diaTracer enables spectrum-centric analysis of diaPASEF proteomics data

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 2, 2025

Abstract Data-independent acquisition has become a widely used strategy for peptide and protein quantification in liquid chromatography-tandem mass spectrometry-based proteomics studies. The integration of ion mobility separation into data-independent analysis, such as the diaPASEF technology available on Bruker’s timsTOF platform, further improves accuracy depth achievable using acquisition. We introduce diaTracer, spectrum-centric computational tool optimized data. diaTracer performs three-dimensional (mass to charge ratio, retention time, mobility) peak tracing feature detection generate precursor-resolved “pseudo-tandem spectra”, facilitating direct (“spectral-library free”) identification from is stand-alone fully integrated FragPipe platform. demonstrate performance data triple-negative breast cancer, cerebrospinal fluid, plasma samples, phosphoproteomics human leukocyte antigens immunopeptidomics experiments, low-input spatial study. also show that enables unrestricted post-translational modifications open/mass-offset searches.

Language: Английский

---

TIMS²Rescore: A Data Dependent Acquisition-Parallel Accumulation and Serial Fragmentation-Optimized Data-Driven Rescoring Pipeline Based on MS²Rescore

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 6, 2025

The high throughput analysis of proteins with mass spectrometry (MS) is highly valuable for understanding human biology, discovering disease biomarkers, identifying therapeutic targets, and exploring pathogen interactions. To achieve these goals, specialized proteomics subfields, including plasma proteomics, immunopeptidomics, metaproteomics, must tackle specific analytical challenges, such as an increased identification ambiguity compared to routine experiments. Technical advancements in MS instrumentation can mitigate issues by acquiring more discerning information at higher sensitivity levels. This exemplified the incorporation ion mobility parallel accumulation serial fragmentation (PASEF) technologies timsTOF instruments. In addition, AI-based bioinformatics solutions help overcome integrating data into workflow. Here, we introduce TIMS2Rescore, a data-driven rescoring workflow optimized DDA-PASEF from platform includes new MS2PIP spectrum prediction models IM2Deep, deep learning-based peptide predictor. Furthermore, fully streamline throughput, TIMS2Rescore directly accepts Bruker raw search results ProteoScape many other engines, Sage PEAKS. We showcase performance on immunopeptidomics (HLA class I II), metaproteomics sets. open-source freely available https://github.com/compomics/tims2rescore.

Language: Английский

---

Proteomics—The State of the Field: The Definition and Analysis of Proteomes Should Be Based in Reality, Not Convenience

Proteomes, Journal Year: 2024, Volume and Issue: 12(2), P. 14 - 14

Published: April 19, 2024

With growing recognition and acknowledgement of the genuine complexity proteomes, we are finally entering post-proteogenomic era. Routine assessment proteomes as inferred correlates gene sequences (i.e., canonical ‘proteins’) cannot provide necessary critical analysis systems-level biology that is needed to understand underlying molecular mechanisms pathways or identify most selective biomarkers therapeutic targets. These requirements demand at level proteoforms/protein species, actual active players. Currently, only highly refined integrated integrative top-down proteomics (iTDP) enables analytical depth routine, comprehensive, quantitative proteome assessments across widest range proteoforms inherent native systems. Here a broad perspective field, taking in historical current realities, establish more balanced understanding where field has come from (in particular during ten years since Proteomes was launched), issues, how things likely need proceed if deep analyses succeed. We base this our firm belief best proteomic reflect, closely possible, sample moment sampling. also seek emphasise future approaches based on exploitation complementarity currently successful approaches. This emphasises continuously evaluate further optimize established approaches, avoid complacency thinking expectations but promote careful development introduction new notably those address proteoforms. Above all, wish rigorous focus quality must override largely values speed; latter would certainly be nice, could thus effectively, routinely, quantitatively assessed. Alas, composed proteoforms, not species can amplified directly mirror genes ‘canonical’). The problem hard, accept it such, payoff playing longer game promise far biomarkers, drug targets, truly personalised even individualised medicine.

Language: Английский

---

Secretome analysis using Affinity Proteomics and Immunoassays: a focus on Tumor Biology

Molecular & Cellular Proteomics, Journal Year: 2024, Volume and Issue: 23(9), P. 100830 - 100830

Published: Aug. 14, 2024

The study of the cellular secretome using proteomic techniques continues to capture attention research community across a broad range topics in biomedical research. Due their untargeted nature, independence from model system used, historically superior depth analysis, as well comparative affordability, mass spectrometry-based approaches traditionally dominate such analyses. More recently, however, affinity-based assays have massively gained analytical depth, which together with high sensitivity, dynamic coverage throughput capabilities render them exquisitely suited analysis. In this review, we revisit challenges implied by secretomics and provide an overview platforms currently available for analyses, tumor example basic translational

Language: Английский

---

Deep Profiling of Plasma Proteoforms with Engineered Nanoparticles for Top-Down Proteomics

Journal of Proteome Research, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 23, 2024

The dynamic range challenge for the detection of proteins and their proteoforms in human plasma has been well documented. Here, we use nanoparticle protein corona approach to enrich low-abundance selectively reproducibly from top-down proteomics quantify differential enrichment 2841 detected 114 proteins. Furthermore, allowed between ∼1 μg/mL ∼10 pg/mL absolute abundance, providing up a 10

Language: Английский

---

Ultra-deep proteomics by Thin-diaPASEF with a 60-cm long column system

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: April 26, 2024

Abstract Recent advances have allowed for the detection of 10,000 proteins from cultured human cell samples, such as HeLa and HEK293T cells in a single-shot proteome analysis. However, deeper analysis remains challenging. Therefore, this study, we aimed to perform deep proteomic using timsTOF HT. To achieve proteomics, developed Thin-diaPASEF, parallel accumulation-serial fragmentation (PASEF) technology featuring thinly divided m/z axis only regions high ion density. Furthermore, 60-cm long C18 column with particle size 1.7 µm, an average 11,698, 11,615 11,019 unique were successfully detected 500 ng HEK293T, K562 digests, respectively, 100 min active gradient. The same system was used analyze Lycopersicon esculentum lectin (LEL) enriched plasma serum. LEL method identified 8,613 4,078 proteins, serum, respectively. Our ultra-deep will be helpful in-depth comparison medical biological research because it enables highly coverage single-shot.

Language: Английский

---

Frontiers in plasma proteome profiling platforms: innovations and applications

Clinical Proteomics, Journal Year: 2024, Volume and Issue: 21(1)

Published: June 21, 2024

Abstract Biomarkers play a crucial role in advancing precision medicine by enabling more targeted and individualized approaches to diagnosis treatment. Various biofluids, including serum, plasma, cerebrospinal fluid (CSF), saliva, tears, pancreatic cyst fluids, urine, have been identified as rich sources of potential for the early detection disease biomarkers conditions such cancer, cardiovascular diseases, neurodegenerative disorders. The analysis plasma serum proteomics research encounters challenges due their high complexity wide dynamic range protein abundance. These factors impede sensitivity, coverage, when employing mass spectrometry, widely utilized technology discovery proteomics. Conventional Neat Plasma workflow are inefficient accurately quantifying low-abundant proteins, those associated with tissue leakage, immune response molecules, interleukins, cytokines, interferons. Moreover, manual nature poses significant hurdle conducting large cohort studies. In this study, our focus is on comparing workflows proteomic profiling establish methodology that not only sensitive reproducible but also applicable studies biomarker discovery. Our investigation revealed Proteograph XT outperforms other terms proteome depth, quantitative accuracy, reproducibility while offering complete automation sample preparation. Notably, demonstrates versatility applying it various types biofluids. Additionally, proteins quantified cover secretory peripheral blood, pathway enriched relevant components necrosis factors, chemokines, B T cell receptors provides valuable insights. often challenging quantify complex biological samples, hold markers thereby contributing improvement patient care quality.

Language: Английский

---

A Technical Evaluation of Plasma Proteomics Technologies

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 13, 2025

Plasma proteomic technologies are rapidly evolving and of critical importance to the field biomedical research. Here we report a technical evaluation six notable plasma - unenriched (Neat), Acid depletion, PreOmics ENRICHplus, Mag-Net, Seer Proteograph XT, Olink Explore HT. The methods were compared on depth, reproducibility, linearity, tolerance lipid interference, limit detection/quantification. In total performed 618 LC-MS/MS experiments 93 HT assays. method achieved greatest depth (∼4,500), while detected ∼2,600 proteins. Other MS-based ranged from ∼500-2,200. Neat, Seer, had strong showed higher variability. All MS good linearity with spiked-in C-Reactive Protein (CRP); CRP was surprisingly not in assay. None affected by interference. more than double number quantifiable proteins (4,800) for both LOD LOQ next best method. comparable Neat Mag-Net LOD, but worse LOQ. Finally, tested applicability these detecting differences between healthy cancer groups non-small cell lung (NSCLC) cohort.

Language: Английский

---

Automated High-Throughput Affinity Capture-Mass Spectrometry Platform with Data-Independent Acquisition

Journal of Proteome Research, Journal Year: 2025, Volume and Issue: unknown

Published: Jan. 27, 2025

Affinity capture (AC) combined with mass spectrometry (MS)-based proteomics is highly utilized throughout the drug discovery pipeline to determine small-molecule target selectivity and engagement. However, tedious sample preparation steps time-consuming MS acquisition process have limited its use in a high-throughput format. Here, we report an automated workflow employing biotinylated probes streptavidin magnetic beads for enrichment 96-well plate format, ending direct sampling from EvoSep Solid Phase Extraction tips liquid chromatography (LC)-tandem (MS/MS) analysis. The streamlined significantly reduced both overall hands-on time needed preparation. Additionally, developed data-independent acquisition-mass (DIA-MS) method establish efficient label-free quantitative chemical proteomic kinome profiling workflow. DIA-MS yielded coverage of ∼380 kinases, > 60% increase compared using data-dependent (DDA)-MS method, provided reproducible kinase inhibitor dasatinib. We further showcased applicability this AC-MS assessing two clinical-stage CDK9 inhibitors against ∼250 probe-enriched kinases. Our study here provides roadmap engagement native cell or tissue lysates AC-MS.

Language: Английский

---

Molecularly-guided spatial proteomics captures single-cell identity and heterogeneity of the nervous system

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 11, 2025

Single-cell proteomics is an emerging field with significant potential to characterize heterogeneity within biological tissues. It offers complementary insights single-cell transcriptomics by revealing unbiased proteomic changes downstream of the transcriptome. Recent advancements have focused on enhancing proteome coverage and depth, mostly in cultured cell lines, a few recent studies explored analyzing tissue micro-samples but were limited homogenous peripheral In this current work, we utilize power spatial single cell-proteomics through immunostaining-guided laser capture microdissection (LCM) coupled LC-MS investigate heterogenous central nervous system. We used method compare neuronal populations from cortex substantia nigra, two brain regions associated motor cognitive function various neurological disorders. Moreover, technique understand neuroimmune stab wound injury. Finally, focus our application system, where myenteric plexus ganglion nerve bundle. This study demonstrates utility neuroscience research toward understanding fundamental biology molecular drivers conditions.

Language: Английский

---