Biology subjects

Lubeck, M.

Publications and source records attributed to Lubeck, M..

11 recordsLinked to original sources

bioRxiv2026

Protein concentrates from pilot-scale biorefining of clover grass: Interfacial properties and their effects on the physical and oxidative stability of fish oil-in-water emulsion

Clover grass blends are promising sources of nutritional and techno-functional proteins, but currently mainly utilized for animal feeding. The application as a physical and oxidative stabilizer in food emulsions remains underexplored. In this study, the stabilizing effects of clover grass proteins (CGPs), produced through a pilot-scale, two-stage membrane filtration process yielding a native GPC concentrate (DC), as well as enzymatic hydrolysate hereof (DCH), were compared with commercial plant proteins (soy and pea) and animal sources (sodium caseinate). Both DC and DCH produced emulsions (0.4% (w/w) protein and 5% fish oil) with smaller size droplets and larger electrostatic repulsion between droplets compared to the other proteins tested. Moreover, DC and DCH exhibited higher protection against the generation of both primary and secondary oxidation products. Furthermore, emulsions stabilized with CGPs were well-protected from off-flavor compounds. Mass spectrometry-based proteomics analysis revealed that DC included a high RuBisCO content (38%) and the membrane process successfully depleted pigment-binding proteins affiliated with grassy color and sensory attributes. Moreover, DC was enriched (compared to the initial green juice) in known antioxidant proteins, constituting 10% of the total protein. In the hydrolysate (DCH), 30% of the total MS1 peptide signal originated from peptides predicted as probable free radical scavengers. These findings demonstrate that refined, native CGP, as well as its hydrolysate, improved both physical and oxidative stability of emulsions compared to plant and animal-based reference proteins due to a high endogenous antioxidant properties of the protein.

bioengineering↗

bioRxiv2026

Techno-functional properties of clover grass protein and the effect of different process technologies

This study investigated the effects of different downstream processes for protein isolation on the bulk properties and composition of clover grass protein prototypes (CGPs). A clarified clover grass juice, obtained using membrane filtration (MF), underwent precipitation by acid (AP), heat (HP), or acid+heat (AHP), or underwent ultra- and diafiltration to produce a concentrate (DC) as well as subsequent tryptic hydrolysis of DC (DCH). HP had the highest protein content (p<0.05) and was whiter than other CGPs, although it showed lower aqueous solubility. In contrast, DC showed excellent solubility across a broad pH range. CGPs efficiently decreased oil-water interfacial tension (16-13 mN/m) and displayed viscoelastic and solid-like interfacial behavior. CGPs-stabilized emulsions displayed low physical stability with larger droplets despite high absolute {zeta}-potentials. CGPs were rich in RuBisCO (37-47%) but had varying levels of other proteins. Despite significant protein-level differences, overall protein composition of CGPs was comparable, highlighting that protein state governs bulk functionality more than subtle compositional changes. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/701969v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@f37756org.highwire.dtl.DTLVardef@1fb5beorg.highwire.dtl.DTLVardef@1d4efe2org.highwire.dtl.DTLVardef@d11ef8_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender.com HighlightsO_LIThe effect of different processes on functional properties of CGPs was explored. C_LIO_LIHeat treatment increased protein purity and whiteness at the expense of solubility. C_LIO_LICGPs efficiently reduced O/W interfacial tension but produced unstable emulsions. C_LIO_LICGPs were found rich in RuBisCO (34-47%) using quantitative proteomics. C_LIO_LIProtein state had larger influence on functionality than protein-level composition. C_LI

bioengineering↗

bioRxiv2025

Selective removal of green pigments and associated proteins from clover-grass protein concentrates: Molecular insight into a non-destructive, two-stage membrane-based biorefinery concept for high-quality food protein production

Green leaves are gaining traction as an emerging protein source and a sustainable alternative to animal-based protein as leaf proteins often possess good nutritional and functional properties. Current methods for producing protein isolates from leaves include partially or fully denaturing conditions, diminishing protein solubility and functionality. Here, we characterize the performance of a multi-stage, membrane-based green biorefinery concept capable of producing a native protein product from clover grasses devoid of attributes such as green color and grassy smell/taste. By sampling at each step along the process, we obtain insights on the fate of proteins and pigments over each unit operation. The process efficiently removes green pigments (>99.9%), when comparing the product stream with the initial feed stream, based on UV/Vis analysis. Using mass spectrometry-based proteomics, two complementary quantification strategies, and subsequent bioinformatic analysis, this can particularly be ascribed to a very selective retention (>99%) of unwanted membrane-associated and pigment-binding proteins in the first-stage filtration. In the second-stage filtration and subsequent diafiltration stage, residual unwanted proteins, fragments, and pigments are efficiently washed out while retaining the overall protein composition. The product maintains the high RuBisCO content of green juice and is furthermore enriched in proteins with known antioxidant properties while depleted in known food allergens. This work presents an in-depth understanding of protein-level selectivity in membrane-based green biorefinery and can help guide process optimization towards improved yields and quality. HighlightsO_LIStreams from a two-stage membrane-based green biorefinery were characterized C_LIO_LI>99.9% pigment-binding proteins were removed primarily in first stage filtration C_LIO_LIThe 2nd filtration and diafiltration removed residual pigment and retained protein C_LIO_LIThe full process efficiently removed >99.9% of chlorophylls and carotenoids C_LIO_LIThe soluble product is rich in RuBisCO and enriched in known antioxidant proteins C_LI

bioengineering↗

bioRxiv2023

Mitochondrial Apolipoprotein MIC26 is a metabolic rheostat regulating central cellular fuel pathways

Mitochondria play central roles in metabolism and metabolic disorders such as type 2 diabetes. MIC26, a MICOS complex subunit, was linked to diabetes and modulation of lipid metabolism. Yet, the functional role of MIC26 in regulating metabolism under hyperglycemia is not understood. We employed a multi-omics approach combined with functional assays using WT and MIC26 KO cells cultured in normoglycemia or hyperglycemia, mimicking altered nutrient availability. We show that MIC26 has an inhibitory role in glycolysis and cholesterol/lipid metabolism under normoglycemic conditions. Under hyperglycemia, this inhibitory role is reversed demonstrating that MIC26 is critical for metabolic adaptations. This is partially mediated by alterations of mitochondrial metabolite transporters. Furthermore, MIC26 deletion led to a major metabolic rewiring of glutamine utilization as well as oxidative phosphorylation. We propose that MIC26 acts as a metabolic rheostat, that modulates mitochondrial metabolite exchange via regulating mitochondrial cristae, allowing cells to cope with nutrient overload.

cell biology↗

bioRxiv2022

Synchro-PASEF allows precursor-specific fragment ion extraction and interference removal in data-independent acquisition

Data-independent acquisition (DIA) methods have become increasingly popular in mass spectrometry (MS)-based proteomics because they enable continuous acquisition of fragment spectra for all precursors simultaneously. However, these advantages come with the challenge of correctly reconstructing the precursor-fragment relationships in these highly convoluted spectra for reliable identification and quantification. Here we introduce a scan mode for the combination of trapped ion mobility spectrometry (TIMS) with parallel accumulation - serial fragmentation (PASEF) that seamlessly and continuously follows the natural shape of the ion cloud in ion mobility and peptide precursor mass dimensions. Termed synchro-PASEF, it increases the detected fragment ion current several-fold at sub-second cycle times. Consecutive quadrupole selection windows move synchronously through the mass and ion mobility range, defining precursor-quadrupole relationships. In this process, the quadrupole slices through the peptide precursors, which separates fragment ion signals of each precursor into adjacent synchro-PASEF scans. This precisely defines precursor - fragment relationships in ion mobility and mass dimensions and effectively deconvolutes the DIA fragment space. Importantly, the partitioned parts of the fragment ion transitions provide a further dimension of specificity via a lock and key mechanism. This is also advantageous for quantification, where signals from interfering precursors in the DIA selection window do not affect all partitions of the fragment ion, allowing to retain only the specific parts for quantification. Overall, we establish the defining features of synchro-PASEF and explore its potential for proteomic analyses.

biochemistry↗

bioRxiv2022

Oxonium Ion-Guided Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro

Spatial separation of ions in the gas-phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro series combines a trapped ion mobility device with a quadrupole, collision-cell and a time-of-flight analyser to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from non-modified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared to non-modified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and non-modified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. This new approach was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation, combined with the focussing of the mass spectrometer, allows for improved extraction of information from the samples, even at shorter LC gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known make-up, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

biochemistry↗

bioRxiv2020

Ultra-high sensitivity mass spectrometry quantifies single-cell proteome changes upon perturbation

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing1-3. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary4,5. Chemical labeling-based single-cell approaches introduce hundreds of cells into the MS, but direct analysis of single cells has not yet reached the necessary sensitivity, robustness and quantitative accuracy to answer biological questions6,7. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography and a novel trapped ion mobility mass spectrometer, resulting in a more than ten-fold improved sensitivity. We accurately and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators such as CDK2NA, the E2 ubiquitin ligase UBE2S, DNA topoisomerases TOP2A/B and the chromatin regulator HMGA1. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable core proteome despite perturbation, while the transcriptome appears volatile. This emphasizes substantial regulation of translation and sets the stage for its elucidation at the single cell level. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material8, posttranslational modifications and small molecule studies to gain unprecedented insights into cellular heterogeneity in health and disease.

systems biology↗

bioRxiv2020

Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry

Ion mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent - termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 [A]2 and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.

biochemistry↗

bioRxiv2019

Parallel accumulation - serial fragmentation combined with data-independent acquisition (diaPASEF): Bottom-up proteomics with near optimal ion usage

Data independent acquisition (DIA) modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection windows collectively cover the entire m/z range, overall only a few percent of all incoming ions are sampled. Making use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro), we here devise a novel scan mode that samples up to 100% of the peptide precursor ion current. We extend an established targeted data extraction workflow by including the ion mobility dimension for both signal extraction and scoring, thereby increasing the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a very high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.

systems biology↗

bioRxiv2019

MaxQuant software for ion mobility enhanced shotgun proteomics

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range. Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides, proteins and posttranslational modification sites in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

bioinformatics↗

bioRxiv2018

Online parallel accumulation - serial fragmentation (PASEF) with a novel trapped ion mobility mass spectrometer

In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, while mass spectra are acquired in about 100 microseconds with time-of-fight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive due to its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 800,000 fragmentation spectra in standard 120 min LC runs are easily achievable, which can be used for near exhaustive precursor selection in complex mixtures or re-sequencing weak precursors. MaxQuant identified more than 6,400 proteins in single run HeLa analyses without matching to a library, and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,900 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

systems biology↗