To Cleave or Not To Cleave in XL-MS?

Cross-linking mass spectrometry (XL-MS) is an efficient technique for uncovering structural features and interactions of the in-solution state of the proteins under investigation. Distance constraints obtained by this technique are highly complementary to classical structural biology approaches like X-ray crystallography and cryo-EM and have successfully been leveraged to shed light on protein structures of increasing size and complexity. To accomplish this, small reagents are used that typically incorporate two amine reactive moieties connected by a spacer arm and that can be applied in solution to protein structures of any size. Over the years, many reagents initially developed for different applications were adopted, and others were specifically developed for XL-MS. This has resulted in a vast array of options, making it difficult to make the right choice for specific experiments. Here, we delve into the previous decade of published XL-MS literature to uncover which workflows have been predominantly applied. We focus on application papers as these represent proof that biologically valid results can be extracted. This ignores some more recent approaches that did not have sufficient time to become more widely applied, for which we supply a separate discussion. From our selection, we extract information on the types of samples, cross-linking reagent, prefractionation, instruments, and data analysis, to highlight widely used workflows. All of the results are summarized in an easy-to-use flow chart defined by selection points resulting from our analysis. Although potentially biased by our own experiences, we expect this overview to be useful for novices stepping into this rapidly expanding field.

Identification of a tumor-specific allo-HLA-restricted γδTCR.

γδT cells are key players in cancer immune surveillance because of their ability to recognize malignant transformed cells, which makes them promising therapeutic tools in the treatment of cancer. However, the biological mechanisms of how γδT-cell receptors (TCRs) interact with their ligands are poorly understood. Within this context, we describe the novel allo-HLA-restricted and CD8α-dependent Vγ5Vδ1TCR. In contrast to the previous assumption of the general allo-HLA reactivity of a minor fraction of γδTCRs, we show that classic anti-HLA-directed, γδTCR-mediated reactivity can selectively act on hematological and solid tumor cells, while not harming healthy tissues in vitro and in vivo. We identified the molecular interface with proximity to the peptide-binding groove of HLA-A*24:02 as the essential determinant for recognition and describe the critical role of CD8 as a coreceptor. We conclude that alloreactive γδT-cell repertoires provide therapeutic opportunities, either within the context of haplotransplantation or as individual γδTCRs for genetic engineering of tumor-reactive T cells.

Probing the biophysical interplay between a viral genome and its capsid.

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

Squeezing protein shells: how continuum elastic models, molecular dynamics simulations, and experiments coalesce at the nanoscale.

The current rapid growth in the use of nanosized particles is fueled in part by our increased understanding of their physical properties and ability to manipulate them, which is essential for achieving optimal functionality. Here we report detailed quantitative measurements of the mechanical response of nanosized protein shells (viral capsids) to large-scale physical deformations and compare them with theoretical descriptions from continuum elastic modeling and molecular dynamics (MD). Specifically, we used nanoindentation by atomic force microscopy to investigate the complex elastic behavior of Hepatitis B virus capsids. These capsids are hollow, approximately 30 nm in diameter, and conform to icosahedral (5-3-2) symmetry. First we show that their indentation behavior, which is symmetry-axis-dependent, cannot be reproduced by a simple model based on Föppl-von Kármán thin-shell elasticity with the fivefold vertices acting as prestressed disclinations. However, we can properly describe the measured nonlinear elastic and orientation-dependent force response with a three-dimensional, topographically detailed, finite-element model. Next, we show that coarse-grained MD simulations also yield good agreement with our nanoindentation measurements, even without any fitting of force-field parameters in the MD model. This study demonstrates that the material properties of viral nanoparticles can be correctly described by both modeling approaches. At the same time, we show that even for large deformations, it suffices to approximate the mechanical behavior of nanosized viral shells with a continuum approach, and ignore specific molecular interactions. This experimental validation of continuum elastic theory provides an example of a situation in which rules of macroscopic physics can apply to nanoscale molecular assemblies.

Amyloid-beta-stimulated plasminogen activation by tissue-type plasminogen activator results in processing of neuroendocrine factors.

Alzheimer's disease brain is characterized by the abundant presence of amyloid deposits. Accumulation of the major constituent of these deposits, amyloid-beta (Abeta), has been associated with decreased neurotransmission, increased neuronal cell death, and with cognitive decline. The mechanisms underlying these phenomena have not yet been fully elucidated. We have previously shown that amyloid peptides like Abeta bind tissue-type plasminogen activator (tPA) and cause enhanced plasmin production. Here we describe the identification of five major neuronal cell-produced Abeta-associated proteins and how Abeta-stimulated plasmin formation affects their processing. These five proteins are all neuroendocrine factors (NEFs): chromogranins A, B and C; truncated chromogranin B; and VGF. Plasminogen caused processing of Abeta-bound (but not soluble) tPA, chromogranin B and VGF and the degradation products were released from Abeta. Processing of the neuroendocrine factors was dependent on tPA as it was largely abrogated in tPA-/- cells or in the presence of a specific tPA-inhibitor. If plasmin indeed produces NEF-derived peptides in vivo, some of these peptides may have biological activity, for instance in regulating neurotransmitter release that may affect the pathology of Alzheimer's disease.

Quantitative analysis of the novel anticancer drug ABT-518, a matrix metalloproteinase inhibitor, plus the screening of six metabolites in human plasma using high-performance liquid chromatography coupled with electrospray tandem mass spectrometry.

A liquid chromatographic/tandem mass spectrometric (LC/MS/MS) assay for the quantitative analysis of the novel anticancer drug ABT-518 and the screening of six potential metabolites in human plasma has been developed and validated to support a phase I study with the drug. ABT-518 is an inhibitor of matrix metalloproteinases, which are associated with tumor growth and development of metastasis. Plasma samples were prepared for analysis using a simple solid-phase extraction method on phenyl cartridges. LC separation was performed on a Zorbax extend C18 column (150 x 2.1 mm i.d., 5 microm particle size) using a mobile phase of methanol-aqueous 10 mM ammonium hydroxide (80:20, v/v) pumped at a flow-rate of 0.2 ml min(-1). An API2000 triple-quadrupole mass spectrometer was used for specific and sensitive detection. The best chromatographic speed (total run time 8 min) and peak shapes were obtained by employing an alkaline mobile phase (pH in aqueous phase approximately 10). Furthermore, an alkaline eluent was favored in order to obtain a better overall sensitivity for the protonated analytes. The dynamic range was from 10 to 1000 ng ml(-1) from 500 microl of plasma for ABT-518 and the metabolites were detected at levels of the same order of magnitude. Inter-assay accuracies for ABT-518 at five concentration levels were between -9.24 and 6.93% and inter-assay precisions were always <10.7%. Analyte stability was not critical during either storage or processing. This method was successfully applied in a phase I clinical study of ABT-518. The active drug, ABT-518, and all of the metabolites included in the assay could be identified in plasma from dosed patients. We believe that the method described in this paper using an alkaline mobile phase in combination with a basic stable analytical column may also be generally useful for the bioanalysis of other basic drugs.

Towards the understanding of molecular mechanisms in the early stages of heat-induced aggregation of beta-lactoglobulin AB.

Heat-induced aggregation of bovine beta-lactoglobulin AB (10 mg/ml) was studied at 68.5 degrees C at two different pH values (6.7, 4.9) using gel electrophoresis techniques and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis under non-reducing and reducing conditions showed that in the early stages of the aggregation of beta-lactoglobulin disulfide linked aggregates were formed on heating at pH 6.7, but not at pH 4.9. We related this result to the pH-dependent activity of the free thiol group at C121. Mass spectrometric analyses were conducted in two steps. The first involved the analysis of intact non-native monomers and dimers following their ultrasonic passive elution into a suitable solvent mixture in order to confirm the identity of the different gel bands. The second step comprises the analysis of in-gel digests for the determination of disulfide patterns in non-native monomers, covalent dimers and trimers. The results of in-gel digestions analyzed by mass spectrometry suggest that non-native dimers could result from the formation of inter-molecular disulfide bonds C121-C66, C160-C160, or C121-C160. Moreover, two inter-molecular bonds C121-C66 and C160-C160 between two and the same monomer units have been detected, which may play an important role in limiting the process of covalent beta-lactoglobulin network formation. The combination of SDS-PAGE and MALDI-TOF MS enables us to understand the mechanism of beta-lactoglobulin aggregation at the macromolecular level.

Determination of the stability of the noncovalent phospholipid transfer protein-lipid complex by electrospray time-of-flight mass spectrometry.

Phosphatidylcholine transfer protein (PC-TP) containing different molecular species of PC and phosphatidylinositol transfer protein alpha (PI-TPalpha) containing either a PI, PC, or PG molecule were identified as intact complexes by nano-electrospray ionization time-of-flight mass spectrometry. The stability of these complexes in the gas phase was determined by elevating the cone voltage (cv) resulting in the appearance of the protein void of lipid. PC-TP containing a PC species carrying an sn-1 palmitoyl chain was less stable than PC-TP containing a PC species carrying an sn-1 stearoyl chain given that these complexes were dissociated for 50% at a cv of roughly 30 and 45 V, respectively. Different acyl chains on the sn-2 position did not lead to significant changes in stability of the complex. In the case of PI-TPalpha, the complexes containing PI and PG were dissociated for 50% at a cv of 100 V as compared to a cv of 40 V for the complex containing PC. We propose that this difference in stability is due to hydrogen bonds between the polar headgroup of PI and PG and the lipid-binding site of PI-TPalpha. This may explain why PI-TPalpha preferentially binds PI from a membrane interface.