Biology subjects

Mueller-Reif, J. B.

Publications and source records attributed to Mueller-Reif, J. B..

9 recordsLinked to original sources

bioRxiv2026

FastaLake selects compact metaproteomic databases from large sequence reservoirs by de novo peptide lookup

Metaproteomics analyses require protein sequence databases, yet the microbial proteins present in complex communities are not known in advance. Searching reference catalogs containing hundreds of millions of candidate sequences reduces identification sensitivity, while generating sample-specific databases through metagenomic sequencing adds time and cost. We introduce FastaLake, a computational workflow that uses full-length de novo peptide predictions by AlphaNovo to retrieve complete proteins from broad sequence resources and selects compact databases for conventional searching. In a defined eight-member community, FastaLake approached reference-search peptide yields without knowing its members. Using public sequence catalogs, FastaLake identified about as many peptides as matched metagenomic databases in two stool cohorts and 20-35% more in intestinal fluid at the same nominal 1% peptide false discovery rate. The resulting proteomes reveal personal functional profiles that persist across intestinal regions despite differences in leading species between intestinal fluid and stool. DNA, RNA and protein profiles agreed on broad differences between patients but only weakly on individual functions. All 726 acquisition-specific databases were smaller than the human reference proteome. Compact databases allowed non-specific searches of all 347 CapScan acquisitions, revealing histidine enrichment at peptide C termini in intestinal fluid. FastaLake enables metaproteomics analysis without matched metagenomic sequencing by constructing search databases from sequence resources and de novo peptide sequencing.

bioinformatics↗

bioRxiv2026

Machine learning-guided discovery of a conserved plasmid proteomic signature enables MALDI-TOF MS detection of pOXA-48-carrying Enterobacterales

OXA-48 carbapenemases are among the most widespread and important resistance mechanisms in Enterobacterales. Yet detecting carbapenemases by conventional workflows necessitates additional testing, thus delaying optimization of therapy and implementation of infection control measures. Here, we present a machine learning approach that identifies the conserved pOXA-48 plasmid directly from routine MALDI-TOF spectra acquired for species identification. The model detects pOXA-48 carriers with an AUROC of 0.96-0.98 across two independent hospital cohorts and instrument platforms, indicating near-perfect discrimination. Using bottom-up proteomics, plasmid conjugation, and plasmid curing, we link the discriminative MALDI-TOF spectral features to proteins encoded on pOXA-48, with DUF1496 domain-containing protein producing the most discriminative spectral feature. Our approach reframes the resistance prediction task from inferring a resistance phenotype to detecting a conserved plasmid through its expressed proteomic signature and has the potential to enable rapid MALDI-TOF MS-based diagnostics for a wide range of plasmid-based resistance determinants.

microbiology↗

bioRxiv2026

In situ polymerized monolith tips for reproducible, format-flexible proteomic sample preparation applied to biofluids

Sample preparation increasingly sets the throughput and reproducibility of mass spectrometry (MS)-based proteomics. StageTips (stop-and-go extraction tips) and variants thereof have long been common implements to purify samples, and we recently extended the concept to solid-phase extraction capture (SPEC) tips, in which the entire digestion takes place in sub-microliter volumes. Here we replace the hand-packed bed with a strong anion-exchange (SAX) monolith photopolymerized directly inside the pipette tip from a defined recipe (pSPEC). A liquid-handling robot casts 384 tunable tips in minutes, at low cost and in any format, adding negligibly to the workflows variance. Across biofluids, pSPEC added [~]20% more identifications than in-solution plasma and reached 3,500 protein groups from a single injection of healthy urine and 4,800 from saliva at 100 samples per day, depths usually requiring depletion or fractionation. The same light-cast chemistry should extend to single cells, affinity capture, and population-scale studies.

biochemistry↗

bioRxiv2026

Preimplantation factor (PIF) is an endogenous inhibitor of potassium channel KV1.3 regulating neutrophil function during pregnancy

Pregnancy is a unique period regarding immune cell regulation. Within the placenta, maternal immune cells play a central role in immune surveillance and tissue remodeling. However, regulatory mechanisms of systemic immunity during pregnancy are less clear. Here, we show that neutrophil function is altered in pregnant mice (E13.5), indicated by increased slow rolling velocity and reduced adhesion. Mechanistically, PreImplantation factor (PIF), a 15 amino acid peptide which is produced by human and murine trophoblast cells of the placenta, is continuously secreted into the maternal circulation and plays a key role in modulating neutrophil function via blocking the voltage-gated potassium channel KV1.3. This resulted in impaired intracellular Ca2+ signaling and subsequently disturbance of neutrophil post-arrest modifications and a higher susceptibility to physiological shear forces in vivo and in vitro. Furthermore, PIF-mediated KV1.3 blockade impaired E-selectin-mediated release of S100A8/A9 and phagocytosis. Taken together, we have identified PIF as an important modulator of neutrophil function during pregnancy suggesting a critical role in regulating innate immune responses throughout gestation.

immunology↗

bioRxiv2025

Exploring chromatographic dimensions for state-of-the-art proteomics applications

The evolution of mass spectrometry (MS)-based proteomics has been driven by continuous technological advances in sample preparation, instrumentation, and data acquisition. While chromatographic separation has historically been considered a critical bottleneck in achieving comprehensive proteome coverage, recent developments in ultra-fast data acquisition fundamentally challenge this paradigm. We investigated whether the traditional paradigm that chromatographic performance directly correlates with proteome depth still holds true. Spanning a matrix of experiments with five distinct stationary phases, including C18 chemistries, C8, and Phenyl-Hexyl, across eight column lengths (40-140 mm), we evaluate protein identification performance using data-independent acquisition (DIA) on the Orbitrap Astral mass spectrometer. Despite substantial chromatographic differences, we observed remarkably convergent proteome coverage metrics. All C18 and C8 phases consistently achieved over 150,000 precursor- and approximately 9,000 protein group identifications, regardless of column length variations. While distinct selectivity fingerprints persisted across chemistries, these chromatographic differences did not translate into meaningful variations in proteome coverage under high-speed acquisition conditions at 200 Hz. We conclude that the analytical bottleneck has fundamentally shifted from chromatographic resolution to mass spectrometric sampling efficiency, where comprehensive peptide identification is now gained through advanced spectral deconvolution rather than physical separation alone. This paradigmatic shift is reflected in modern proteomics by method development priorities being directed beyond traditional separation optimization, with greater emphasis placed on operational robustness, analytical throughput, and reproducibility.

biochemistry↗

bioRxiv2025

Pre-Analytical Drivers of Bias in Bead-Enriched Plasma Proteomics

Bead-based enrichment has emerged as a promising strategy to improve depth in plasma proteomics by overcoming the dynamic range barrier. However, its robustness against pre-analytical variation has not been sufficiently characterized. Here, we systematically evaluate five plasma proteomics workflows--including three bead-based methods and two conventional protocols--using controlled spike-ins of low-abundance proteins and defined cellular contaminants. We find that bead-based approaches enhance detection of low-abundance proteins but can be highly susceptible to systematic bias from platelet and PBMC contamination, even at low levels. This can easily inflate results by thousands of proteins, likely accounting for some of the very high literature-reported numbers. In contrast, a perchloric acid-based workflow shows notable resistance to erythrocyte and platelet-derived contamination. We further investigate how centrifugation conditions, anticoagulant choice, and buffer-bead combinations modulate contamination profiles and demonstrate that bias can partially be mitigated by optimized sample handling. In total, we identify more than 13,000 different protein groups from various conditions, including cellular components from the circulating proteome. Our results provide a quantitative framework for assessing workflow performance under variable sample quality and offer guidance for both biomarker discovery and quality control in clinical proteomics studies.

biochemistry↗

bioRxiv2025

A simplified perchloric acid workflow with neutralization (PCA-N) for democratizing deep plasma proteomics at population scale

Large scale plasma proteomics studies offer tremendous potential for biomarker discovery but face significant challenges in balancing analytical depth, throughput and cost-effectiveness. We present an optimized perchloric acid-based workflow with neutralization - PCA-N - that addresses these limitations. By introducing a neutralization step following protein precipitation, PCA-N enables direct enzymatic digestion without additional purification steps, reducing sample volume requirements to only 5 {micro}L of plasma while maintaining deep plasma proteome coverage. The streamlined protocol allows preparation of over 10,000 samples per day using 384-well formats at costs comparable to undepleted plasma analysis (NEAT). Rigorous validation according to the recently introduced CLSI C64 guideline demonstrated that despite somewhat higher technical variability compared to NEAT, PCA-N maintained excellent biological resolution and reproducibility. We confirmed the workflows exceptional stability through analysis of 1,500 quality control samples systematically interspersed among 36,000 plasma samples measured continuously over 311 days. Technical performance remained consistent across multiple instruments, sample preparation batches and nearly a year of measurements. Compared to NEAT plasma proteomics, PCA-N doubled the proteomic depth while maintaining comparable reagent costs and throughput. The minimal sample reequipments, operational simplicity while using only common laboratory chemical and exceptional scalability positions PCA-N as an attractive approach for population-level plasma proteomics, democratizing access to deep plasma proteome analysis. HighlightsO_LIPerchloric acid with neutralization (PCA-N) allows drastic upscaling of plasma proteomics C_LIO_LIMinimal sample input (5 {micro}L) in 384-well format enables thousands of samples processed daily C_LIO_LIPCA-N democratizes deep plasma proteomics using only common chemicals C_LIO_LIValidation per CLSI C64 demonstrates excellent repeatability and reproducibility C_LIO_LIPCA-N showed exceptional stability across 311 days of continuous MS acquisition C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=106 HEIGHT=200 SRC="FIGDIR/small/645089v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@11eb315org.highwire.dtl.DTLVardef@7f4f41org.highwire.dtl.DTLVardef@2c41feorg.highwire.dtl.DTLVardef@1a09925_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

A novel hybrid high speed mass spectrometer allows rapid translation from biomarker candidates to targeted clinical tests using 15N labeled proteins

Recent developments in affinity binder or mass spectrometry (MS)-based plasma proteomics are now producing panels of potential biomarker candidates for diagnosis or prognosis. However, clinical validation and implementation of these biomarkers remain limited by the reliance on dated triple quadrupole MS technology. Here, we evaluate a novel hybrid high-speed mass spectrometer, Stellar MS, which integrates the robustness of triple quadrupoles with the enhanced capabilities of an advanced linear ion trap analyzer. This instrument allows for extremely rapid and sensitive parallel reaction monitoring (PRM) and MS3 targeting. The Stellar MS allowed targeting thousands of peptides originally measured on Orbitrap Astral MS, achieving high reproducibility and low coefficients of variation (CV) as well as sensitivity and specificity sufficient for more than the top 1000 plasma proteins. Furthermore, we developed targeted assays for alcohol-related liver disease (ALD) biomarkers, showcasing the potential of Stellar MS in clinical applications. Absolute quantification is typically a requirement for clinical assays and we explore the use of 15 N labeled protein standards in a rapid, streamlined and generic manner. Our results indicate that Stellar MS can bridge the gap between proteomics discovery and routine clinical testing, enhancing the diagnostic and prognostic utility of protein biomarkers.

biochemistry↗

bioRxiv2021

A new parallel high-pressure packing system enables rapid multiplexed production of capillary columns

Reversed-phase high performance liquid chromatography (HPLC) is the most commonly applied peptide separation technique in mass spectrometry (MS)-based proteomics. Particle-packed capillary columns are predominantly used in nano-flow HPLC systems. Despite being the broadly applied standard for many years capillary columns are still expensive and suffer from short lifetimes, particularly in combination with ultra-high-pressure chromatography systems. For this reason, and to achieve maximum performance, many laboratories produce their own in-house packed columns. This typically requires a considerable amount of time and trained personnel. Here, we present a new packing system for capillary columns enabling rapid, multiplexed column production with pressures reaching up to 3000 bar. Requiring only a conventional gas pressure supply and methanol as driving fluid, our system replaces the traditional setup of helium pressured packing bombs. By using 10x multiplexing, we have reduced the production time to just under 2 minutes for several 50 cm columns with 1.9 {micro}m particle size, speeding up the process of column production 40 to 800 times. We compare capillary columns with various inner diameters (ID) and length packed under different pressure conditions with our newly designed, broadly accessible high-pressure packing station. One sentence summaryA newly constructed parallel high-pressure packing system enables the rapid multiplexed production of capillary columns. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/433033v2_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@4d224forg.highwire.dtl.DTLVardef@79dd3org.highwire.dtl.DTLVardef@aa047org.highwire.dtl.DTLVardef@1a0b837_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗