Search

Selecting a suitable nano-liquid chromatography system (LC), ionization source and mass spectrometer for LC–tandem mass spectrometry (MS-MS) studies is complicated by numerous competing technologies. This study compares four popular nano-LC systems, four ionization sources and three MS facilities that use completely different LC–MS-MS systems. Statistically significant differences in LC performance were identified with similarly performing Proxeon, Waters and Eksigent nanoLC-Ultra systems [retention time routinely at 0.7–0.9% relative standard deviation (RSD)], and all outperformed the Eksigent nanoLC-2D (RSD ∼2%). In addition, compatibility issues were identified between the Bruker HCT ion trap mass spectrometer and both the Eksigent nanoLC-2D and the Bruker nanoelectrospray source. The electrospray source itself had an unexpected and striking effect on chromatographic reproducibility on the Bruker HCT ion trap. The New Objective nanospray source significantly outperformed the Bruker nanospray source in retention time RSD (1% RSD versus 14% RSD, respectively); and the Bruker nebulized nanospray source outperformed both of these traditional, non-nebulized sources (0.5% RSD in retention time). Finally, to provide useful benchmarks for overall proteomics sensitivity, different LC–MS-MS platforms were compared by analyzing a range of concentrations of tryptic digests of bovine serum albumin at three MS facilities. The results indicate that similar sensitivity can be realized with a Bruker HCT-Ultra ion trap, a Thermo LTQ-Velos Linear ion trap and a Thermo LTQ-Orbitrap XL-ETD. [ABSTRACT FROM PUBLISHER]

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 [ABSTRACT FROM AUTHOR]

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein. [ABSTRACT FROM AUTHOR]

The nature of the “toxic gain of function” that results from amyotrophic lateral sclerosis (ALS)-, Parkinson-, and Alzheimer-related mutations is a matter of debate. As a result no adequate model of any neurodegenerative disease etiology exists. We demonstrate that two synergistic properties, namely, increased protein aggregation propensity (increased likelihood that an unfolded protein will aggregate) and decreased protein stability (increased likelihood that a protein will unfold), are central to ALS etiology. Taken together these properties account for 69% of the variability in mutant Cu/Zn-superoxide-dismutase-linked familial ALS patient survival times. Aggregation is a concentration-dependent process, and spinal cord motor neurons have higher concentrations of Cu/Zn-superoxide dismutase than the surrounding cells. Protein aggregation therefore is expected to contribute to the selective vulnerability of motor neurons in familial ALS.

Ras dimerization is critical for Raf activation. Here we show that the Ras binding domain of Raf (Raf-RBD) induces robust Ras dimerization at low surface densities on supported lipid bilayers and, to a lesser extent, in solution as observed by size exclusion chromatography and confirmed by SAXS. Community network analysis based on molecular dynamics simulations shows robust allosteric connections linking the two Raf-RBD D113 residues located in the Galectin scaffold protein binding site of each Raf-RBD molecule and 85 Å apart on opposite ends of the dimer complex. Our results suggest that Raf-RBD binding and Ras dimerization are concerted events that lead to a high-affinity signaling complex at the membrane that we propose is an essential unit in the macromolecular assembly of higher order Ras/Raf/Galectin complexes important for signaling through the Ras/Raf/MEK/ERK pathway. [ABSTRACT FROM AUTHOR]

The combination of native electrospray ionization with top-down fragmentation in mass spectrometry (MS) allows simultaneous determination of the stoichiometry of noncovalent complexes and identification of their component proteoforms and cofactors. Although this approach is powerful, both native MS and top-down MS are not yet well standardized, and only a limited number of laboratories regularly carry out this type of research. To address this challenge, the Consortium for Top-Down Proteomics initiated a study to develop and test protocols for native MS combined with top-down fragmentation of proteins and protein complexes across 11 instruments in nine laboratories. Here we report the summary of the outcomes to provide robust benchmarks and a valuable entry point for the scientific community., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Mutations in the gene encoding Cu-Zn superoxide dismutase 1 (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS) cases. A shared effect of these mutations is that SOD1, which is normally a stable dimer, dissociates into toxic monomers that seed toxic aggregates. Considerable research effort has been devoted to developing compounds that stabilize the dimer of fALS SOD1 variants, but unfortunately, this has not yet resulted in a treatment. We hypothesized that cyclic thiosulfinate cross-linkers, which selectively target a rare, 2 cysteine-containing motif, can stabilize fALS-causing SOD1 variants in vivo. We created a library of chemically diverse cyclic thiosulfinates and determined structure-cross-linking-activity relationships. A pre-lead compound, "S-XL6," was selected based upon its cross-linking rate and drug-like properties. Co-crystallographic structure clearly establishes the binding of S-XL6 at Cys 111 bridging the monomers and stabilizing the SOD1 dimer. Biophysical studies reveal that the degree of stabilization afforded by S-XL6 (up to 24°C) is unprecedented for fALS, and to our knowledge, for any protein target of any kinetic stabilizer. Gene silencing and protein degrading therapeutic approaches require careful dose titration to balance the benefit of diminished fALS SOD1 expression with the toxic loss-of-enzymatic function. We show that S-XL6 does not share this liability because it rescues the activity of fALS SOD1 variants. No pharmacological agent has been proven to bind to SOD1 in vivo. Here, using a fALS mouse model, we demonstrate oral bioavailability; rapid engagement of SOD1G93A by S-XL6 that increases SOD1G93A's in vivo half-life; and that S-XL6 crosses the blood-brain barrier. S-XL6 demonstrated a degree of selectivity by avoiding off-target binding to plasma proteins. Taken together, our results indicate that cyclic thiosulfinate-mediated SOD1 stabilization should receive further attention as a potential therapeutic approach for fALS., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hossain et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Proteoforms, which arise from post-translational modifications, genetic polymorphisms and RNA splice variants, play a pivotal role as drivers in biology. Understanding proteoforms is essential to unravel the intricacies of biological systems and bridge the gap between genotypes and phenotypes. By analysing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and can decipher protein function, uncover disease mechanisms and advance precision medicine. This Primer explores TDP, including the underlying principles, recent advances and an outlook on the future. The experimental section discusses instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques and data collection. The results section looks at how to decipher raw data, visualize intact protein spectra and unravel data analysis. Additionally, proteoform identification, characterization and quantification are summarized, alongside approaches for statistical analysis. Various applications are described, including the human proteoform project and biomedical, biopharmaceutical and clinical sciences. These are complemented by discussions on measurement reproducibility, limitations and a forward-looking perspective that outlines areas where the field can advance, including potential future applications., Competing Interests: Competing interests J.A.L., J.C.-R., J.N.A., L.P.-T., L.M.S. and Y.G. are currently board members of Consortium for Top-down Proteomics. Y.O.T. is an employee of Spectroswiss, a company that develops data acquisition systems and data processing software for mass spectrometry. X.L. has a project contract with Bioinformatics Solutions Inc., a company that develops data processing software for mass spectrometry. D.S.R. and Y.G. are named as inventors for the patent application US Patent App. 17/786,482. L.P.-T. is named as an inventor for the US Patent App. 17/954,834. Y.G. is named as an inventor for the US Patent App. 18/069,005; US Patent App. 17/978,793; US Patent App. 18/451,614; and US Patent 11,567,085. S.W. declares no competing interests.

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS.

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Top-down mass spectrometry enables the analysis of intact proteins and proteoforms. Here members of the Consortium for Top-Down Proteomics provide a decision tree that guides researchers to robust protocols for mass analysis of intact proteins (antibodies, membrane proteins and others) from mixtures of varying complexity. We also present cross-platform analytical benchmarks using a protein standard sample, to allow users to gauge their proficiency.

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human-readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) is used for analyzing protein dynamics, protein folding/unfolding, and molecular interactions. Until this study, HDX MS experiments employed mass spectral resolving powers that afforded only one peak per nominal mass in a given peptide's isotope distribution, and HDX MS data analysis methods were developed accordingly. A level of complexity that is inherent to HDX MS remained unaddressed, namely, various combinations of natural abundance heavy isotopes and exchanged deuterium shared the same nominal mass and overlapped at previous resolving powers. For example, an A + 2 peak is comprised of (among other isotopomers) a two-(2)H-exchanged/zero-(13)C isotopomer, a one-(2)H-exchanged/one-(13)C isotopomer, and a zero-(2)H-exchanged/two-(13)C isotopomer. Notably, such isotopomers differ slightly in mass as a result of the ∼3 mDa mass defect between (2)H and (13)C atoms. Previous HDX MS methods did not resolve these isotopomers, requiring a natural-abundance-only (before HDX or "time zero") spectrum and data processing to remove its contribution. It is demonstrated here that high-resolution mass spectrometry can be used to detect isotopic fine structure, such as in the A + 2 profile example above, deconvolving the isotopomer species resulting from deuterium incorporation. Resolving isotopic fine structure during HDX MS therefore permits direct monitoring of HDX, which can be calculated as the sum of the fractional peak magnitudes of the deuterium-exchanged isotopomers. This obviates both the need for a time zero spectrum as well as data processing to account for natural abundance heavy isotopes, saving instrument and analysis time.

The small quantities of protein required for mass spectrometry (MS) make it a powerful tool to detect binding (protein-protein, protein-small molecule, etc.) of proteins that are difficult to express in large quantities, as is the case for many intrinsically disordered proteins. Chemical cross-linking, proteolysis, and MS analysis, combined, are a powerful tool for the identification of binding domains. Here, we present a traditional approach to determine protein-protein interaction binding sites using heavy water ((18)O) as a label. This technique is relatively inexpensive and can be performed on any mass spectrometer without specialized software.

The resolution of MALDI MS imaging is limited by the displacement of analytes during matrix deposition or by laser focal diameter. Here we present three methods that minimize the displacement of analytes during matrix deposition, including a method where image resolution is not limited by the laser focal diameter. The first method, matrix solution fixation, simultaneously fixes tissue while depositing matrix and is optimal for analyzing proteins and for applications requiring a fast preparation time. This method is characterized by compatibility with histology methods and laser focal diameter-limited resolution. The second method, a sensor controlled aerosol, is characterized by aerosol droplet size-limited resolution and is optimal for small molecules, including lipids, peptides, and drug-like molecules. The third method, microinjection with matrix, selectively deposits matrix upon cells of interest, offers cellular resolution and is compatible with most analytes. A flow chart summarizing methods is provided so that users may design a tissue preparation strategy based upon their resources and experimental goals.