An automated workflow based on data independent acquisition for practical and high-throughput personalized assay development and minimal residual disease monitoring in multiple myeloma patients.

OBJECTIVES: Minimal residual disease (MRD) status in multiple myeloma (MM) is an important prognostic biomarker. Personalized blood-based targeted mass spectrometry detecting M-proteins (MS-MRD) was shown to provide a sensitive and minimally invasive alternative to MRD-assessment in bone marrow. However, MS-MRD still comprises of manual steps that hamper upscaling of MS-MRD testing. Here, we introduce a proof-of-concept for a novel workflow using data independent acquisition-parallel accumulation and serial fragmentation (dia-PASEF) and automated data processing. METHODS: Using automated data processing of dia-PASEF measurements, we developed a workflow that identified unique targets from MM patient sera and personalized protein sequence databases. We generated patient-specific libraries linked to dia-PASEF methods and subsequently quantitated and reported M-protein concentrations in MM patient follow-up samples. Assay performance of parallel reaction monitoring (prm)-PASEF and dia-PASEF workflows were compared and we tested mixing patient intake sera for multiplexed target selection. RESULTS: No significant differences were observed in lowest detectable concentration, linearity, and slope coefficient when comparing prm-PASEF and dia-PASEF measurements of serial dilutions of patient sera. To improve assay development times, we tested multiplexing patient intake sera for target selection which resulted in the selection of identical clonotypic peptides for both simplex and multiplex dia-PASEF. Furthermore, assay development times improved up to 25× when measuring multiplexed samples for peptide selection compared to simplex. CONCLUSIONS: Dia-PASEF technology combined with automated data processing and multiplexed target selection facilitated the development of a faster MS-MRD workflow which benefits upscaling and is an important step towards the clinical implementation of MS-MRD.

The GlycoPaSER Prototype as a Real-Time N-Glycopeptide Identification Tool Based on the PaSER Parallel Computing Platform.

Real-time database searching allows for simpler and automated proteomics workflows as it eliminates technical bottlenecks in high-throughput experiments. Most importantly, it enables results-dependent acquisition (RDA), where search results can be used to guide data acquisition during acquisition. This is especially beneficial for glycoproteomics since the wide range of physicochemical properties of glycopeptides lead to a wide range of optimal acquisition parameters. We established here the GlycoPaSER prototype by extending the Parallel Search Engine in Real-time (PaSER) functionality for real-time glycopeptide identification from fragmentation spectra. Glycopeptide fragmentation spectra were decomposed into peptide and glycan moiety spectra using common N-glycan fragments. Each moiety was subsequently identified by a specialized algorithm running in real-time. GlycoPaSER can keep up with the rate of data acquisition for real-time analysis with similar performance to other glycoproteomics software and produces results that are in line with the literature reference data. The GlycoPaSER prototype presented here provides the first proof-of-concept for real-time glycopeptide identification that unlocks the future development of RDA technology to transcend data acquisition.

Peptide Interference with APP and Tau Association: Relevance to Alzheimer's Disease Amelioration.

The two major proteins involved in Alzheimer's disease (AD) are the amyloid precursor protein (APP) and Tau. Here, we demonstrate that these two proteins can bind to each other. Four possible peptides APP1 (390-412), APP2 (713-730), Tau1 (19-34) and Tau2 (331-348), were predicted to be involved in this interaction, with actual binding confirmed for APP1 and Tau1. In vivo studies were performed in an Alzheimer Disease animal model-APP double transgenic (Tg) 5xFAD-as well as in 5xFAD crossed with Tau transgenic 5xFADXTau (FT), which exhibit declined cognitive reduction at four months of age. Nasal administration of APP1 and Tau1 mixture, three times a week for four or five months, reduced amyloid plaque burden as well as the level of soluble Aß 1-42 in the brain. The treatment prevented the deterioration of cognitive functions when initiated at the age of three months, before cognitive deficiency was evident, and also at the age of six months, when such deficiencies are already observed, leading to a full regain of cognitive function.

Cross-linking reveals laminin coiled-coil architecture.

Laminin, an ∼800-kDa heterotrimeric protein, is a major functional component of the extracellular matrix, contributing to tissue development and maintenance. The unique architecture of laminin is not currently amenable to determination at high resolution, as its flexible and narrow segments complicate both crystallization and single-particle reconstruction by electron microscopy. Therefore, we used cross-linking and MS, evaluated using computational methods, to address key questions regarding laminin quaternary structure. This approach was particularly well suited to the ∼750-Šcoiled coil that mediates trimer assembly, and our results support revision of the subunit order typically presented in laminin schematics. Furthermore, information on the subunit register in the coiled coil and cross-links to downstream domains provide insights into the self-assembly required for interaction with other extracellular matrix and cell surface proteins.

Identification and Rationalization of Kinetic Folding Intermediates for a Low-Density Lipoprotein Receptor Ligand-Binding Module.

Many mutations that cause familial hypercholesterolemia localize to ligand-binding domain 5 (LA5) of the low-density lipoprotein receptor, motivating investigation of the folding and misfolding of this small, disulfide-rich, calcium-binding domain. LA5 folding is known to involve non-native disulfide isomers, yet these folding intermediates have not been structurally characterized. To provide insight into these intermediates, we used nuclear magnetic resonance (NMR) to follow LA5 folding in real time. We demonstrate that misfolded or partially folded disulfide intermediates are indistinguishable from the unfolded state when focusing on the backbone NMR signals, which provide information on the formation of only the final, native state. However, 13C labeling of cysteine side chains differentiated transient intermediates from the unfolded and native states and reported on disulfide bond formation in real time. The cysteine pairings in a dominant intermediate were identified using 13C-edited three-dimensional NMR, and coarse-grained molecular dynamics simulations were used to investigate the preference of this disulfide set over other non-native arrangements. The transient population of LA5 species with particular non-native cysteine connectitivies during folding supports the conclusion that cysteine pairing is not random and that there is a bias toward certain structural ensembles during the folding process, even prior to the binding of calcium.

Medication detection by a combinatorial fluorescent molecular sensor.

Working together to uncover the truth: A molecule-sized diagnostic system combining several recognition elements and four fluorescence-emission channels enabled the identification of a wide range of pharmaceuticals on the basis of distinct photophysical processes. The molecular sensor (see simplified representation; ID = identification) was also used to analyze drug concentrations and combinations in urine samples in a high-throughput manner.

Characterization of protein complexes in extracellular vesicles by intact extracellular vesicle crosslinking mass spectrometry (iEVXL).

Extracellular vesicles (EVs) are blood-borne messengers that coordinate signalling between different tissues and organs in the body. The specificity of such crosstalk is determined by preferential EV docking to target sites, as mediated through protein-protein interactions. As such, the need to structurally characterize the EV surface precedes further understanding of docking selectivity and recipient-cell uptake mechanisms. Here, we describe an intact extracellular vesicle crosslinking mass spectrometry (iEVXL) method that can be applied for structural characterization of protein complexes in EVs. By using a partially membrane-permeable disuccinimidyl suberate crosslinker, proteins on the EV outer-surface and inside EVs can be immobilized together with their interacting partners. This not only provides covalent stabilization of protein complexes before extraction from the membrane-enclosed environment, but also generates a set of crosslinking distance restraints that can be used for structural modelling and comparative screening of changes in EV protein assemblies. Here we demonstrate iEVXL as a powerful approach to reveal high-resolution information, about protein determinants that govern EV docking and signalling, and as a crucial aid in modelling docking interactions.

Unexpected influence of stereochemistry on the cytotoxicity of highly efficient Ti(IV) salan complexes: new mechanistic insights.

The effect of stereochemistry on the cytotoxicity of highly active and hydrolytically stable N-methylated Ti(IV) salan complexes is reported. Four bis(isopropoxo) complexes incorporating N-methylated salan ligands with different aromatic substitution patterns have been prepared in racemic and optically active forms for the first time by ligand-to-metal chiral induction from trans-diaminocyclohexyl-based chiral ligands. The configuration of the metal center that derives from that of the ligand has an enormous influence on cytotoxicity, with the racemic mixture mostly being more active than the single enantiomers that are of either similar or different activity. This implies that the active species is a salan-bound heterochiral polynuclear compound, interacting with a chiral target. Four additional complexes of achiral salan and chiral labile sec-butoxo ligands, analyzed as racemic and as homochiral, revealed no influence of stereochemistry, supporting early dissociation of the labile ligands to give the polynuclear products.

New insights on the active species and mechanism of cytotoxicity of salan-Ti(IV) complexes: a stereochemical study.

Following the discovery of cisplatin, much effort has been devoted to the exploration of transition metal complexes as cytotoxic agents. We have recently introduced the highly efficient C(2)-symmetrical salan-Ti(IV) family of complexes, demonstrating high cytotoxicity toward colon and ovarian cells and enhanced hydrolytic stability in mixed organic/water solutions. The effect of stereochemistry is hereby reported, by comparing the cytotoxic activity and hydrolysis of pure enantiomers and their racemic mixture for four complexes of this family with different aromatic substitutions: para-Me, para-Cl, ortho-Cl, and ortho-OMe. These complexes include the trans-diaminocyclohexyl bridge, which enables ligand-to-metal chiral induction to give solely the Δ isomer when starting from the R,R ligand and vice versa. Different activity is obtained for the different stereochemical forms (Δ, Λ, and rac) in two of the four complexes, where for the other two either all forms are inactive or all are highly active. Additionally, where not all are of similar activity, the racemic mixture is the least active of the three. We therefore conclude that the salan ligand is essential for the fruitful biological interaction, which probably involves a chiral cellular target. The activity of the racemate differing from that expected from a simple mixture of enantiomers operating separately may be explained by the involvement of a polynuclear active species, where different metal centers might be of different configurations. This is particularly supported by the different polynuclear products of hydrolysis obtained from an optically pure complex and from the racemic one, as analyzed crystallographically. The former is an all-R,R chiral C(1)-symmetrical homodimer, while the latter is an achiral R,R-S,SC(i)-symmetrical heterodimer obtained through chiral recognition.

Extracellular crosslinking mass spectrometry reveals HLA class I - HLA class II interactions on the cell surface.

Human Leukocyte Antigen (HLA) complexes are critical cell-surface protein assemblies that facilitate T-cell surveillance of almost all cell types in the body. While T-cell receptor binding to HLA class I and class II complexes is well-described with detailed structural information, the nature of cis HLA interactions within the plasma membrane of the surveyed cells remains to be better characterized, as protein-protein interactions in the membrane environment are technically challenging to profile. Here we performed extracellular chemical crosslinking on intact antigen presenting cells to specifically elucidate protein-protein interactions present in the external plasma membrane. We found that the crosslink dataset was dominated by inter- and intra-protein crosslinks involving HLA molecules, which enabled not only the construction of an HLA-centric plasma membrane protein interaction map, but also revealed multiple modes of HLA class I - HLA class II interactions with further structural modeling based on crosslinker distance restraints. Collectively, our data demonstrate that HLA molecules colocalize and can be densely packed on the plasma membrane.

3D mapping of native extracellular matrix reveals cellular responses to the microenvironment.

Cells and extracellular matrix (ECM) are mutually interdependent: cells guide self-assembly of ECM precursors, and the resulting ECM architecture supports and instructs cells. Though bidirectional signaling between ECM and cells is fundamental to cell biology, it is challenging to gain high-resolution structural information on cellular responses to the matrix microenvironment. Here we used cryo-scanning transmission electron tomography (CSTET) to reveal the nanometer- to micron-scale organization of major fibroblast ECM components in a native-like context, while simultaneously visualizing internal cell ultrastructure including organelles and cytoskeleton. In addition to extending current models for collagen VI fibril organization, three-dimensional views of thick cell regions and surrounding matrix showed how ECM networks impact the structures and dynamics of intracellular organelles and how cells remodel ECM. Collagen VI and fibronectin were seen to distribute in fundamentally different ways in the cell microenvironment and perform distinct roles in supporting and interacting with cells. This work demonstrates that CSTET provides a new perspective for the study of ECM in cell biology, highlighting labeled extracellular elements against a backdrop of unlabeled but morphologically identifiable cellular features with nanometer resolution detail.

Single-molecule spectroscopy exposes hidden states in an enzymatic electron relay.

The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.