Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder.

Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited.

Advances in ion mobility spectrometry-mass spectrometry reveal key insights into amyloid assembly.

Interfacing ion mobility spectrometry to mass spectrometry (IMS-MS) has enabled mass spectrometric analyses to extend into an extra dimension, providing unrivalled separation and structural characterization of lowly populated species in heterogeneous mixtures. One biological system that has benefitted significantly from such advances is that of amyloid formation. Using IMS-MS, progress has been made into identifying transiently populated monomeric and oligomeric species for a number of different amyloid systems and has led to an enhanced understanding of the mechanism by which small molecules modulate amyloid formation. This review highlights recent advances in this field, which have been accelerated by the commercial availability of IMS-MS instruments. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.

Development of a urinary biomarker of human exposure to deoxynivalenol.

Deoxynivalenol (DON) is a mycotoxin frequently found as a contaminant of cereal crops and may be etiologically associated with adverse health effects in developing countries where considerable quantities of contaminated crops are consumed. We investigated the metabolism of DON in rats as a basis to establish methodology for a candidate biomarker of human exposure to this toxin and tested this methodology on urine samples from a potentially highly exposed population. Sprague-Dawley rats received a single dose of [14C]DON (5.0+/-0.1 mg/kg body weight, 5.5+/-0.1 microCi/kg) and the distribution of DON in body fluids was investigated over 72 h. DON and its metabolites were detectable in the plasma of rats with the highest levels at 8 h, at which time approximately 9% was bound to plasma protein. A total of 37% of the administered DON was excreted in the urine and DON-glucuronide was implicated as the major urinary metabolite based on reverse-phase HPLC analysis of beta-glucuronidase- and sulphatase-treated samples. An immunoaffinity column (IAC)-HPLC method was subsequently developed to measure urinary metabolites, with a view to establishing a urine-based human biomarker. Urine samples were collected from female inhabitants of Linxian County, China, a high risk region for oesophageal cancer (OC) and an area of potentially high DON exposure, and Gejiu, a low risk region in China. DON was detected in all 15 samples following beta-glucuronidase treatment and IAC enrichment with the identity of DON being confirmed by mass spectrometry. The mean levels of DON from the suspected high and low exposure regions of China were 37 ng/ml (range 14-94 ng/ml) and 12 ng/ml (range 4-18 ng/ml), respectively. This is estimated to correspond to daily exposures of 1.1-7.4 microg/kg/day and 0.3-1.4 microg/kg/day, respectively. This is the first reported measurement of a urinary biomarker for DON in both animals and humans and should facilitate epidemiological studies of disease associations with this mycotoxin.

A study of human coagulation factor XIII A-subunit by electrospray ionisation mass spectrometry.

A study of factor XIII A-subunit has been made by electrospray ionisation mass spectrometry. Factor XIII has been isolated and rigorously purified, and the intact A-subunit protein has been analysed to confirm its molecular weight accurately for the first time. Thrombin proteolysis of the A-subunit has been monitored and the resulting cleavage products, the activated A-subunit protein and the Activation Peptide, have been analysed by electrospray ionisation mass spectrometry to confirm the site of proteolysis. Facile and rapid separation of the Activation Peptide from the activated A-subunit protein has been achieved using simple on-line separation techniques. The molecular weight measurements have been repeated numerous times to provide an indication of the precision and variability of the analyses.

Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.

Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.

RNA aptamers for the MS2 bacteriophage coat protein and the wild-type RNA operator have similar solution behaviour.

We have probed the effects of altering buffer conditions on the behaviour of two aptamer RNAs for the bacterio-phage MS2 coat protein using site-specific substitution of 2'-deoxy-2-aminopurine nucleotides at key adenosine positions. These have been compared to the wild-type operator stem-loop oligonucleotide, which is the natural target for the coat protein. The fluorescence emission spectra show a position and oligonucleotide sequence dependence which appears to reflect local conformational changes. These are largely similar between the differing oligonucleotides and deviations can be explained by the individual features of each sequence. Recognition by coat protein is enhanced, unaffected or decreased depending on the site of substitution, consistent with the known protein-RNA contacts seen in crystal structures of the complexes. These data suggest that the detailed conformational dynamics of aptamers and wild-type RNA ligands for the same protein target are remarkably similar.

GroEL accelerates the refolding of hen lysozyme without changing its folding mechanism.

The chaperonin GroEL binds folding intermediates of four-disulfidehen lysozyme transiently within its central cavity. Using stopped flow fluorescence we show that GroEL binds early intermediates in folding and accelerates the slow kinetic phase that reflects the reversal of non-native interactions involving tryptophan residues and the formation of the native state. Pulsed hydrogen exchange monitored by electrospray ionization mass spectrometry demonstrates that GroEL does not alter the folding mechanism, nor are protected species unfolded by the chaperonin. The data suggest a mechanism for GroEL-assisted folding in which the reorganization of non-native tertiary interactions is facilitated but domain folding is unperturbed.

A near-native state on the slow refolding pathway of hen lysozyme.

The refolding of four disulfide lysozyme (at pH 5.2, 20 degrees C) involves parallel pathways, which have been proposed to merge at a near-native state. This species contains stable structure in the alpha- and beta-domains but lacks a functional active site. Although previous experiments have demonstrated that the near-native state is populated on the fast refolding pathway, its relevance to slow refolding molecules could not be directly determined from previous experiments. In this paper, we describe experiments that investigate the effect of added salts on the refolding pathway of lysozyme at pH 5.2, 20 degrees C. We show, using stopped flow tryptophan fluorescence, inhibitor binding, and circular dichroism (CD), that the rate of formation of native lysozyme on the slow refolding track is significantly reduced in solutions of high ionic strength in a manner dependent on the position of the anion in the Hofmeister series. By contrast, the rate of evolution of hydrogen exchange (HX) protection monitored by electrospray ionization mass spectrometry (ESI MS) is unchanged under the refolding conditions studied. The data show, therefore, that at high ionic strengths beta-domain stabilization and native state formation on the slow refolding pathway become kinetically decoupled such that the near-native state becomes significantly populated. Thus, by changing the energy landscape with the addition of salts new insights into the relevance of intermediate states in lysozyme refolding are revealed.

The dhnA gene of Escherichia coli encodes a class I fructose bisphosphate aldolase.

The gene encoding the Escherichia coli Class I fructose-1, 6-bisphosphate aldolase (FBP aldolase) has been cloned and the protein overproduced in high amounts. This gene sequence has previously been identified as encoding an E. coli dehydrin in the GenBanktrade mark database [gene dhnA; entry code U73760; Close and Choi (1996) Submission to GenBanktrade mark]. However, the purified protein overproduced from the dhnA gene shares all its properties with those known for the E. coli Class I FBP aldolase. The protein is an 8-10-mer with a native molecular mass of approx. 340 kDa, each subunit consisting of 349 amino acids. The Class I enzyme shows low sequence identity with other known FBP aldolases, both Class I and Class II (in the order of 20%), which may be reflected by some novel properties of this FBP aldolase. The active-site peptide has been isolated and the Schiff-base-forming lysine residue (Lys236) has been identified by a combination of site-directed mutagenesis, kinetics and electrospray-ionization MS. A second lysine residue (Lys238) has been implicated in substrate binding. The cloning of this gene and the high levels of overexpression obtained will facilitate future structure-function studies.

Analysis of a standard peptide mixture by capillary electrophoresis with mass spectrometry and with tandem mass spectrometry.

The combination of capillary electrophoresis with mass spectrometry allows efficient separation and identification of components in mixtures with greater specificity than can be obtained using capillary electrophoresis and ultra violet detection alone. For the mixture of peptides analysed in this application, molecular mass information was obtained on all of the components in the initial analysis. On-line capillary electrophoresis/tandem mass spectrometry was then used to generate amino acid sequence information for each of these peptides, at the pmole level, in the form of collision-induced production-ion spectra which were recorded only over the relevant time windows.

On the analysis of bovine trypsin by electrospray-mass spectrometry.

In this paper the results obtained from an electrospray mass spectrometric (ES-MS) analytical study of commercial grade bovine trypsin are presented and discussed. It is proven, somewhat contrary to an earlier report, that ES-MS analysis may be performed routinely on a triple quadrupole mass spectrometer using the normal ES-MS raw data transformation procedures to identify and quantify the three forms of trypsin, namely, beta, alpha and psi, present in the samples. Further, it was found that all of the samples analysed contained small amounts of two peptides of M(r) = 5447 and 17,882 Da, which are postulated to originate from catalytic cleavage of alpha-trypsin by beta-trypsin.

Continuous-flow fast atom bombardment mass spectrometry.

The continuous-flow fast atom bombardment probe performs equally well with or without a high-performance liquid chromatography column producing clean spectra containing little or no background noise. Its function as a liquid chromatography-mass spectrometry interface for labile and involatile samples has been illustrated with reference to dansylated amino acids. The versatility of the new probe has been exemplified by on-line enzymatic peptide sequencing.