We investigated the predictive and prognostic effects of VeriStrat®, a serum or plasma based assay, on response and survival in a subset of patients enrolled on the NCIC Clinical Trials Group (CTG) BR.21 phase III trial of erlotinib versus placebo in previously treated advanced non-small cell lung cancer (NSCLC) patients.
Pretreatment plasma samples were available for 441 of 731 enrolled patients and were provided as anonymized aliquots to Biodesix. The VeriStrat test was performed in a CLIA-accredited laboratory at Biodesix, Inc. Results (Good, Poor) were returned to NCIC CTG, who performed all statistical analyses.
VeriStrat testing was successful in 436 samples (98.9%), with 61% classified as Good. VeriStrat was prognostic for overall survival in both erlotinib-treated patients and those on placebo, independent of clinical covariates. For VeriStrat Good patients, the median survival was 10.5 months on erlotinib vs. 6.6 months for placebo (HR 0.63, 95% C.I. 0.47–0.85, P=0.002). For VeriStrat Poor patients, the median survival was 4 months for patients receiving erlotinib, and 3.1 months for placebo (HR: 0.77, 95% C.I. 0.55–1.06, P=0.11). VeriStrat was predictive for objective response (P =0.002), but was not able to predict for differential survival benefit from erlotinib (interaction p-value 0.48). Similar results were found for progression-free survival (PFS).
We were able to confirm that VeriStrat is predictive of objective response to erlotinib. VeriStrat is prognostic for both OS and PFS, independent of clinical features, but is not predictive of differential survival benefit vs. placebo.
erlotinib; proteomics; metastatic non-small cell lung cancer; biomarkers
Primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC) and autoimmune hepatitis (AIH) are the major forms of autoimmune liver diseases each characterized by the destruction of a specific liver cell type and the presence of differing auto-antibodies. We took a proteomic approach utilizing in situ matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) to obtain profiles directly from liver samples of patients with PBC, PSC, AIH and controls. The ability to precisely localize the region for acquisition of MALDI MS allowed us to obtain profiles from bile ducts, inflammatory infiltrates and hepatocytes from each biopsy sample. Analysis tools developed to identify peaks and compare peaks across diseases and cell types were used to develop models to classify the samples. Using an initial set of testing samples from PBC patients and controls, we identified unique peaks present in bile ducts, inflammatory infiltrates and hepatocytes that could classify samples in a validation cohort with 88–91% accuracy. Interestingly, profiles of PSC and AIH did not differ significantly from PBC. Identification of proteins in these peaks may represent novel autoantigens or effector molecules. These findings illustrate the potential of a proteomic approach to autoimmune diseases with in situ MALDI MS.
autoimmune hepatitis; mass spectrometry; primary biliary cirrhosis; primary sclerosing cholangitis
Apomyolgobin (apoMb) is an important model for understanding the folding mechanism of helical proteins. This study focuses on a partially structured state of sperm whale apoMb populated at pH 4.2 (M-state), which structurally resembles a late kinetic intermediate in the formation of the native state (N) at higher pH. The thermodynamics and cooperativity of apoMb folding at pH 4.2 and 6.2 were studied by global analysis of the urea-induced unfolding transitions monitored by tryptophan fluorescence and circular dichroism. The kinetics of folding and unfolding of apoMb at pH 4.2 was measured over a time window from 40 to 850 μs, using fluorescence-detected continuous-flow measurements. Our observation of biphasic kinetics provides clear evidence for rapid (<100 μs) accumulation of previously unresolved intermediate states in both refolding and unfolding experiments. Quantitative kinetic modeling of the results, using a four-state mechanism with two intermediates on a direct route between the unfolded and folded states (U↔I↔L↔M), gave new insight into the conformational states and barriers that precede the rate-limiting step in the formation of the N-state of apoMb.
protein folding; myoglobin; rapid mixing; continuous flow; fluorescence; circular dichroism
The interaction of a protein antigen, horse cytochrome c (cyt c), with a monoclonal antibody has been studied by hydrogen-deuterium (H-D) exchange labeling and two-dimensional nuclear magnetic resonance (2D NMR) methods. The H-exchange rate of residues in three discontiguous regions of the cyt c polypeptide backbone was slowed by factors up to 340-fold in the antibody-antigen complex compared with free cyt c. The protected residues, 36 to 38, 59, 60, 64 to 67, 100, and 101, and their hydrogen-bond acceptors, are brought together in the three-dimensional structure to form a contiguous, largely exposed protein surface with an area of about 750 square angstroms. The interaction site determined in this way is consistent with prior epitope mapping studies and includes several residues that were not previously identified. The hydrogen exchange labeling approach can be used to map binding sites on small proteins in antibody-antigen complexes and may be applicable to protein-protein and protein-ligand interactions in general.
To understand the process of protein folding, it will be necessary to obtain detailed structural information on folding intermediates. This difficult problem is being studied by using hydrogen exchange and rapid mixing to label transient structural intermediates, with subsequent analysis of the proton-labelling pattern by two-dimensional nuclear magnetic resonance spectroscopy. Results for cytochrome c show that the method provides the spatial and temporal resolution necessary to monitor structure formation at many defined sites along the polypeptide chain on a timescale ranging from milliseconds to minutes.
The 195 amino acid basic protein (δAg) of hepatitis delta virus (HDV) is essential for replication of the HDV RNA genome. Numerous properties have been mapped to full-length δAg and attempts made to link these to secondary, tertiary and quaternary structures. Here, for the full-size δAg, extensive intrinsic disorder was predicted using PONDR-FIT, a meta-predictor of intrinsic disorder, and evidenced by circular dichroism measurements. Most δAg amino acids are in disordered configurations with no more than 30% adopting a α-helical structure. In addition, dynamic light scattering studies indicated that purified δAg assembled into structures of as large as dodecamers. Cross-linking followed by denaturing polyacrylamide gel electrophoresis revealed hexamers to octamers for this purified δAg and at least this size for δAg found in virus-like particles. Oligomers of purified δAg were resistant to elevated NaCl and urea concentrations, and bound without specificity to RNA and single- and double-stranded DNAs.
hepatitis delta virus; delta antigen; intrinsic disorder; protein oligomerization; nucleic acid binding
We applied an established and commercially available serum proteomic classifier for survival after treatment with erlotinib (VeriStrat®) in a blinded manner to pre-treatment sera obtained from recurrent advanced NSCLC patients before treatment with the combination of erlotinib plus bevacizumab. We found that VeriStrat® could classify these patients into two groups with significantly better or worse outcomes and may enable rational selection of patients more likely to benefit from this costly and potentially toxic regimen.
Lung Cancer; erlotinib; bevacizumab; serum biomarker
Serum proteomics and mutations in the epidermal growth factor receptor (EGFR) and KRAS have been associated with benefit after therapy with EGFR-targeted therapies in non-small cell lung cancer, but all three have not been evaluated in any one study.
Pretreatment serum proteomics predicts survival in Western advanced non-small cell lung cancer patients with wild-type EGFR and independent of KRAS mutation status.
We analyzed available biospecimens from Eastern Cooperative Oncology Group 3503, a single-arm phase II study of erlotinib in first-line advanced lung cancer, for proteomics signatures in the previously described serum matrix-assisted laser desorption ionization proteomic classifier (VeriStrat) as well as for KRAS and EGFR mutations.
Out of 137 enrolled patients, analyzable biologic samples were available on 102. Nine of 41 (22%) demonstrated KRAS mutations and 3 of 41 (7%) harbored EGFR mutations. VeriStrat classification identified 64 of 88 (73%) as predicted to have “good” and 24 of 88 (27%) predicted to have “poor” outcomes. A statistically significant correlation of VeriStrat status (p < 0.001) was found with survival. EGFR mutations, but not KRAS mutations, also correlated with survival.
The previously defined matrix-assisted laser desorption ionization predictor remains a potent and highly clinically significant predictor of survival after first-line treatment with erlotinib in patients with wild-type EGFR and independent of mutations in KRAS.
Lung cancer; Proteomics; Erlotinib; KRAS; EGFR
We hypothesized that a serum proteomic profile predictive of survival benefit in non–small cell lung cancer patients treated with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI) reflects tumor EGFR dependency regardless of site of origin or class of therapeutic agent.
Pretreatment serum or plasma from 230 patients treated with cetuximab, EGFR-TKIs, or chemotherapy for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) or colorectal cancer (CRC) were analyzed by mass spectrometry. Each sample was classified into “good” or “poor” groups using VeriStrat, and survival analyses of each cohort were done based on this classification. For the CRC cohort, this classification was correlated with the tumor EGFR ligand levels and KRAS mutation status.
In the EGFR inhibitor–treated cohorts, the classification predicted survival (HNSCC: gefitinib, P = 0.007 and erlotinib/bevacizumab, P = 0.02; CRC: cetuximab, P = 0.0065) whereas the chemotherapy cohort showed no survival difference. For CRC patients, tumor EGFR ligand RNA levels were significantly associated with the proteomic classification, and combined KRAS and proteomic classification provided improved survival classification.
Serum proteomic profiling can detect clinically significant tumor dependence on the EGFR pathway in non–small cell lung cancer, HNSCC, and CRC patients treated with either EGFR-TKIs or cetuximab. This classification is correlated with tumor EGFR ligand levels and provides a clinically practical way to identify patients with diverse cancer types most likely to benefit from EGFR inhibitors. Prospective studies are necessary to confirm these findings.
Na+/H+ exchanger regulatory factor (NHERF1) is a signaling adaptor protein comprising two PDZ domains and a C-terminal ezrin-binding (EB) motif. To understand the role of intramolecular interactions in regulating its binding properties, we characterized the complex between the second PDZ domain PDZ2 and the C-terminal 242–358 fragment of NHERF1 using NMR and fluorescence methods. NMR chemical shift and relaxation data implicate 11 C-terminal residues in binding and, together with a thermodynamic analysis of mutant proteins, indicate that the EB region becomes helical when bound to PDZ2. Both specific contacts between PDZ2 and EB as well as non-specific interactions involving a 100-residue flexible linker contribute to stabilizing two structurally distinct closed conformations of NHERF1. The affinity of mutant proteins for an extrinsic ligand is inversely related to the helix-forming propensity of the EB motif. The findings provide a structural framework for understanding how autoinhibitory interactions modulated the binding properties of NHERF1.
An important step towards understanding the mechanism of the PrPC to PrPSc conversion is to elucidate the folding pathway(s) of the prion protein. Based on stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the sub-millisecond time-scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous flow measurements using a capillary mixing system with a time resolution of ~ 100 μs. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90–231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of ~50 μs), followed by a rate-limiting folding step (with a time constant of ~700 μs). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for Gerstmann-Straussler-Scheinker disease- associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrPSc oligomer.
The competition between intramolecular histidine-heme loop formation and ligand-mediated oligomer formation in the denatured state is investigated for two yeast iso-1-cytochrome c variants, AcH26I52 and AcA25H26I52. Besides the native His 18 heme ligand, both variants contain a single His at position 26. The AcA25H26I52 variant has Pro 25 mutated to Ala. The concentration dependence of the apparent pKa for His 26-heme binding in 3 M gdnHCl indicates that the P25A mutation disfavors oligomerization mediated by intermolecular heme ligation by 10-fold. Single and double pH jump stopped-flow experiments with the AcH26I52 variant show that fast phases for His-heme bond formation and breakage are due to intramolecular loop formation and slow phases for His-heme bond formation and breakage are due to intermolecular aggregation. The presence of two closely-spaced slow phases in the kinetics of loop formation for both variants suggests that intermolecular His 26-heme ligation results in both dimers and higher order aggregates. The P25A mutation slows formation and speeds breakdown of an initial dimer, demonstrating a strong effect of local sequence on aggregation. Analysis of the kinetic data yields equilibrium constants for intramolecular loop formation and intermolecular dimerization at pH 7.1 and indicates that the rate constant for intermolecular aggregation is very fast at this pH (107 to 108 M−1s−1). In light of the very fast rates of aggregation in the denatured state, comparison of models involving reversible or irreversible oligomerization steps suggest that equilibrium control of the partitioning between folding and aggregation is advantageous for productive protein folding in vivo.
Despite close structural similarity, the ferric and ferrous forms of cytochrome c (cyt c) differ greatly in terms of their ligand binding properties, stability, folding and dynamics. The reduced heme iron binds diatomic ligands such as CO only under destabilizing conditions that promote weakening or disruption of the native methionine-iron linkage. This makes CO a useful conformational probe for detecting partially structured states that cannot be observed in the absence of endogenous ligands. Heme absorbance, circular dichroism and NMR were used to characterize the denaturant-induced unfolding equilibrium of Fe2+ cyt c in the presence and absence of CO. In addition to the native state (N), which does not bind CO, and the unfolded CO-complex (U-CO), a structurally distinct CO-bound form (M-CO) accumulates to high levels (~75% of the population) at intermediate guanidine hydrochloride concentrations. Comparison of the unfolding transition for different conformational probes reveals that M-CO is a compact state containing a native-like helical core and regions of local disorder in the segment containing the native Met80 ligand and adjacent loops. Kinetic measurements of CO binding and dissociation under native, partially denaturing and fully unfolded conditions indicate that a state, M, that is structurally analogous to M-CO is populated even in the absence of CO. The binding energy of the CO ligand lowers the free energy of this high-energy state to such an extent that it accumulates even under mildly denaturing equilibrium conditions. The thermodynamic and kinetic parameters obtained in this study provide a fully self-consistent description of the linked unfolding/CO-binding equilibria of reduced cyt c.
protein folding; denaturation; heme; ligand binding; NMR
We assessed proteomic patterns in breast cancer using MALDI MS and laser capture microdissected cells. Protein and peptide expression in invasive mammary carcinoma versus normal mammary epithelium and estrogen-receptor positive versus estrogen-receptor negative tumors were compared. Biomarker candidates were identified by statistical analysis and classifiers were developed and validated in blinded test sets. Several of the m/z features used in the classifiers were identified by LC–MS/MS and two were confirmed by immunohistochemistry.
MALDI MS; laser capture microdissection; breast cancer
This review summarizes the essential characteristics of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF MS), especially as they relate to its applications in quantitative analysis. Approaches to quantification by MALDI-TOF MS are presented and published applications are critically reviewed.
quantification; quantitative analysis; MALDI; mass spectrometry; biomarkers
We describe the computational design of a single-chain four-helix bundle that noncovalently self-assembles with fully synthetic non-natural porphyrin cofactors. With this strategy, both the electronic structure of the cofactor as well as its protein environment may be varied to explore and modulate the functional and photophysical properties of the assembly. Solution characterization (NMR, UV/vis) of the protein showed that it bound with high specificity to the desired cofactors, suggesting that a uniquely structured protein and well-defined site had indeed been created. This provides a genetically expressed single-chain protein scaffold that will allow highly facile, flexible, and asymmetric variations to enable selective incorporation of different cofactors, surface-immobilization and introduction of spectroscopic probes.
To monitor the development of tertiary structural contacts during folding, a unique tryptophan residue was introduced at seven partially buried locations (residues 15, 27, 61, 76, 91, 102 and 121) of a tryptophan-free variant of staphylococcal nuclease (P47G/P117G/H124L/W140H). Thermal unfolding measurements by circular dichroism indicate that the variants are destabilized, but maintain the ability to fold into a native-like structure. For the variants with Trp at positions 15, 27 and 61, the intrinsic fluorescence is significantly quenched in the native state due to close contact with polar side chains that act as intramolecular quenchers. All other variants exhibit enhanced fluorescence under native conditions consistent with burial of the tryptophans in an apolar environment. The kinetics of folding was observed by continuous- and stopped-flow fluorescence measurements over refolding times ranging from 100 μs to 10 s. The folding kinetics of all variants is quantitatively described by a mechanism involving a major pathway with a series of intermediate states and a minor parallel channel. The engineered tryptophans in the β-barrel and the N-terminal part of the α-helical domain become partially shielded from the solvent at an early stage (< 1 ms), indicating that this region of the protein undergoes a rapid specific collapse and remains uncoupled from the rest of the α-helical domain until the late stages of folding. For several variants, a major increase in fluorescence coincides with the rate-limiting step of folding on the 100 ms time scale, indicating that these tryptophans reach their buried native environment only during the late stages of folding. Other variants show more complex behavior with a transient increase in fluorescence during the 10 ms phase followed by a decrease during the rate-limiting phase. These observations are consistent with burial of these probes in a collapsed, but loosely packed intermediate, followed by the rate-limiting formation of the densely packed native core, which brings the tryptophans into close contact with intramolecular quenchers.
protein folding; continuous-flow; stopped-flow; fluorescence; circular dichroism
The blood coagulation protein factor XI (FXI) consists of a pair of disulfide-linked chains each containing four apple domains and a catalytic domain. The apple 4 domain (A4; F272-E362) mediates noncovalent homodimer formation even when the cysteine involved in an intersubunit disulfide is mutated to serine (C321S). To understand the role of noncovalent interactions stabilizing the FXI dimer, equilibrium unfolding of wild-type A4 and its C321S variant was monitored by circular dichroism, intrinsic tyrosine fluorescence and dynamic light scattering measurements as a function of guanidine hydrochloride concentration. Global analysis of the unimolecular unfolding transition of wild-type A4 revealed a partially unfolded equilibrium intermediate at low to moderate denaturant concentrations. The optically detected equilibrium of C321S A4 also fits best to a three-state model in which the native dimer unfolds via a monomeric intermediate state. Dimer dissociation is characterized by a dissociation constant, Kd, of ~90 nM (in terms of monomer), which is in agreement with the dissociation constant measured independently using fluorescence anisotropy. The results imply that FXI folding occurs via a monomeric equilibrium intermediate. This observation sheds light on the effect of certain naturally occurring mutations, such as F283L, which lead to intracellular accumulation of non-native forms of FXI. To investigate the structural and energetic consequences of the F283L mutation, which perturbs a cluster of aromatic side chains within the core of the A4 monomer, it was introduced into the dissociable dimer, C321S A4. NMR chemical shift analysis confirmed that the mutant can assume a native-like dimeric structure. However, equilibrium unfolding measurements show that the mutation causes a four-fold increase in the Kd for dissociation of the native dimer and a 1 kcal/mol stabilization of the monomer, resulting in a highly populated intermediate. Since the F283 side chain does not directly participate in the dimer interface, we propose that the F283L mutation leads to increased dimer dissociation by stabilizing a monomeric state with altered side chain packing that is unfavorable for homodimer formation.
FXI; blood coagulation; protein folding; NMR; fluorescence; CD