Explore the vast range of titles available.

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology. The characterization of biomacromolecule structural vibrations has been impeded by a broad continuous vibrational density of states obscuring molecule specific vibrations. A terahertz microscopy system using polarization control produces signatures to dynamically fingerprint proteins and a RNA G-quadruplex.

Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure.Graphical abstractPeptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data.

The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic ‘fingerprints’ associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore. Single amino acids and peptides can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules and measuring the electron tunnelling current across the junction.

Assessing past foodways, subsistence strategies, and environments depends on the accurate identification of animals in the archaeological record. The high rates of fragmentation and often poor preservation of animal bones at many archaeological sites across sub-Saharan Africa have rendered archaeofaunal specimens unidentifiable beyond broad categories, such as “large mammal” or “medium bovid”. Identification of archaeofaunal specimens through Zooarchaeology by Mass Spectrometry (ZooMS), or peptide mass fingerprinting of bone collagen, offers an avenue for identification of morphologically ambiguous or unidentifiable bone fragments from such assemblages. However, application of ZooMS analysis has been hindered by a lack of complete reference peptide markers for African taxa, particularly bovids. Here we present the complete set of confirmed ZooMS peptide markers for members of all African bovid tribes. We also identify two novel peptide markers that can be used to further distinguish between bovid groups. We demonstrate that nearly all African bovid subfamilies are distinguishable using ZooMS methods, and some differences exist between tribes or sub-tribes, as is the case for Bovina (cattle) vs. Bubalina (African buffalo) within the subfamily Bovinae. We use ZooMS analysis to identify specimens from extremely fragmented faunal assemblages from six Late Holocene archaeological sites in Zambia. ZooMS-based identifications reveal greater taxonomic richness than analyses based solely on morphology, and these new identifications illuminate Iron Age subsistence economies c. 2200–500 cal BP. While the Iron Age in Zambia is associated with the transition from hunting and foraging to the development of farming and herding, our results demonstrate the continued reliance on wild bovids among Iron Age communities in central and southwestern Zambia Iron Age and herding focused primarily on cattle. We also outline further potential applications of ZooMS in African archaeology.

Proteins are the primary functional actors of the cell. While proteoform diversity is known to be highly biologically relevant, current protein analysis methods are of limited use for distinguishing proteoforms. Mass spectrometric methods, in particular, often provide only ambiguous information on post-translational modification sites, and sequences of co-existing modifications may not be resolved. Here we demonstrate fluorescence resonance energy transfer (FRET)-based single-molecule protein fingerprinting to map the location of individual amino acids and post-translational modifications within single full-length protein molecules. Our data show that both intrinsically disordered proteins and folded globular proteins can be fingerprinted with a subnanometer resolution, achieved by probing the amino acids one by one using single-molecule FRET via DNA exchange. This capability was demonstrated through the analysis of alpha-synuclein, an intrinsically disordered protein, by accurately quantifying isoforms in mixtures using a machine learning classifier, and by determining the locations of two O -GlcNAc moieties. Furthermore, we demonstrate fingerprinting of the globular proteins Bcl-2-like protein 1, procalcitonin and S100A9. We anticipate that our ability to perform proteoform identification with the ultimate sensitivity may unlock exciting new venues in proteomics research and biomarker-based diagnosis. Distinguishing proteoforms and post-translational modifications has remained a challenge. Here the authors explore single-molecule fluorescence resonance energy transfer to probe amino acids via DNA exchange and map the location of individual amino acids and post-translational modifications within single full-length protein molecules.

Oligonucleotide mapping via liquid chromatography with UV detection coupled to tandem mass spectrometry (LC-UV-MS/MS) was recently developed to support development of Comirnaty, the world’s first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with 100% maximum sequence coverage, and a microheterogeneity assessment of 5′ terminus capping and 3′ terminus poly(A)-tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.

Peptide mapping analysis is a regulatory expectation to verify the primary structure of a recombinant product sequence and to monitor post-translational modifications (PTMs). Although proteolytic digestion has been used for decades, it remains a labour-intensive procedure that can be challenging to accurately reproduce. Here, we describe a fast and reproducible protocol for protease digestion that is automated using immobilised trypsin on magnetic beads, which has been incorporated into an optimised peptide mapping workflow to show method transferability across laboratories. The complete workflow has the potential for use within a multi-attribute method (MAM) approach in drug development, production and QC laboratories. The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe. LC-MS/MS along with database searching was used to characterise the protein and determine relevant product quality attributes (PQAs) for further testing. A list of relevant critical quality attributes (CQAs) was then established by creating a peptide workbook containing the specific mass-to-charge (m/z) ratios of the modified and unmodified peptides of the selected CQAs, to be monitored in a subsequent test using LC-MS analysis. Data is provided that shows robust digestion efficiency and low levels of protocol induced PTMs.

Purposes In the peptide mapping reduction process for monoclonal antibodies (mAbs) and other proteins, the conventional reducing reagents β-mercaptoethanol (β-ME) and dithiothreitol (DTT) pose challenges due to their strong odor and toxicity at high concentrations. Cysteine (Cys), an essential amino acid for new protein synthesis, is an overlooked, nontoxic, and odorless reducing agent. This study presents a novel peptide mapping method using Cys as the reducing agent. Methods We explored the reducing effect of Cys at different concentrations and pH levels for peptide mapping analysis of a specific mAb (mAb-1), using DTT as a positive control. RP-HPLC analysis, including UV chromatogram comparison and overall similarity calculation, was conducted for comparison. LC–MS analysis was subsequently utilized to characterize the primary sequence of mAb-1. We also applied the method to other mAbs or proteins to demonstrate its wide applicability. Results The UV chromatogram and overall similarity of Cys as a reducing agent at concentrations ranging from 10 to 40 mM and pH levels between 7.0 and 11.0 were consistent with those of the positive control. Reduced concentrations of Cys or lower pH levels compromised reducing efficacy. This novel reducing method proficiently characterized the primary sequence of mAb-1, achieving an overall sequence coverage of 97%. In the analysis of other mAbs or proteins, the peptide mapping results also showed high consistency. Conclusions Cys exhibits a reducing ability comparable to DTT and possesses the advantageous characteristics of being nontoxic and odorless, making it a potential alternative for disulfide bond reduction and peptide mapping analysis of proteins and mAbs.

A multilaboratory analysis characterized the ability of 27 different labs to identify 20 proteins at equimolar concentrations in a highly purified test sample mixture using mass spectrometry. The results show that while the technology is reproducible, many common experimental problems arise, and improved search engines and databases are still needed. We performed a test sample study to try to identify errors leading to irreproducibility, including incompleteness of peptide sampling, in liquid chromatography–mass spectrometry–based proteomics. We distributed an equimolar test sample, comprising 20 highly purified recombinant human proteins, to 27 laboratories. Each protein contained one or more unique tryptic peptides of 1,250 Da to test for ion selection and sampling in the mass spectrometer. Of the 27 labs, members of only 7 labs initially reported all 20 proteins correctly, and members of only 1 lab reported all tryptic peptides of 1,250 Da. Centralized analysis of the raw data, however, revealed that all 20 proteins and most of the 1,250 Da peptides had been detected in all 27 labs. Our centralized analysis determined missed identifications (false negatives), environmental contamination, database matching and curation of protein identifications as sources of problems. Improved search engines and databases are needed for mass spectrometry–based proteomics.

Although dysfunctional protein homeostasis (proteostasis) is a key factor in many age‐related diseases, the untargeted identification of structurally modified proteins remains challenging. Peptide location fingerprinting is a proteomic analysis technique capable of identifying structural modification‐associated differences in mass spectrometry (MS) data sets of complex biological samples. A new webtool (Manchester Peptide Location Fingerprinter), applied to photoaged and intrinsically aged skin proteomes, can relatively quantify peptides and map statistically significant differences to regions within protein structures. New photoageing biomarker candidates were identified in multiple pathways including extracellular matrix organisation (collagens and proteoglycans), protein synthesis and folding (ribosomal proteins and TRiC complex subunits), cornification (keratins) and hemidesmosome assembly (plectin and integrin α6β4). Crucially, peptide location fingerprinting uniquely identified 120 protein biomarker candidates in the dermis and 71 in the epidermis which were modified as a consequence of photoageing but did not differ significantly in relative abundance (measured by MS1 ion intensity). By applying peptide location fingerprinting to published MS data sets, (identifying biomarker candidates including collagen V and versican in ageing tendon) we demonstrate the potential of the MPLF webtool for biomarker discovery. Peptide location fingerprinting is a proteomic mass spectrometry tool capable of detecting localised statistically significant changes in peptide yield along the structures of proteins in complex, whole tissue lysates. In this study, peptide location fingerprinting revealed novel biomarker candidates of skin photoageing undetectable by conventional relative quantification.