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23rd Human Proteome Organization World Congress

Prof

Repurposed 3D printer allows economical and programmable fraction collection for proteomics of nanogram scale samples

New Technology: Chromatography

IntroductionTwo-dimensional liquid chromatography (2DLC) is a powerful tool for the analysis of complex proteomes. However, it suffers from sample losses during transfer between separation steps, compromising sensitivity and quantitative accuracy in LC-MS analysis, restricting the use of 2DLC to studies where abundant material is readily available. For studies involving samples with limited availability, achieving adequate analytical depth while minimizing sample losses presents a significant challenge. To address this issue, in this work, we built and applied a low loss fractionation system comprised of a reversibly reconfigured Evosep One LC system for the first dimension and a repurposed 3D-printer as a fraction collector in a range of Proteomics workflows covering both data dependent and data independent approaches as well as phosphoproteomics experiment.MethodsPeptides from human cell lysates were analysed by 2D RP-RP. An Evosep One LC system was used for the first-dimension separation in combination with a modified 3D printer as a fraction collector. Fractionated and non-fractionated samples were analysed by low-pH LC-MS/MS on an Ultimate 3000 RSLCnano system coupled to an Orbitrap Ascend Tribrid mass spectrometer (Thermo Fisher Scientific) using Data Dependent (DDA), Data Independent (DIA) or Wide Window Acquisition (WWA) modes. The resulting MS raw data were analysed by FragPipe, MaxQuant, DIA-NN or Proteome Discoverer. Data analysis was performed using the tools available in Perseus and GraphpadPrism.ResultsThe 2D RP-RP system used in this work demonstrated superior proteome coverage over single-shot experiments, achieving high sensitivity at peptide level for low amount of input material. More importantly, increase in protein level intensity was observed in 2D RP-RP, which indicates the complexity reduction afforded by the system outweighs the sample losses endured. The application of DIA and WWA to fractionated samples allowed further improvements in peptide and protein IDs in comparison to DDA, demonstrating that the use of data independent acquisition methods also benefit from the complexity reduction caused by fractionation. The utility of the 2D system was further investigated in phosphoproteomics workflow, showing significant improvements in phosphosites and phosphoproteins over single-shot analysis.ConclusionsOur 2D workflow showed significant increases in peak capacity and identification rates over single-shot experiments in the analysis of human cell lysates, exhibiting superior proteome coverage using limited sample amounts. Moreover, substantial improvements in coverage were also observed when the workflow was applied to the analysis of subproteomes. We envision that the 2D system showed here will have broad applicability across various proteomics workflows and could be particularly useful for isobaric multiplexed experiments applied to small cell populations.

Poster presentation

Versatile and bidirectional use of microfluidic LC columns, optimized configuration for achieving significant increases in proteome coverage at low nanoLC flow rates

LC-MS-based proteomics relies on HRAM mass spectrometers for resolution, speed, and sensitivity, critical for accurate identification and quantification after LC separation. To match the increasing speed of MS instruments, nanoLC column development has focused on reducing dead volume and ensuring optimal elution, particularly at ultra-low flow rates. Pillar array technology, with its precise order and high permeability, demonstrates outstanding performance, although connectivity quality can significantly impact results. An optimal strategy is proposed to enhance connectivity and diminish post-column dead volumes, resulting in notable performance enhancements and enabling more thorough proteomic analysis. Moreover, its unique capabilities facilitate high-throughput, ultra-sensitive analysis by swiftly adapting to programmed flow rates, enabling 100 samples per day at elution rates as low as 100 nL/min.Utilizing a column heating device directly attached to the ESI source of the mass spectrometer enabled close integration of the nanoLC column, reducing the post-column volume by a factor of 3 to as little as 50 nL. Ensuring a low dead volume connection from the outlet fitting to a suitable nanoelectrospray emitter preserved the "on-chip" separation performance, resulting in significantly improved chromatography and deeper proteome coverage across all tested conditions.In our initial assessment, we explored the benefits for conventional bottom-up proteomics sample loads (50-500 ng of tryptic digest injected) using customized LC methods. These methods achieved sample throughput rates of 60 and 30 samples per day, respectively, with peptide elution at conventional nanoLC flow rates (250 nL/min), reaching instrument productivity levels of up to 85%. From triplicates at a throughput rate of 60 samples per day, we identified 5970 protein groups, with 94% quantified below a coefficient of variation of 20%. High-resolution LC separation and reproducible elution allowed for quantification of significantly more protein groups (7% below 20% CV and up to 30% below 10% CV) compared to conventional pulled-tip emitter nanoLC column types.Anticipating greater performance gains at lower flow rates, we conducted benchmarking with sample loads ranging from 50 pg to 10 ng. Methods were optimized for elution at 100 nL/min, achieving sample throughput rates of 96, 72, and 48 samples per day with instrument productivities of 70%, 77.5%, and 85%, respectively. Post-column volume reduction led to a performance increase of up to 25% (reducing average FWHM by 1.5s), increasing proteome coverage by up to 22% on the protein group level and identifying up to 2413 protein groups from as little as 250 pg.

Improved proteome coverage and reproducibility in large cohort analyses using packed emitter columns

Proteomic analyses of large cohorts of human patient samples are used to understand the biological mechanisms of disease and to discover novel biomarkers for diagnosis and response to treatment. Deep proteome coverage and reproducibility of results is crucial for the success of these studies. We developed a novel packed emitter chromatography column, the IonOpticks Aurora Rapid 8x150 (8 cm length x 150 μm inner diameter, 1.7 μm C18), that improves the results from patient cohort analyses by ensuring high and consistent proteome coverage and reproducible quantitation. This study evaluates the efficiency of the column for high-throughput proteomic analysis of large patient plasma cohorts coupled with an Evosep One and a Thermo Fisher Orbitrap Astral mass spectrometer. As part of this study, patient plasma samples were analysed using a 100 samples per day method with approximately 700 samples run between quality control (QC) injections. To ensure consistent performance and reproducibility, the system underwent weekly QC injections of HeLa tryptic peptides using a 60 samples per day method. The system achieved robust protein identifications, with over 9,300 proteins and 100,000 unique peptides consistently identified per QC run across multiple columns. To assess the reproducibility of protein quantification across multiple QC runs and columns, protein intensities were used to calculate a coefficient of variation (CV) and Pearson correlation, revealing uniform performance with low CV values and high correlation between runs and between columns. This consistent performance, along with consistently narrow peak widths averaging 2.9 seconds FWHM, indicates good column performance and reproducibility, important for large patient cohort analyses. Furthermore, to ensure reliable data analysis, we evaluated retention time stability. This analysis demonstrated a high level of consistency, with a 2.52% median CV of retention times. Additionally, a four-month experiment on 16 selected peptides demonstrated stable retention times. Taken together, these results demonstrate that our novel packed emitter column combined with the Evosep One and Thermo Fisher Orbitrap Astral Mass Spectrometer enabled maximum protein and peptide identifications whilst also ensuring robust and reproducible protein quantification across a large patient plasma cohort analysis.

High-throughput plasma proteomics with cap-flow LC separation and dia-PASEF-MS/MS detection

Plasma represents a major source for clinical diagnosis and research which is obtained minimally invasive. Proteomics analysis of plasma is challenged by the high dynamic range of protein abundance as well as the variability between individual donations and donors. Major issues that hinder widespread application of mass spectrometry-based plasma proteome analysis is the low sample throughput of nanoLC-MS applications and lower sensitivity of HPLC separation at high flow rates. Capillary flow rate chromatography (1-10ul/min) provides a good option to combine high sensitivity and high sample throughput. The protein depth of cap-LC is similar to that observed with nanoLC applications using similar column dimensions and gradient length, however durations for sample loading and column equilibration are minimized due to the increased flow rate.Two sample preparations of plasma proteins were performed for 15 individual plasma donations, direct trypsination for a "neat" proteome composition and the ENRICH-iST kit for a deep plasma proteome. HPLC and MS settings were optimized at a flow rate of 2 µl/min to surpass 110 samples/day. MS acquisition parameters for dia-PASEF on the TimsTOF HT mass spectrometer were adapted to maintain reliable quantitation and to maximize the number of peptide and protein hits. For cap-LC separation, the separation gradient and the injection amount were programmed for highest sample throughput at highest level of data coverage as well as minimal sample carry-over and column loading and equilibration periods.A pooled sample was generated for LC method optimization. First, dia-PASEF parameters were optimized with nanoLC separation by probing the number of IM ramps, the position and extraction width of the windows within a 600 Th range for precursor isolation and limiting the ion mobility range to allow for optimal positioning of the windows or the precursor ion cloud. Using the optimized dia-PASEF cycle, injection amount and gradient length were tested for nanoLC and cap-LC flow rates. For nanoLC, we aimed at highest proteome coverage achievable to generate a reliable precursor library for subsequent fast gradient analysis. For cap-LC measurements, gradients for 75 and 140 samples/day were programmed and the reproducibility and throughput were confirmed. Results were searched in Spectronaut 19 against the spectral library generated by nanoLC analysis as well as in the directDIA+ node. For neat plasma, more than 400 protein groups with 140 SPD were quantified reliably. Enriched plasma samples covered more than 2000 protein groups in 7 min active gradient time.

Long-term stability and reproducibility of nano-, capillary- and micro-flow LC-MS separations

Introduction Nano-, capillary- and micro-flow LC hyphenation with MS detection is irreplaceable in a high-sensitivityanalysis of complex bio-samples. But concerns remain around long-term stability, day-to-day as well assystem-to-system results reproducibility. The transition of low-flow LC-MS applications from academic andR&amp;D to translational research laboratories demands to prove the suitability of LC and MS technologies forthe analysis of large sample cohorts. In addition, the consistency of results across multiple laboratories andoperators is essential for pooling datasets for joint statistical analysis. Here we describe the output ofcomprehensive evaluation of proteomics data collected over several months of continuous operation atdifferent laboratories and discuss the impact of hardware and consumables on the reproducibility of results. Methods Low-flow LC-MS and/or LC-UV data were collected in Germering (Germany), Bremen (Germany), and SanJose (USA) by different operators using identical LC and MS hardware and different batches of separationcolumns. The Vanquish Neo UHPLC system was used for peptides separation on 75 μm ID EASY-SprayPepMap Neo columns with 15, 50, and 75 cm length and 1 mm ID columns with 15 cm length. OrbitrapExploris 480 and Orbitrap Exploris 240 MS instruments were used to detect and fragment peptides anddata-dependent acquisition mode. Data were processed with and without resolving of chimeric spectra andreported with 1% FDR on peptide and protein level. Preliminary Data Long-term robustness. The continuous injections of bovine serum albumin protein digest under typical fornanoLC-MS bottom-up proteomics experimental conditions were conducted for at least 6 months in twolaboratories. The longevity results of column backpressure, retention time variation, and full-width half-max(FWHM) for selected peptide peaks showed the suitability of low-flow separations LC for the uninterruptedlong-term analysis. Novel Aspect A comprehensive evaluation of analytical variability in low-flow LC-MS analysis provides guidelines forachieving reproducible results across instruments and laboratories

A tandem capillary and micro-flow LC workflow for high-throughput quantitative proteomics at near 100% mass spectrometer utilization

Introduction Pushing the limits of low-flow LC for high-throughput quantitative workflows often comes at the cost of decreasing mass spectrometer utilization, data quality (e.g. proteome depth, column carryover and quantitative accuracy/precision), and method robustness. For fast methods, such trade-offs must be made to maximize acquisition time and minimize method overhead. Here, we present a novel tandem LC workflow for high-throughput quantitative proteomics that eliminates method overhead while improving on traditional column washing and equilibration cycles.Methods A Vanquish Neo UHPLC system was configured for the tandem direct injection workflow coupled to either an Orbitrap Exploris 480, TSQ Altis Plus Triple Quadrupole or Stellar mass spectrometer for ultra-fast DDA, DIA and PRM analysis. Human plasma and cell lysates were utilized to demonstrate the performance in bottom-up proteomics at flow rates of 1.5 - 100 µL/min with 150 µm – 1 mm ID columns connecting to only one emitter, offering minimal analytical variation.Preliminary results The tandem LC-MS workflow was tested using gradients down to 8 min (corresponding to 180 samples/day) to demonstrate throughput, run-to-run reproducibility, and close to 100% MS utilization for quantitative proteomics. Using the 150 µm x 15 cm column operated at 1.5 µL/min, we reproducibly identified &gt;1,200 protein groups in DDA at 180 samples/day throughput with high quantification accuracy between columns. While moving to the PRM analysis, the tandem direct injection workflow along with the Stellar mass spectrometer directly supports the expansion of large-scale targeted experiments covering &gt; 3500 peptides and &gt;60 samples per day from 300 ng human plasma digest with negligible carryover permitting high-confidence peptide verification. Overall, this new workflow is a promising approach to improving instrument productivity offering high sensitivity, high sample throughput, and maximum quantification confidence.(For Research Use/Purposes Only – Not for Diagnostic Procedures)

Achieving close-to-complete proteome coverage in single-shot deep dive DIA workflows

Throughout the history of LC-MS based proteomics, comprehensive identification and quantification of all proteoforms present in biological samples has been the ultimate ambition. Several approaches have been close to achieving this goal, with the deepest coverage commonly obtained by fractionation of digested proteins using a separation mechanism orthogonal to low-pH reversed phase separation (SCX, high-pH C18) and subsequent LC-MS analysis of isobaric labeled peptide samples. As recent innovations in LC-MS instrumentation have leveraged significant increases in analysis speed, resolution and sensitivity, we investigate the possibility to achieve similar coverage using single-shot label-free analyses with ultra-high peak capacity LC-MS. Hereby significantly reducing the labor-intensiveness, overall cost and required analytical skill needed to achieve close to fulll proteome coverage.Human proteome samples were analyzed using an Orbitrap mass spectrometer coupled to a Vanquish Neo UHPLC instrument. For spectral library generation, protein digest samples were fractionated into 8 concatenated fractions at pH 5.5. For single-shot analyses, lyophilized and unfractionated cell digests were resuspended in 40 µl 0.1 % FA to obtain a stock solution of 500 ng/µl. Reversed phase LC separation was performed on a 110 cm µPAC Neo column with gradient lengths up to 180 min. Both DDA and DIA acquisition strategies were used to collect data. LC-MS data were searched using Spectronaut 17 or a beta version of Thermo Scientific Proteome Discoverer 3.1.In single-shot deep dive proteomic experiments, highest possible peak capacities are key to reduce sample complexity as much as possible prior to MS/MS analysis. To increase separation performance for long gradient analyses, a 110 cm long microfluidic channel uniformly filled with 2.5 µm pillars was used, significantly increasing peak capacities to well above 1600 in single-shot analyses. Median peak widths as low as 4.6 and 7.2 s have been obtained for respective gradient lengths of 90 and 180 min, which is 30% better than reported up to now.Evaluating the effect of increased separation performance on proteome depth for single species tryptic digests,following systematic optimization of MS acquisition settings, demonstrated that significant increases in depth were achieved by operating in DIA mode, with up 20% deeper coverage as compared to the best performing DDA method we tested. Using a library free approach, close to 10.000 protein groups were consistently identified with 180 min gradient times. We will further optimize LC-MS methods towards increased throughput and evaluate the impact of high-field asymmetric waveform ion mobility spectrometry (FAIMS) on the proteome coverage that can be achieved within single-shot LC-MS analyses.

A novel selective comprehensive two-dimensional online nanoLC-CZE-MS platform for proteoform characterization

CE-MS has been used to separate proteoforms on intact protein levels, however, selectivity and sensitivity are often not sufficient for complex biological samples. We therefore developed a two-dimensional (2D) heart-cut nanoLC-CZE-MS platform and have shown that this allows the pre-separation of intact proteins from a complex matrix and a 280-fold increased sensitivity compared to a one-dimensional CZE-MS approach. Furthermore, with this 2D approach, a higher proteoform selectivity can be gained [1]. While the transfer of a peak from the first to the second dimension is efficient using our two-dimensional heart-cut approach, the characterization of multiple chromatographic peaks or partly separated proteoforms is time-consuming, requires high sample amounts, and might lead to incomplete proteoform characterization. Therefore, we present here a novel nanoLC-CZE-MS platform to perform selective comprehensive nanoLC-CZE-MS. There, the RPLC column is connected to a storage capillary by a 10-port valve to decouple the storage capillary from the first dimension while performing the second dimension analysis. This allows the storage of a fraction from the first dimension for subsequent 2D analysis. The volume of the stored fraction depends on the volume of the storage capillary and can be easily adjusted. The storage capillary is also connected to an 8-port nanoliter valve with four 20 nL internal loops. Hence, 20 nL fractions can be transferred from the storage capillary to the CZE as the second dimension. Here, we show the essential parameters of our selective comprehensive two-dimensional nanoLC-CZE-MS platform. In addition, we show initial results from the analysis of a cell lysate using our selective comprehensive two-dimensional online nanoLC-CZE-MS platform, demonstrating its highly selective and sensitive analysis of proteoforms.References[1] A. Stolz, C. Neusüß, Characterisation of a new online nanoLC-CZE-MS platform and application for the glycosylation profiling of alpha-1-acid glycoprotein, Anal. Bioanal. Chem. 414 (2022) 1745–1757.

Enhancing sample throughput for deep dive proteomics with Tandem LC

Introduction Low-flow UHPLC coupled with mass spectrometry (MS) is the gold standard for deep quantitative analysis of complex proteomes. However, the unmatched sensitivity of low-flow LC-MS typically comes at the cost of low MS utilization and sample throughput. A high proportion of idle MS time results from long sample injection, loading, column washing, and equilibration cycles. Here, we present a novel, facile, and robust workflow for deep dive proteomics that eliminates idle MS time by employing a tandem LC.Methods The novel LC-MS configuration, comprising a Vanquish Neo UHPLC system configured for tandem direct injection, was coupled to either an Orbitrap Exploris 240/480 or an Orbitrap Astral mass spectrometer for deep proteome profiling using library-free DIA. Human cell lysates were analyzed to demonstrate the performance of untargeted bottom-up TMT and label-free proteomics on 75 µm (≤ 300 nL/min) and 150 µm ID (1.5 µL/min) columns with throughput up to 180 samples/day. To minimize post-column dispersion the Sonation double-barrel column oven was used for all experiments.Preliminary results The tandem LC-MS workflow was tested using gradients from 8 to 120 min (corresponding to 180 to 12 samples/day, respectively) to demonstrate high throughput, run-to-run reproducibility, and close to 100% MS utilization for discovery proteomics. For example, a 65-min gradient with a 75 µm x 75 cm (2 µm particles) column at 300 nL/min enabled the identification of &gt;11,000 protein groups per run in DIA from 200 ng HeLa digest with &gt; 91% MS utilization on the Orbitrap Astral MS. With more than 90% of protein groups overlapped among runs and &lt;5% overhead time, this configuration delivers excellent reproducibility for large-cohort deep-dive proteomics analysis.(For Research Use/Purposes Only – Not for Diagnostic Procedures)

New Technology: Chromatography

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