Blood to Biomarker Quantitation in Under One Hour with Rapid Proteomics using a Hyperthermoacidic Protease.

Multi-step multi-hour tryptic proteolysis has limited the utility of bottom-up proteomics for cases that require immediate quantitative information. The recently available hyperthermoacidic (HTA) protease "Krakatoa" digests samples in a single 5 to 30-minute step at pH 3 and >80 °C; conditions that disrupt most cells and tissues, denature proteins, and block disulfide reformation. The combination of quick single-step sample preparation with high throughput dual trapping column single analytical column (DTSC) liquid chromatography-mass spectrometry (LC-MS) achieves "Rapid Proteomics" in which the time from sample collection to actionable data is less than 1 hour. The presented development and systematic evaluation of this methodology found reproducible quantitation of over 160 proteins from just 1 microliter of whole blood. Furthermore, the preference of the HTA-protease for intact proteins over peptides allows for sensitive targeted quantitation of the Angiotensin I and II bioactive peptides in under half an hour. With these methods we analyzed serum and plasma from 53 individuals and quantified Angiotensin and proteins that were not detected with trypsin. This assessment of Rapid Proteomics suggests that concentration of circulating protein and peptide biomarkers could be measured in almost real-time by LC-MS. TOC Figure: Rapid proteomics enables near real-time monitoring of circulating blood biomarkers. One microliter of blood is collected every 8 minutes, digested for 20 minutes, and then analyzed by targeted mass spectrometry for 8 minutes. This results in a 30-minute delay with datapoints every 8 minutes.

An inflection point in high-throughput proteomics with Orbitrap Astral: analysis of biofluids, cells, and tissues.

This technical note presents a comprehensive proteomics workflow for the new combination of Orbitrap and Astral mass analyzers across biofluids, cells, and tissues. Central to our workflow is the integration of Adaptive Focused Acoustics (AFA) technology for cells and tissue lysis, to ensure robust and reproducible sample preparation in a high-throughput manner. Furthermore, we automated the detergent-compatible single-pot, solid-phase-enhanced sample Preparation (SP3) method for protein digestion, a technique that streamlines the process by combining purification and digestion steps, thereby reducing sample loss and improving efficiency. The synergy of these advanced methodologies facilitates a robust and high-throughput approach for cells and tissue analysis, an important consideration in translational research. This work disseminates our platform workflow, analyzes the effectiveness, demonstrates reproducibility of the results, and highlights the potential of these technologies in biomarker discovery and disease pathology. For cells and tissues (heart, liver, lung, and intestine) proteomics analysis by data-independent acquisition mode, identifications exceeding 10,000 proteins can be achieved with a 24-minute active gradient. In 200ng injections of HeLa digest across multiple gradients, an average of more than 80% of proteins have a CV less than 20%, and a 45-minute run covers ~90% of the expressed proteome. In plasma samples including naive, depleted, perchloric acid precipitated, and Seer nanoparticle captured, all with a 24-minute gradient length, we identified 87, 108, 96 and 137 out of 216 FDA approved circulating protein biomarkers, respectively. This complete workflow allows for large swaths of the proteome to be identified and is compatible across diverse sample types.

Proteomes of Micro- and Nanosized Carriers Engineered from Red Blood Cells.

Red blood cell (RBC)-derived systems offer a potential platform for delivery of biomedical cargos. Although the importance of specific proteins associated with the biodistribution and pharmacokinetics of these particles has been recognized, it remains to be explored whether some of the key transmembrane and cytoskeletal proteins responsible for immune-modulatory effects and mechanical integrity of the particles are retained. Herein, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quantitative tandem mass tag mass spectrometry in conjunction with bioinformatics analysis, we have examined the proteomes of micro- and nanosized erythrocyte ghosts doped with indocyanine green and compared them with those of RBCs. We identified a total of 884 proteins in each set of RBCs, micro-, and nanosized particles, of which 8 and 45 proteins were expressed at significantly different relative abundances when comparing micro-sized particles vs RBCs and nanosized particles vs RBCs, respectively. We found greater differences in relative abundances of some mechano-modulatory proteins, such as band 3 and protein 4.2, and immunomodulatory proteins like CD44, CD47, and CD55 in nanosized particles as compared to RBCs. Our findings highlight that the methods utilized in fabricating RBC-based systems can induce substantial effects on their proteomes. Mass spectrometry data are available at ProteomeXchange with the identifier PXD038780.

High-Resolution Mapping of Amino Acid Residues in DNA-Protein Cross-Links Enabled by Ribonucleotide-Containing DNA.

DNA-protein cross-links have broad applications in mapping DNA-protein interactions and provide structural insights into macromolecular structures. However, high-resolution mapping of DNA-interacting amino acid residues with tandem mass spectrometry remains challenging due to difficulties in sample preparation and data analysis. Herein, we developed a method for identifying cross-linking amino residues in DNA-protein cross-links at single amino acid resolution. We leveraged the alkaline lability of ribonucleotides and designed ribonucleotide-containing DNA to produce structurally defined nucleic acid-peptide cross-links under our optimized ribonucleotide cleavage conditions. The structurally defined oligonucleotide-peptide heteroconjugates improved ionization, reduced the database search space, and facilitated the identification of cross-linking residues in peptides. We applied the workflow to identifying abasic (AP) site-interacting residues in human mitochondrial transcription factor A (TFAM)-DNA cross-links. With sub-nmol sample input, we obtained high-quality fragmentation spectra for nucleic acid-peptide cross-links and identified 14 cross-linked lysine residues with the home-built AP_CrosslinkFinder program. Semi-quantification based on integrated peak areas revealed that K186 of TFAM is the major cross-linking residue, consistent with K186 being the closest (to the AP modification) lysine residue in solved TFAM:DNA crystal structures. Additional cross-linking lysine residues (K69, K76, K136, K154) support the dynamic characteristics of TFAM:DNA complexes. Overall, our combined workflow using ribonucleotide as a chemically cleavable DNA modification together with optimized sample preparation and data analysis offers a simple yet powerful approach for mapping cross-linking sites in DNA-protein cross-links. The method is amendable to other chemical or photo-cross-linking systems and can be extended to complex biological samples.

Structural Effects of Solvation by 18-Crown-6 on Gaseous Peptides and TrpCage after Electrospray Ionization.

Significant effort is being employed to utilize the inherent speed and sensitivity of mass spectrometry for rapid structural determination of proteins; however, a thorough understanding of factors influencing the transition from solution to gas phase is critical for correct interpretation of the results from such experiments. It was previously shown that combined use of action excitation energy transfer (EET) and simulated annealing can reveal detailed structural information about gaseous peptide ions. Herein, we utilize this method to study microsolvation of charged groups by retention of 18-crown-6 (18C6) in the gas phase. In the case of GTP (CEGNVRVSRE LAGHTGY), solvation of the 2+ charge state leads to reduced EET, whereas the opposite result is obtained for the 3+ ion. For the mini-protein C-Trpcage, solvation by 18C6 leads to dramatic increase in EET for the 3+ ion. Examination of structural details probed by molecular dynamics calculations illustrate that solvation by 18C6 alleviates the tendency of charged side chains to seek intramolecular solvation, potentially preserving native-like structures in the gas phase. These results suggest that microsolvation may be an important tool for facilitating examination of native-like protein structures in gas phase experiments. Graphical Abstract ᅟ.

Leveraging ultraviolet photodissociation and spectroscopy to investigate peptide and protein three-dimensional structure with mass spectrometry.

Recent advances in mass spectrometry and lasers have facilitated the development of novel experiments combining the benefits of both technologies. This minireview focuses on the coupling of visible/ultraviolet photons with mass spectrometry for analysis of peptide and protein three-dimensional structure. Practical aspects of instrument design and the relationship between experiment and theory are discussed. Experiments utilizing spectroscopy, action spectroscopy, excitation energy transfer, photodissociation, and photoactivated radical chemistry are described. The strengths and weaknesses of each approach are discussed in relation to the type of information typically obtained. A significant body of data suggests that under appropriate source conditions, kinetically trapped structures are observed in these experiments rather than true gas phase minima, suggesting retention of solution phase structural features is possible. Further refinement and exploration of these methods promises to accelerate protein structure discovery in the near future.

Two-step energy transfer enables use of phenylalanine in action-EET for distance constraint determination in gaseous biomolecules.

Two-step energy transfer is potentially useful for exploring macromolecular structure, but it has not been observed previously in the gas-phase. Single step excitation energy transfer (EET) has been recently documented for tyrosine and tryptophan containing peptides, but not for phenylalanine. Herein, we report sequential energy transfer from phenylalanine to tyrosine to a disulfide, resulting in homolytic cleavage of a sulfur-sulfur bond. Interestingly, energy transfer from phenylalanine is only observed in the presence of tyrosine and only occurs within certain distance constraints. Isolated, electronically excited phenylalanine is known to have an extremely long lifetime in the gas phase, potentially suggesting quicker relaxation occurs via energy transfer to tyrosine. Alternatively, the direct overlap of states between phenylalanine and disulfide bonds is predicted to be poor, in which case tyrosine would serve to bridge the gap. In either case, the distance constraints imposed by this two-step EET are shown to be useful for evaluation and determination of gaseous biomolecular structure.

Characterizing gaseous peptide structure with action-EET and simulated annealing.

Evaluation of biomolecular structure in the gas phase is challenging, but worthwhile due to advantages in sensitivity and speed relative to traditional condensed phase approaches. Herein, we demonstrate that a recently developed method utilizing energy transfer to establish distance constraints can be combined with molecular dynamics calculations to rapidly and accurately reveal gaseous peptide structures. Three peptides in various charge states are examined. The influence of increasing charge state on peptide structure is easily observed. The presence of multiple conformations can be detected. Furthermore, the method is demonstrated to aid the assignment of charge, which is frequently nontrivial for peptides containing numerous acidic and basic residues that could adopt a variety of conformers of equal charge state. Comparison with ion mobility reveals that many low energy structures that are distinguishable by distance constraints would not be resolvable by collision cross section. Action-EET is demonstrated to be a powerful new tool for structure elucidation.

Bond-specific dissociation following excitation energy transfer for distance constraint determination in the gas phase.

Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase.