Integrated mapping of pharmacokinetics and pharmacodynamics in a patient-derived xenograft model of glioblastoma.

Therapeutic options for the treatment of glioblastoma remain inadequate despite concerted research efforts in drug development. Therapeutic failure can result from poor permeability of the blood-brain barrier, heterogeneous drug distribution, and development of resistance. Elucidation of relationships among such parameters could enable the development of predictive models of drug response in patients and inform drug development. Complementary analyses were applied to a glioblastoma patient-derived xenograft model in order to quantitatively map distribution and resulting cellular response to the EGFR inhibitor erlotinib. Mass spectrometry images of erlotinib were registered to histology and magnetic resonance images in order to correlate drug distribution with tumor characteristics. Phosphoproteomics and immunohistochemistry were used to assess protein signaling in response to drug, and integrated with transcriptional response using mRNA sequencing. This comprehensive dataset provides simultaneous insight into pharmacokinetics and pharmacodynamics and indicates that erlotinib delivery to intracranial tumors is insufficient to inhibit EGFR tyrosine kinase signaling.

Direct evidence for cancer-cell-autonomous extracellular protein catabolism in pancreatic tumors.

Mammalian tissues rely on a variety of nutrients to support their physiological functions. It is known that altered metabolism is involved in the pathogenesis of cancer, but which nutrients support the inappropriate growth of intact malignant tumors is incompletely understood. Amino acids are essential nutrients for many cancer cells that can be obtained through the scavenging and catabolism of extracellular protein via macropinocytosis. In particular, macropinocytosis can be a nutrient source for pancreatic cancer cells, but it is not fully understood how the tumor environment influences metabolic phenotypes and whether macropinocytosis supports the maintenance of amino acid levels within pancreatic tumors. Here we utilize miniaturized plasma exchange to deliver labeled albumin to tissues in live mice, and we demonstrate that breakdown of albumin contributes to the supply of free amino acids in pancreatic tumors. We also deliver albumin directly into tumors using an implantable microdevice, which was adapted and modified from ref. 9. Following implantation, we directly observe protein catabolism and macropinocytosis in situ by pancreatic cancer cells, but not by adjacent, non-cancerous pancreatic tissue. In addition, we find that intratumoral inhibition of macropinocytosis decreases amino acid levels. Taken together, these data suggest that pancreatic cancer cells consume extracellular protein, including albumin, and that this consumption serves as an important source of amino acids for pancreatic cancer cells in vivo.

Evidence of histidine and aspartic acid phosphorylation in human prostate cancer cells.

We have developed a method to identify previously undetected histidine and aspartic acid phosphorylations in a human prostate cancer progression model. A phosphoproteome of our cell line model is presented, with correlation of modified protein expression between the three states of cancer: non-tumorigenic, tumorigenic, and metastatic cells. With the described interaction proteins potentially phosphorylated by NM23-H1, cellular responses to motility and conformational change stimuli would be achievable. We detect 20 novel histidine-phosphorylated (pHis) and 80 novel aspartic acid-phosphorylated (pAsp) proteins with diverse functions, such as metabolism, protein folding, and motility. Our data indicate that pHis and pAsp are much more prevalent than previously appreciated and may provide insight into the role of NM23-H1 and signaling events that are critical for metastasis. Using the described method for detecting histidine and aspartic acid phosphorylations and our prostate cancer progression cell system, the potential function of NM23-H1 in suppressing metastasis with a two-component regulation system is discussed.

Molecular imaging of drug transit through the blood-brain barrier with MALDI mass spectrometry imaging.

Drug transit through the blood-brain barrier (BBB) is essential for therapeutic responses in malignant glioma. Conventional methods for assessment of BBB penetrance require synthesis of isotopically labeled drug derivatives. Here, we report a new methodology using matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) to visualize drug penetration in brain tissue without molecular labeling. In studies summarized here, we first validate heme as a simple and robust MALDI MSI marker for the lumen of blood vessels in the brain. We go on to provide three examples of how MALDI MSI can provide chemical and biological insights into BBB penetrance and metabolism of small molecule signal transduction inhibitors in the brain - insights that would be difficult or impossible to extract by use of radiolabeled compounds.

4-HNE adduct stability characterized by collision-induced dissociation and electron transfer dissociation mass spectrometry.

4-Hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH4) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring.

Conformational lability in the class II MHC 310 helix and adjacent extended strand dictate HLA-DM susceptibility and peptide exchange.

HLA-DM is required for efficient peptide exchange on class II MHC molecules, but its mechanism of action is controversial. We trapped an intermediate state of class II MHC HLA-DR1 by substitution of αF54, resulting in a protein with increased HLA-DM binding affinity, weakened MHC-peptide hydrogen bonding as measured by hydrogen-deuterium exchange mass spectrometry, and increased susceptibility to DM-mediated peptide exchange. Structural analysis revealed a set of concerted conformational alterations at the N-terminal end of the peptide-binding site. These results suggest that interaction with HLA-DM is driven by a conformational change of the MHC II protein in the region of the α-subunit 3(10) helix and adjacent extended strand region, and provide a model for the mechanism of DM-mediated peptide exchange.

Toward building a database of bifunctional probes for the MS3D investigation of nucleic acids structures.

This report illustrates the approaches employed to investigate critical aspects of the activity of crosslinking reagents toward nucleic acid substrates, which should be evaluated to identify candidate probes for mass spectrometric 3D (MS3D) investigations of biomolecules and macromolecular complexes. Representative members of different classes of bifunctional reagents were taken into consideration, including bikethoxal and phenyl-diglyoxal [bis-(1,2-dicarbonyls)], cisplatin (coordinative binding agents), chlorambucil and nitrogen mustard [bis-(2-chloroethyl)amines], and sym-triazine trichloride (triazines). Nanospray-Fourier transform mass spectrometry (FTMS) was applied without desalting or separation procedures to characterize the covalent products obtained by probing dinucleotide and trinucleotide substrates under a variety of experimental conditions in vitro. The carefully controlled composition of these substrates enabled us to obtain valid comparisons of probe activity toward individual nucleotides and evaluate possible base-specific effects, including the stability of the different adducts in solution under the selected reaction conditions. The gas-phase behavior of the observed products was investigated using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) to obtain valuable information for guiding the design of sequencing experiments and helping the data interpretation. Structured RNA substrates, such as HIV-1 stemloop 1, were finally employed to investigate the structural determinant of adduct formation and highlight the different nature of the spatial information provided by the various candidate probes.

Decreased asialotransferrin in cerebrospinal fluid of patients with childhood-onset ataxia and central nervous system hypomyelination/vanishing white matter disease.

BACKGROUND: A biomarker for the diagnosis of childhood-onset ataxia and central nervous system hypomyelination (CACH)/vanishing white matter disease (VWM) would have clinical utility and pathophysiologic significance. METHODS: We used 2-dimensional gel electrophoresis/mass spectrometry to compare the cerebrospinal fluid proteome of patients with mutation-confirmed CACH/VWM with that of unaffected controls. We characterized selected spots by in-gel digestion, matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, and nanospray Fourier transform mass spectrometry. RESULTS: A specific transferrin spot pattern was detected in the CSF samples of the CACH/VWM group (n = 7), distinguishing them from the control group (n = 23) and revealing that patients with CACH/VWM have a deficiency of the asialo form of transferrin usually present in healthy cerebrospinal fluid. The glycopeptide structure, determined from isolated transferrin spots by use of in-gel digestion and extraction, was found to be consistent with earlier reports. CONCLUSIONS: The transferrin isoform abnormality in the cerebrospinal fluid of patients with CACH/VWM appears unique and is a potential clinical diagnostic biomarker. The rapid, efficient diagnosis of this disorder would have a significant impact on clinical studies exploring new strategies for the management and treatment of this disease.

Atmospheric pressure MALDI-FTMS of normal and chemically modified RNA.

Atmospheric pressure (AP) MALDI has been combined with Fourier transform mass spectrometry (FTMS) to obtain the unambiguous characterization of RNA samples modified by solvent accessibility reagents used in structural studies of RNA and protein-RNA complexes. The formation of cation adducts typical of MS analysis of nucleic acids was effectively reduced by extensive washing of the anionic analytes retained onto the probe surface by strong interactions with a cationic layer of poly(diallyldimethylammonium chloride) (PADMAC). This rapid desalting procedure allowed for the detection of DNA and RNA samples in high femtomole quantities distributed over a 4 x 4 mm sample well. AP MALDI-FTMS was shown to provide high-resolution spectra for analytes as large as approximately 6.4 kDa with little or no evidence of metastable decomposition. The absence of significant metastable decay observed for precursor ions selected for tandem experiments offered a further measure of the low energy content typical of ions generated by AP MALDI. This feature proved to be very beneficial in the characterization of chemically modified RNA samples, which become particularly prone to base losses upon alkylation. The high resolution offered by FTMS enabled the application of a data-reduction algorithm capable of rejecting any signal devoid of plausible isotopic distribution, thus facilitating the analysis of complex analyte mixtures produced by nuclease treatment of RNA substrates. Proper selection of nucleases and digestion conditions can ensure the production of hydrolytic fragments of manageable size, which could extend the range of applicability of this bottom-up strategy to the structural investigation of very large RNA and protein-RNA complexes.

Atmospheric pressure MALDI-fourier transform mass spectrometry.

The coupling of atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) with Fourier transform mass spectrometry (FTMS) is described, and its significance for the high-resolution analysis of complex peptide mixtures is demonstrated. High kinetic energy and extensive metastable decay characteristic of ions generated by vacuum MALDI have been known to constitute a possible obstacle to high-resolution analysis by FTMS. Since the initial coupling of laser desorption techniques with FTMS was realized two decades ago, several different solutions have been proposed to control the energy of the ions and fulfill the promise of high sensitivity and high resolution offered by this analytical method. Initial results obtained on quadrupole time-of-flight and ion trap analyzers have shown that ions generated by MALDI at atmospheric pressure are intrinsically less energetic than those provided by vacuum MALDI. Our report indicates that this characteristic is particularly beneficial for FTMS applications in which a sharp reduction of metastable decay can make larger ion currents available for detection and possible tandem experiments. In our hands, AP MALDI-FTMS has enabled the analysis of complex peptide mixtures with resolution and accuracy comparable to those obtained by analogous electrospray ionization-FTMS experiments, with no evidence of either metastable decomposition or significant formation of matrix adducts. Analysis of a trypsin digest of bovine serum albumin provided signal-to-noise ratios and limits of detection similar to those obtained by ion trap analyzers, but with unmatched resolution and accuracy. AP MALDI has been shown to provide stable precursor ions in amounts that allowed for informative tandem experiments. Finally, the potential of AP MALDI-FTMS for the high-resolution screening of complex mixtures was demonstrated by the analysis of isobaric peptides differing in mass by less than 0.04 Da.

Top-down characterization of nucleic acids modified by structural probes using high-resolution tandem mass spectrometry and automated data interpretation.

A top-down approach based on sustained off-resonance irradiation collision-induced dissociation (SORI-CID) has been implemented on an electrospray ionization (ESI) Fourier transform mass spectrometer (FTMS) to characterize nucleic acid substrates modified by structural probes. Solvent accessibility reagents, such as dimethyl sulfate (DMS), 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMCT), and beta-ethoxy-alpha-ketobutyraldehyde (kethoxal, KT) are widely employed to reveal the position of single- vs double-stranded regions and obtain the footprint of bound proteins onto nucleic acids structures. Established methods require end-labeling of the nucleic acid constructs, probe-specific chemistry to produce strand cleavage at the modified nucleotides, and analysis by polyacrylamide gel electrophoresis to determine the position of the susceptible sites. However, these labor-intensive procedures can be avoided when mass spectrometry is used to identify the probe-induced modifications from their characteristic mass signatures. In particular, ESI-FTMS can be directly employed to monitor the conditions of probe application to avoid excessive alkylation, which could induce unwanted distortion or defolding of the substrate of interest. The sequence position of the covalent modifications can be subsequently obtained from classic tandem techniques, which allow for the analysis of individual target adducts present in complex reaction mixtures with no need for separation techniques. Selection and activation by SORI-CID has been employed to reveal the position of adducts in nucleic acid substrates in excess of 6 kDa. The stability of the different covalent modifications under SORI-CID conditions was investigated. Multiple stages of isolation and activation were employed in MS(n)() experiments to obtain the desired sequence information whenever the adduct stability was not particularly favorable, and SORI-CID induced the facile loss of the modified base. A new program called MS2Links was developed for the automated reduction and interpretation of fragmentation data obtained from modified nucleic acids. Based on an algorithm that searches for plausible isotopic patterns, the data reduction module is capable of discriminating legitimate signals from noise spikes of comparable intensity. The fragment identification module calculates the monoisotopic mass of ion products expected from a certain sequence and user-defined covalent modifications, which are finally matched with the signals selected by the data reduction program. Considering that MS2Links can generate similar fragment libraries for peptides and their covalent conjugates with other peptides or nucleic acids, this program provides an integrated platform for the structural investigation of protein-nucleic acid complexes based on cross-linking strategies and top-down ESI-FTMS.