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23rd Human Proteome Organization World Congress

Maximizing LC-MS sensitivity and throughput in single-cell proteomics

New Technology: Chromatography

Introduction Proteomics has recently shifted towards profiling smaller sample quantities, including single-cell analysis. Although state-of-the-art liquid chromatography-mass spectrometry (LC-MS) technology has demonstrated unprecedented performance in investigating proteome changes, it lacks sensitivity for low-abundance peptides in single-cell proteomics (SCP) applications. Naturally, LC-MS sensitivity can be enhanced by utilizing ultralow LC flow rates (&lt;20 nL/min) with small inner diameter columns (20-30 µm) and emitters, but this comes at the cost of decreased sample throughput and restricting the possibility to analyze large sample cohorts. Therefore, a balance between sensitivity and sample throughput is the prerequisite for large-scale single-cell sample profiling. To address this dilemma, we successfully developed a low-nanoflow UHPLC separation setup with a wide-window data-independent acquisition (WW-DIA) strategy to achieve deep proteome profiling in a high-throughput fashion.Methods We established a novel low nano flow LC-MS configuration running gradients at 100 nL/min on a Vanquish Neo UHPLC system with an Acclaim PepMap 100 C18 50 µm ID x 15 cm column (2 um dp) in direct injection and trap-and-elute workflows. Electrospray ionization was carried out via a 10 µm ID glass emitter into a FAIMS Pro interface operated at a single compensation voltage to reduce background ion interference, followed by data acquisition in an Orbitrap Exploris 480 or an Orbitrap Astral mass spectrometer.Preliminary results Seven chromatography methods were developed to permit fast sample loading, efficient column washing, and equilibration while maintaining a stable 100 nL/min flow during the gradient. This permits the analysis of up to 120 samples/day with a 8-min peptide elution window (i.e., 67% MS utilization), whilst also offering extremely high levels of sensitivity for routine SCP analysis. Using this optimized front-end configuration, we identified ~2,200 protein groups from 250 pg of standard protein digest using the WW-DIA strategy in a library-free search without booster channels. A proof of principle study indicated that &gt;1,700 protein groups were identified and quantified from single-cell samples in a label-free experiment with negligible carryover at 100 cells/day (CPD) throughput. Furthermore, employing the Orbitrap Astral mass spectrometer significantly improved performance by doubling the protein identifications and quantification.In conclusion, we established a high-performance LC-MS configuration for SCP by balancing the sensitivity and throughput, thus enabling the investigation of cellular heterogeneity in clinically and biologically relevant cellular populations.

Oral presentation

Combining Proteome Integral Solubility Alteration (PISA) assay and one-tip workflow for a streamlined and high-throughput drug target deconvolution

New Technology: Sample Preparation

The Proteome Integral Solubility Alteration (PISA) assay is a well-established mass spectrometry (MS)-based proteomics methodology. It identifies changes in protein-ligand complexes based on their solubility variation under different treatment conditions based on thermal probing at several temperatures and is applicable to living cells and cell lysates. PISA usually necessitates relatively high sample input, but biomedically-relevant material available for analysis is often limited. This is the case for patient derived samples or challenging and costly cell cultures, and thus reducing sample requirements would increase the widespread applicability of PISA. Utilizing the One-Tip workflow, a novel sample preparation technique based on the Evotip, streamlines analysis of low sample input due to direct protein digestion on top of the C18-disk in the tip, followed by desalting, concentration and injection into the Evosep One LC system.The addition of 0.2% n-Dodecyl-Beta-Maltoside (DDM) during the freeze-thaw cell lysis after the PISA assay allows for a swift transition to the One-Tip method. Since DDM is a MS compatible detergent, there is no need to perform a buffer exchange of the protein sample prior to enzymatic Lys-C/trypsin digestion, thus streamlining the method by decreasing sample preparation time as well as minimizing variability thanks to fewer pipetting steps. To test the sensitivity of the integration of PISA with the One-Tip workflow, we assayed the cellular targets of staurosporine, a broadband kinase inhibitor, in different quantities of HeLa cells. The PISA samples were analyzed on the Orbitrap Astral mass spectrometer using narrow-window DIA. As a readout, we investigated the correlation between cell number input and subsequent protein IDs and identified kinase targets. This showed that One-Tip-PISA can be performed on very low cell inputs, as low as 2500 HeLa cells for each temperature point sampled. This yielded reproducible results showing a strong correlation with known staurosporine targets, surpassing the protein depth and sample reproducibility of comparable methods like the Protein Aggregation Capture (PAC) protocol, when using a limited sample amount. Furthermore, the One-Tip-PISA method enabled us to prepare and analyze a full 96-well plate of HeLa cells treated with 11 different kinase inhibitors at 2 different concentrations within a single day. The resulting injection-ready Evotips can be analyzed within a subsequent day by fast LC-gradients. The One-Tip-PISA method is promising for higher throughput and lower sample input compared to the existing PISA analysis, with potential applications in a wide range of research areas.

Professor

Enhanced secretomics workflow reveals organ-dependent secreted protein landscape across multiple murine tissues

Exercise exerts benefits across multiple organ systems and is associated with prevention and treatment of many diseases, including cardiovascular disease, pre-eclampsia, and type 2 diabetes mellitus, among others. Despite this, the identity, origin, and mechanism(s) of action of molecules facilitating these beneficial outcomes are relatively unexplored. The interrogation of the exercise- and organ-dependent secretome is essential to understanding the origin, destination, and therapeutic potential of exerkine-mediated inter-organ signaling. However, comprehensive organ-dependent secretomics faces several analytical challenges, including large dynamic range, blood contamination, and risk of cell burst during preparation. Further, unconventional protein secretion pathways make bioinformatic prediction of all secreted proteins challenging, resulting in poor proteome annotation and requiring robust validation of secreted candidates.We have developed a robust workflow for the interrogation of organ-dependent secretomes from murine tissue. Tissues are dissected within minutes of animal sacrifice and are rapidly rinsed with PBS buffer to remove residual blood. We implement a low-speed filtered centrifugation to remove the extracellular fluid. Fluid is centrifuged to remove cells followed by albumin/IGG depletion, and then further separated via ultra-centrifugation or magnetic beads to distinguish between extracellular vesicles and released proteins. All samples are analyzed with LC-MS/MS and quantified via TMT.We applied this protocol to iWAT, skeletal muscle, lung, and placenta. Both the lung and placenta produce the highest volume of EF of 10 tested murine tissues; both fluids had protein concentrations of &gt;50 ug/uL, producing &gt;700 ug of protein per sample, on average. While the lung is amenable to our secretomics method without modification, the placenta required a slightly modified procedure with extra perfusion. Similarly, the iWAT tissue required a brief fixation in paraformaldehyde. Western blots show that these fluids are distinct from organ lysates and lack markers that would indicate cell breakage and/or other contaminant infiltration.LC-MS/MS analysis revealed &gt;3000 proteins in the extracellular fluid as well as the extracellular vesicles, with unique proteins present in each fraction that reinforce the need for extracellular fluid fractionation. Comparing sedentary and exercised mice revealed lung specific cytokines present only in the lung EF, including the CXL15, an inflammation-induced neutrophil trafficking protein. Additionally, MCK-PGC1alpha transgenic mice show distinctions in secreted proteomes among their muscle, lungs, and placenta, emphasizing the advantage of our methods for the characterization of physiology-dependent signaling. Overall, this optimized secretome isolation procedure has the ability to reveal dynamic changes in organ-dependent signaling that can further enhance therapeutic discovery efforts.

A complete and automated end-to-end sample preparation strategy for high-throughput and standardized proteomics with high sensitivity

Proteomics is going through a significant paradigm shift and is rapidly evolving towards higher throughput and robust easier-to-use technologies. The use of short LC gradients combined with fast and sensitive mass spectrometers has been a key aspect in this development. The Evosep One is designed for high-throughput and high-volume applications, where standardized methods with increasing robustness enable routine analysis of hundreds of samples per instrument per day. This has moved the bottleneck towards sample preparation, which requires standardized and fully automated end-to-end workflows.We have developed an automated end-to-end workflow for proteomics sample preparation, designed for fast and robust protein digestion capable of processing up to 500 samples on one AssayMap Bravo in a single workday. The workflow results in ready-to-use samples for LC-MS as tryptic peptides are loaded directly onto Evotips in a complete hands-free manner, starting from protein lysate. It is optimized for speed and is completed within just 90 minutes. To illustrate the robustness and reproducibility, 500 samples were prepared in one day and analyzed with the corresponding high-throughput 500 samples per day method in a randomized order.At this level of throughput, the cost per sample is an important consideration. Therefore, we scaled down the use of consumables and designed the workflow to digest only what is needed for one LC-MS injection to minimize enzyme usage. To examine the sensitivity of our sample preparation workflow, we scaled down to even lower starting input materials and observed significant sensitivity down to just 1 ng protein starting amount.

Novel data-independent acquisition and targeted quantitation on Orbitrap Tribrid instruments, unleashing the power of Hybrid-DIA technology

New Technology: MS-based Proteomics

For any proteomic analysis using mass spectrometry, the desired outcome defines the methodology of choice. However, each methodology has tradeoffs, and relying on a single approach may be insufficient to the task. For example, rapid chromatography increases sample throughput, but limits the sensitivity and quantitative capabilities of the sample analysis. Tandem mass tagging of peptides multiplexes sample analysis, but requires high resolution and data-dependent methods. Targeted methods focus on a small set of peptides, leaving much of the sample unexplored. To overcome this challenge, sample analysis requires serially applied methodologies, a costly redundancy if sample or instrument availability is limited.Hybrid-DIA MS technology offers a novel solution by intelligently integrating targeted peptide analysis and discovery-based data independent acquisition (DIA), simultaneously maximizing instrument utilization and increasing sample throughput. Hybrid-DIA MS operates through the Thermo Fisher Scientific Moonshot application, so far only available to the Exploris 480 Orbitrap mass spectrometer via the Instrument Application Programming Interface (IAPI).We present Moonshot Pro, a redevelopment of the Moonshot application for the Tribrid series of Orbitrap mass spectrometers, with greatly improved functionality, visual output, and performance of Hybrid-DIA MS technology. The application analyzes DIA mass spectra in real-time to detect peptide transitions of interest. These precursor peptides are then targeted for quantitation on the fly. Targeted scans are fully customizable for resolution, injection time, and ion accumulation to maximize sensitivity.Moonshot Pro combines various targeted methodologies with DIA, leveraging a transition mass inclusion list for peptides of interest. By scanning DIA windows for candidate peptides, Moonshot Pro enhances the sensitivity and specificity alongside survey scanning based on the high-resolution accurate precursor mass. Quantitative PRM scans only occur after observation of the correct transitions for a peptide, allowing DIA to remain largely uninhibited to maximize scan frequency and sample coverage. Moonshot Pro performs SureQuant analysis with addition of heavy synthetic peptides to trigger the targeting of endogenous peptide counterparts. Customizing targeted scan settings for sensitivity allows endogenous peptide quantification even when abundances are below detection in DIA. MSx mode simultaneously fragments synthetic and endogenous peptides, enhancing quantification. Additionally, samples labeled with TMTpro18 and synthetic peptides labeled with Super Heavy TMTpro provide a means to multiplex the analysis and quantitation. This dramatically increases sample throughput by quantifying target peptides from multiple samples in a single discovery-based DIA analysis, enhancing data completeness for targets of interest, with potential for use in proteogenomic and translational research studies.

Session
From sample preparation to analysis by mass spectrometry

Appointment

Chairs

Program

SessionFrom sample preparation to analysis by mass spectrometry

Appointment

Chairs

Program

Session
From sample preparation to analysis by mass spectrometry