphate, deoxyadenosine-5′-monophosphate, adenosine-5′-monophosphate, and adenosine-3′-monophosphate. Semiempirical calculations demonstrate the presence of hydrogen bonding upon protonation of the purine mononucleotides which may lead to incomplete H/D exchange. H/D exchange rates differed between the deoxymononucleotides and the ribomononucleotides, suggesting that the 2′-OH group plays an important role in the exchange process. Reactions of nucleosides and mononucleotides with D2O demonstrate that a structure-specific long-lived ion—molecule complex between D2O and the mononucleotide involving the phosphate group is necessary for exchange to overcome the high-energy activation barrier. In contrast, a structure-specific long-lived ion—molecule complex between the mononucleotides and ND3 is not required for exchange to occur.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry of Hydrophobic Peptides

Hydrophobic peptides, especially those with acid-labile protecting groups, are difficult to characterize using mass spectrometric methods. We have developed a new procedure for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometric analysis of such samples. Hydrophobic peptides, which are insoluble in aqueous solutions, are dissolved in chloroform and combined with matrixes prepared in chloroform or chloroform/methanol solutions. The use of a common solvent for the matrix and the analyte improves the analyte isolation step in MALDI mass spectrometry. The lack of acidic solutions previously used for electrospray ionization or MALDI mass spectrometry of hydrophobic peptides extends this methodology to cyclic or protected hydrophobic peptides. Conventional peptide matrixes, such as 2,5-dihydroxybenzoic acid and sinapinic acid, as well as 3-indoleacrylic acid are shown to be suitable for hydrophobic peptides. Cyclic hydrophobic peptides and linear hydrophobic peptides with b...

Top-down characterization of a native highly intralinked protein: concurrent cleavages of disulfide and protein backbone bonds.

Top-down analysis of proteins has developed rapidly in recent years. However, its application to disulfide-bonded proteins is still limited. Using native chicken lysozyme as a model, we studied the characteristics of collision-induced dissociation (CID) of disulfide-bonded proteins on an LTQ Orbitrap mass spectrometer with electrospray ionization (ESI) in positive mode. For low-charged protein precursor ions with no or limited mobile protons, product ions generated from CID correspond to the concurrent cleavages of disulfide and protein backbone bonds. Up to three disulfide bonds could be easily cleaved with four possible dissociation pathways for each disulfide bond. That led to modifications of the corresponding cysteine residues through addition or subtraction of a hydrogen atom or sulfhydryl group. The protein backbone cleavages mainly occurred at the amide bonds from C-terminal to aspartic acid residues (e.g., ion series of b(18), b(48), y(10), and y(28)), N-C(alpha) bonds from N-terminal to cysteine residues (e.g., c(5), ion series of c(29) and c(63)), and amide bonds from C-terminal to glutamic acid residues (e.g., ion series of b(35)). The characteristics of the top-down analysis for this highly knotted protein will help to understand the general dissociation pattern of disulfide-bonded proteins, which in turn will help to avoid time-consuming bottom-up procedures for the identification of proteins and their modifications.

Interactions between sodium dodecyl sulfate micelles and peptides during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of proteolytic digests

Although sodium dodecyl sulfate (SDS) is routinely used as a denaturing agent for proteins, its presence is highly detrimental on the analysis of peptides and proteins by mass spectrometry. It has been found, however, that when SDS is present in concentrations near to or above its critical micelle concentration (CMC), improvements in the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of peptide mixtures or hydrophobic proteins are obtained. To elucidate possible explanations for such improvements, here we have undertaken a study examining the effect of SDS micelles on peptide mixtures. Fluorescently labeled peptides were used as probes to determine whether hydrophobic or hydrophilic peptides interact exclusively with SDS micelles. In addition, four globular proteins were digested with trypsin and then various amounts of SDS were added before MALDI mass spectrometry. To examine the role of mixture complexity on the mass spectral results, the tryptic digest of bovine serum albumin was also fractionated according to hydrophobicity before SDS treatment. Results from these experiments suggest that micelle-peptide interactions increase peptide-matrix cocrystallization irrespective of analyte hydrophobicity. As these studies were performed using the dried-droplet method of sample spotting, the presence of micelles is also hypothesized to reduce Marangoni effects during the crystallization process.

Author Response: On the Presence of (O-Acyl)-Omega-Hydroxy Fatty Acids and Their Esters in Human Meibomian Gland Secretions

In the wake of our response1 to his comments2 on our recent paper,3 Butovich followed with a second letter.4 The title and contents of Butovichs letter relate to the presence of (O-acyl)-omega-hydroxy fatty acids (OAHFAs) and their esters in human meibum. He also discussed and criticized the shotgun mass spectrometry approach for meibum analysis. We would like to respond to these concerns. n nSince the main topic of Butovichs comments was OAHFAs and their esters, we would like to discuss that issue first. We agree that there is no question of the existence of free OAHFAs. Our present study showed that complex lipids including cholesteryl esters were stable under our experimental conditions,3 which suggests that the OAHFAs we detected were not from an in-source dissociation of ω-type I-St diesters or any other types of esterified OAHFAs (e-OAHFAs). We also found that the complex lipids of meibum were very stable in solutions with 1 mM ammonium acetate, and no significant hydrolysis was observed. It is known that esters can undergo hydrolysis in acidic or basic solutions. Although the stability of meibum lipids in ammonium hydroxide solution was not systematically studied, our analysis of meibum samples with ammonium hydroxide as the additive did show results similar to those with 1 mM ammonium acetate. For this analysis, we used a low concentration (10 ppm to 0.1%) of ammonium hydroxide as the additive and prepared the solution immediately before the analysis, to minimize possible hydrolysis. Butovichs new results shown in Figure 1A of his letter4 confirmed our hypothesis1 that OAHFAs can be formed from dissociation of e-OAHFAs under the same condition that fatty acids form from dissociation of cholesteryl esters.5 Although Butovich showed that peaks of free OAHFAs were separated from those of OAHFAs dissociated from e-OAHFAs,4 one question that remains from Butovichs results is whether there was a significant amount of free OAHFAs resulting from hydrolysis of e-OAHFAs during the injection. It seems unlikely, since the mobile phases were flowing through the column continuously, and the continuous hydrolysis would have left a long tail of chromatographic peaks. However, the hydrolysis may be affected by the solution composition and could be most significant at the beginning of the gradient. n nOur recently reported estimate of free fatty acids (FFAs)3 has been criticized by Butovich2 in the previous letter as well as the current one and was used as evidence of the weakness of the shotgun mass spectrometry approach.4 On the basis of his experience, Butovich suspected that the FFAs we detected were from in-source fragmentation of complex lipids. However, our experimental setup was different from his, including the ionization modes. He used atmospheric pressure chemical ionization (APCI), while we used electrospray ionization (ESI)—a softer ionization method.6 Another important difference was the temperature used. We used 100°C to 150°C to help the ionization, whereas Butovich typically used 350°C. The dissociation of cholesteryl esters during chemical ionization has been reported previously, either in positive or negative mode.7,8 Whether the APCI process has played a role in the dissociation is unknown; however, it is known that high temperature can cause significant fragmentation of intact lipids,9,10 which may be one reason that Butovich observed significant dissociation in his experiments.2 In contrast, in the experimental condition with much lower temperature and ESI, our recent study with standard esters including triolein, behenyl olate, cholesteryl stearate, and cholesteryl oleate showed that the dissociation was insignificant (<0.5%, if any). In addition, the estimated amount of FFAs reported by us (i.e., 3% of total lipids in meibum)3 is in line with the 1% to 3% previously reported by three different groups.11–13 The low concentration of FFAs claimed by Butovich (<0.1%)2 may be due to the insensitive detection at the shorter retention time in their experiments (Fig. 1 of Ref. 5). Although the pattern of FFAs detected in negative mode seems to be similar to that of the fatty acid moieties in cholesteryl esters,3 it does not necessarily suggest that these fatty acids must be from the dissociation. In fact, the pattern of the free OAHFAs that Butovich reported is also similar to that of the e-OAHFAs,4 with the consideration of the high detection efficiency in negative mode at the later elution time (Fig. 1, Ref. 5). The similarity between the patterns may have occurred because these fatty acids were the precursors from which the esters were synthesized. n nIt is challenging to work with complex samples such as meibum. Both the shotgun method and LC-MS method have strengths and weaknesses. The LC-MS method has the advantage of adding one more parameter for identifying lipids; however, it takes a long time for separation and data analysis. In contrast, shotgun analysis is very fast and highly sensitive if performed appropriately. In the past, the species of complex samples could not be separated by mass spectrometry alone and had to be combined with separation approaches, such as GC and HPLC. The development of modern mass spectrometers makes it possible to detect different species directly. Compared to the ion-trap mass spectrometer used by Butovich, the Q-TOF mass spectrometer used by us has much higher resolution and mass accuracy, which makes the shotgun method appropriate. Therefore, investigators using mass spectrometers to analyze the lipids in meibum should take into consideration the resolution of the instrument used and experimental conditions when comparing results. In addition, modern mass spectrometers can add one more dimension, ion mobility, to separate ions based on their cross sections,14 which may be of interest when analyzing complex samples such as meibum. Last, contrary to Butovichs claim, a direct-infusion system has fewer components than an LC-MS system; thus, we found it much easier to clean. n nWe are glad to see that Butovich4 confirmed our observations that C16:1-based wax esters in some cases are of higher concentrations than C18:1-based wax esters with his LC-MS method. The trend of the higher ratio of C16:1-based wax esters for a low m/z wax ester may have some implications for the function of meibum, although the rationale is unknown at this time. n nIn summary, both free OAHFAs and FFAs, which could be important for maintaining the stability of tear film, exist in appreciable amount in meibum samples. High temperature may cause problems in the analysis of meibum lipids, and shotgun mass spectrometry has its own advantages for the analysis of meibum samples.

Investigation into surfactant-aided matrix-assisted laser desorption/ionization-mass spectrometry for peptide mapping

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is a popular analytical tool for the analysis of large biomolecules such as proteins, oligonucleotides and oligosaccharides. This method has proven to be extremely useful for the analysis of water soluble proteins. However, analysis of hydrophobic proteins using MALDI-MS has been more of a challenge. The difficulty arises due to the limited solubility of these compounds in the aqueous solutions used to prepare the MALDI matrix. This study has investigated the effect of surfactants on the MALDI-MS data obtained from hydrophobic peptides and tryptic digests of hydrophobic and hydrophilic proteins. The surfactant sodium dodecylsulfate was found to improve the information content obtained during MALDI-MS analysis of such molecules. Preliminary results which examine the applicability of surfactant-aided MALDI-TOFMS for the characterization of proteolytic digests of proteins will be presented, and the potential use of this analytical strategy in a proteomics-based examination of intrinsic membrane proteins will be discussed.

Nucleic Acids and Nucleotides Studied Using Mass Spectrometry

The applications of mass spectrometry to the study of the structure and sequence information of nucleic acids (DNA and RNA) and their building blocks (oligonucleotides) are surveyed. In addition, studies on individual nucleobases, nucleosides, and nucleotides are also covered. The main techniques used are electrospray ionization (ESI) and matrix-assisted laser desorption (MALDI).