Biology subjects

Jensen, O. N.

Publications and source records attributed to Jensen, O. N..

At least 19 recordsLinked to original sources

bioRxiv2026

Ganglioside profiling by ion mobility and mass spectrometry imaging identifies distinct biomolecular isomers in Niemann-Pick Disease

Gangliosides are glycosphingolipids implicated in the neurodegenerative pathology of Niemann-Pick disease type C2 (NPC2 disease), where accumulation of GM2 and GM3 is a hallmark of disrupted lipid trafficking and disease progression. Some gangliosides are structural isomers with identical molecular composition and mass, making them difficult to distinguish by mass spectrometry imaging (MSI) alone and leaving their spatial distributions unresolved. To address this challenge, we combined ion mobility spectrometry with mass spectrometry imaging for isomer-resolved ganglioside analysis. We assessed ganglioside complexity in wild-type (Npc2+/+) and NPC2-deficient mice with or without AAV-BR1-mediated NPC2 gene therapy (Npc2-/- AAV-BR1-NPC2 and Npc2-/- vehicle) using matrix-assisted laser desorption ionization (MALDI) MSI, and imaging-parallel reaction monitoring-parallel accumulation serial fragmentation (iPRM-PASEF-MS/MS) for in situ brain tissue analysis. Trapped ion mobility spectrometry (TIMS) enabled gas-phase separation of isomeric gangliosides, while iPRM-PASEF-MS/MS facilitated structural discrimination based on diagnostic fragment ions and identification of molecular modifications. Brain sections were analyzed by MALDI-MSI at 20m spatial resolution, with selected regions examined using post-ionization imaging by MALDI-2 at 5m. 59 mobility-resolved ganglioside features representing 23 putative annotations were identified, including separation and spatial mapping of GM1a/GM1b and GD1a/GD1b isomers. Notably, unreported modifications of gangliosides were identified, including GM1 fucosylation in Npc2-/- mice and GD1 O-acetylation exclusively detected in Npc2+/+ mice. Spatial ganglioside profiling by MSI demonstrated that gene therapy of Npc2-/- mice partially restored normal ganglioside localization in brain, indicating modulation of lipid storage. This study establishes isomer-resolved lipid imaging for investigating ganglioside alterations in disease models and the effects of therapeutic intervention.

biochemistry↗

bioRxiv2026

Near-Zero Missed Cleavages with a High-Fidelity Recombinant Arg-C Zero for Mass Spectrometry-Based Proteomics

Proteolytic digestion remains a critical step in bottom-up proteomics workflows, with enzyme specificity and efficiency directly impacting peptide identification and protein sequence coverage. Here, we present the comprehensive characterization of Arg-C Zero, a recombinant arginyl endopeptidase derived from Porphyromonas gingivalis that exhibits exceptional fidelity in cleaving specifically at the C-terminus of arginine residues. Unlike conventional serine proteases such as Trypsin, Arg-C Zero utilizes a histidine-cysteine catalytic dyad mechanism, achieving near-zero missed cleavage rates (>99% efficiency) under standard proteomics conditions. Through systematic evaluation using HeLa protein extracts, we demonstrate that Arg-C Zero maintains consistent performance across varying digestion times. The enzyme shows robust activity across a broad pH range and tolerates up to 4M urea, making it ideally suitable for a diverse range of proteomics sample preparation workflows. While Trypsin/LysC combinations remain superior for comprehensive proteome coverage, Arg-C Zero offers unique advantages for applications requiring high specificity and reproducible arginine-specific cleavage patterns, particularly for analysis of post-translational modifications (PTMs). Here, we demonstrate how Arg-C Zero aids comprehensive mapping of histone PTMs, and when used in low-pH workflows help preserve labile ADP-ribosylation sites, expanding the analytical capabilities of mass spectrometry for characterizing these challenging modifications. The enzymes resistance to proline-adjacent cleavage sites and compatibility with standard mass spectrometry buffers position it as a valuable addition to the proteomics enzyme toolkit.

biochemistry↗

bioRxiv2026

Top-down Sequencing of Intact Proteoforms using the timsOmni mass spectrometer: Accurate Determination of Co-occurring Histone Modifications

Deep characterization of intact proteoforms remains an analytical challenge in functional proteomics, particularly for heterogenous multi-site post-translational modifications at distinct amino acid residues. Histones are among the most dynamically and diversely post-translationally modified proteins in eukaryote cells, carrying multiple, co-occurring and reversible modifications that can give rise to isomeric proteoform species. Tandem mass spectrometry with multimodal fragmentation capabilities is a promising approach for deep characterization of intact proteoforms, such as modified histones. We applied the novel timsOmni mass spectrometer, which incorporates the Omnitrap platform enabling multimodal MS workflows, for residue-level mapping of histone modifications, including acetylation and methylation. Recombinant histones H3.1 and H4 were in vitro acetylated by enzymes GCN5, PCAF and p300 to generate mono- and multi-acetylated proteoforms. Complementary MS2 electron- and collision-based dissociation (ECD, EID, RCID and ECciD), together with MS3 strategies, produced complete or near-complete backbone fragmentation of intact protein ions (>92% amino acid sequence coverage). For monoacetylated species generated by the more site-selective lysine acetyltransferases, the dominant proteoform matched the known catalytic preferences of the enzymes (H3.1K14ac for GCN5 and PCAF, and H4K8ac for PCAF), while minor positional isomers were also identified and their relative abundance estimated. In contrast, the broader substrate specificity of p300 produced a wide distribution of H4 proteoforms bearing up to seven acetylated lysine residues. Species carrying six and seven acetylations were characterized by multimodal MS2/MS3 experiments, enabling localization of individual acetylation sites and discrimination of positional isomers. Finally, endogenous histone proteoforms from liver extracts were analyzed, yielding sequence coverages of 92-93% for the most abundant species and enabling confident localization of multiple PTMs (acetylation and methylation). These results illustrate that multimodal MSn fragmentation of intact proteins supports residue-level assignment of combinatorial histone marks and coexisting positional isomers. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/722147v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@387ab5org.highwire.dtl.DTLVardef@2410org.highwire.dtl.DTLVardef@13fc392org.highwire.dtl.DTLVardef@140e054_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIMultimodal MS{superscript 2}/MS3 maps histone PTMs on intact proteins. C_LIO_LIECD, EID, RCID, and ECciD provide complete or near-complete sequence coverage. C_LIO_LIMS3 localizes acetylation sites, distinguishes positional isomers. C_LIO_LIEndogenous H4 proteoforms are assigned with site-specific PTM mapping. C_LI

biochemistry↗

bioRxiv2026

Batch correction for large-scale mass spectrometry imaging experiments

We assess batch correction methods for MALDI mass spectrometry imaging experiments. ComBAT reduced batch-related technical variance, maintained biological variation, and improved the overall score by 19.4%. Availability and implementationMethods are available in R. comBAT is used through the "sva" package while Harmony, CCA, FastMNN are available in the "Seurat" package https://github.com/satijalab/seurat. scVI, scANVI are Scanorama are available in the Python programming language and through github: https://github.com/scverse/scvi-tools.

bioinformatics↗

bioRxiv2026

Ion Mobility Separation of Isomeric Acyl-lysine Marks in Peptides

Isomeric post-translational modifications (PTMs) on proteins challenge proteomic analyses due to their identical mass and fragmentation patterns. We evaluate high-resolution ion mobility spectrometry (IMS) for separating three naturally occurring acyl-lysine isomer pairs on histones: crotonyl/methacryl, butyryl/isobutyryl and L-/D-lactyl. These PTMs were chemically installed on lysine residues 9 and 18 (K9, K18) of synthetic histone H3 peptides (residues 3-15 and 3-25). Using trapped IMS (TIMS) we observe half-height separation of H3[3-15] peptides possessing crotonyl/methacryl and L/D-lactyl marks, and the lactyl isomers of H3[3-25] can be distinguished. In contrast, multi-pass cyclic IMS (cIM) achieves baseline or near-baseline resolution for every pair, except the longest butyryl/isobutyryl peptide isomers, despite their collision-cross-section differences of {approx}1%. We show that resolution increased with the square root of cIM pass number, allowing baseline separation within 300 ms. Beyond separation, structure-mobility relationships emerge: branched modifications (isobutyryl, methacryl) yield more compact gas-phase conformations than their linear analogs (butyryl, crotonyl). For the doubly crotonylated/methacrylated peptides studied, both PTM identity and site determine the mobility. These results demonstrate IMS as a sensitive method for elucidation of acyl-modified histone peptide fine structure by resolving isomeric PTM ambiguity. This addresses a persistent analytical bottleneck and should be included in routine proteomics MS-based workflows

biochemistry↗

bioRxiv2025

A Charge-reversal Point Mutation Completely Depletes Flavin Chromophore from European Robin Cryptochrome 4a Protein

Cryptochrome 4a (Cry4a) is a magnetically sensitive protein that could enable night-migratory birds to sense the geomagnetic field for navigation. The key to the protein magnetic sensitivity is the flavin adenine dinucleotide (FAD) cofactor, which initiates the electron transfer within the protein leading to a spin-correlated radical pair. De-spite its importance, the mechanism of FAD binding in avian Cry4a proteins remains unclear. Here we show that point mutagenesis of positively charged arginine residue at position 356 to negatively charged glutamic acid completely depletes FAD binding from European robin (Erithacus rubecula) Cry4a. The result indicates that electrostatic in-teractions constitute the primary driving force that enables FAD binding in European robin Cry4a. The finding provides new structural insight into the molecular basis of FAD binding in Cry4 and advances our understanding on the biophysical underpinnings of bird magnetoreception. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/690116v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@93344forg.highwire.dtl.DTLVardef@4f642corg.highwire.dtl.DTLVardef@39912corg.highwire.dtl.DTLVardef@169e1de_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

PAF15-PCNA assembly exhaustion governs lagging strand replication and replisome integrity

Genome replication in eukaryotic cells is surveyed by the S-phase checkpoint, which orchestrates sequential replication origin activation to avoid exhaustion of hitherto poorly defined rate-limiting replisome components. Here, we find that excessive activation of replication origins depletes chromatin-bound PCNA and lagging strand components, thereby limiting additional PCNA loading at new origins when checkpoint control is disrupted. PAF15 (PCNA-associated factor 15) emerges as a dosage-sensitive regulator of PCNA, delineating the dynamic range of global genome duplication and defining distinct roles for PCNA on the leading and lagging strands. Through its high-affinity PIP motif and interaction within the DNA encircling channel of PCNA, PAF15 stabilizes PCNA exclusively on the lagging strand, optimizing and rate-limiting lagging strand processing. On the other hand, misregulation of PAF15--whether by overexpression or mislocalization to the leading strand--impairs replication fork progression and leads to cell death. These defects are mitigated by TIMELESS and CLASPIN, which restrain PAF15-PCNA interactions beyond the lagging strand. E2F4-mediated repression orchestrates PAF15 expression in normal and cancer cells, maintaining its optimal dosage for lagging strand-specific interactions with PCNA. Thus, the S-phase checkpoint functions in concert to restrict origin activation when lagging strand PAF15-PCNA assembly is exhausted, linking a previously concealed strand-specific rate limitation to overall replication dynamics.

biochemistry↗

bioRxiv2025

Cryptochrome 4b protein is likely irrelevant for the radical pair based magnetoreception in the European robin

Avian cryptochrome 4 (Cry4) protein is a putative magnetosensitive molecule facilitating precise long-distance navigation in migratory birds. Two splice variants of Cry4 were reported in European robin (Erithacus rubecula), namely ErCry4a and ErCry4b. It is known that ErCry4a protein exhibits electron transfer between the flavin adenine dinucleotide (FAD) cofactor and tryptophan residues that generates magnetically sensitive radical pairs for magnetoreception. However, little is known about the ErCry4b isoform. We therefore characterized the properties of ErCry4b to see whether it fulfills prerequisites to be a radical pair magnetic sensor molecule. Our results show that ErCry4b protein does not bind FAD in vitro. Computational structure simulations revealed that the FAD non-binding in ErCry4b is likely due to protein structure dynamics. Furthermore, ErCry4b protein abundance in the robin retina, cerebellum and liver is below the detection limit of immunoprecipitation assays coupled with mass spectrometry. Meanwhile, transcript analyses show that ErCRY4b mRNA abundance is 10 times less than ErCRY4b in the retina. In conclusion, ErCry4b does not fulfill the prerequisites to be a radical pair based magnetic sensing molecule due to the lack of FAD binding, and it might not even be expressed as a functional protein in the European robin.

biochemistry↗

bioRxiv2024

Anubis: a multi-level authentication scale for ancient proteins using random forest classification

Deamidation is a time-dependent, post-translational modification with wide applications in clinical proteomics and palaeoproteomics as ageing indicators and authentication markers. Despite its significance, the use of deamidation for authenticating ancient proteins remains controversial, primarily due to variability in deamidation kinetics and context-specific protein preservation. We propose Anubis, an open-source, Python-based pipeline to evaluate relative deamidation patterns and authenticate ancient proteins. Anubis leverages random forest classification based on a range of tandem mass spectrometry-based features and physico-chemical characteristics. We use a combination of ancient and experimentally degraded beta-lactoglobulin to demonstrate that Anubis can authenticate a putative ancient dairy protein using position-specific deamidation patterns, relative deamidation (asparagine vs. glutamine), and overall modification patterns using trypsin processed in the same batch as a baseline. This multi-level approach addresses the complexities of ancient protein deamidation and paves the way for automated, reliable authentication across different archaeological substrates.

bioinformatics↗

bioRxiv2024

Mind Your Spectra: Points to be Aware of when Validating the Identification of Isobaric Histone Peptidoforms

Mass spectrometry (MS) has become a central technique to identify and quantify post-translational modifications (PTMs), overcoming limitations of antibody-based methods. Histones get dynamically modified by diverse chemical groups, particularly on their numerous lysine residues, to fine-tune all DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual data curation. This study focused on the Lys27-Arg40 stretch of histone H3, considered four sequence variants, an increasing number of lysine PTMs and artifacts coming from histone sample processing, which resulted in many peptides with the same atomic composition. Our analysis revealed the value of low-mass b1 and cyclic immonium fragment ions to validate identification of the distinct peptidoforms. We examined how MS/MS spectra are transformed by common software tools during the conversion of RAW files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We established the fragmentation profiles and retention times for forty H3 K27-R40 variantxPTM combinations, including the mouse-specific variants H3mm7 and H3mm13, and targeted their detection in histone samples extracted from mouse testis and brain via a scheduled parallel reaction monitoring (PRM) analysis. The transcripts of these two mousespecific variants were reported to be highly abundant in these tissues and the corresponding proteins may seem to be identified by data-dependent analyses. However, we only detected very low levels of the unmodified form of H3mm7 and found no trace of H3mm13 by PRM. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.

biochemistry↗

bioRxiv2024

How to deal with internal fragment ions?

1Tandem mass spectrometry of peptides and proteins generates mass spectra of their gas-phase fragmentation product ions, including N-terminal, C-terminal, and internal fragment ions. Whereas N- and C-terminal ions are routinely assigned and identified using computational methods, internal fragment ions are often difficult to annotate correctly. They become particularly relevant for long peptides and full proteoforms where the peptide backbone is more likely to be fragmented multiple times. Internal fragment ions potentially offer tremendous information regarding amino acid sequences and positions of post-translational modifications of peptides and intact proteins. However, their practical application is challenged by the vast number of theoretical internal fragments that exist for long amino acid sequences, leading to a high risk of false-positive annotations. We analyze the mass spectral contributions of internal fragment ions in spectra from middle-down and top-down experiments and introduce a novel graph-based annotation approach designed to manage the complexity of internal fragments. Our graph-based representation allows us to compare multiple candidate proteoforms in a single graph, and to assess different candidate annotations in a fragment ion spectrum. We demonstrate cases from middle-down and top-down data where internal ions enhance amino acid sequence coverage of polypeptides and proteins and accurate localization of post-translational modifications. We conclude that our graph-based method provides a general approach to process complex tandem mass spectra, enhance annotation of internal fragment ions, and improve proteoform sequencing and characterization by mass spectrometry.

bioinformatics↗

bioRxiv2023

Propionate reinforces epithelial identity and reduces aggressiveness of non-small cell lung carcinoma via chromatin remodelling

Epithelial to mesenchymal transition (EMT) is a developmental cellular program driving metastasis and chemo-resistance in cancer, but its pharmacological treatment has been so far challenging. Targeting deregulated metabolic processes in cancer is emerging as a realistic therapeutic strategy. Here, we used an EMT-focussed integrative functional genomic approach and identified negative association of the short-chain fatty acids propionate and butanoate with EMT in non-small cell lung cancer (NSCLC) patients. Strikingly, in vitro treatment of lung cancer cell lines with propionate reinforced the epithelial transcriptional program promoting cell adhesion and reverting the aggressive and chemoresistant EMT phenotype. Propionate treatment reduced cells metastatic ability in nude mice and limited lymph nodal spread in a genetic NSCLC mouse model. Further analyses indicated chromatin remodeling via H3K27 acetylation (p300-mediated) as the mechanism shifting the EMT balance towards epithelial state upon propionate. Propionate administration could be tested in the clinic for reducing NSCLC aggressiveness. HighlightsAn EMT-centric investigation of metabolic processes in a comprehensive lung cancer transcriptome profiles identified negative associations between EMT and SCFAs (propionate and butyrate) Propionate enhances the epithelial features both at the molecular and cellular levels Pre-treatment of cells with propionate inhibits EMT associated processes including migration and sensitizes the cells to chemotherapeutic drug cisplatin Oral administration of propionate inhibits EMT-mediated lung colonization ability of NSCLC cells, and lymph node metastasis in a genetic mouse NSCLC model Molecular mechanistic investigation of propionate revealed chromatin remodelling through p300-mediated histone acetylation in E-cadherin gene regulation along with epithelial features reinforcement

cancer biology↗

bioRxiv2023

MYC enhancer invasion promotes prognostic cancer type-specific gene programs through an epigenetic switch

The transcription factor MYC is overexpressed in most cancers, where it drives multiple hallmarks of cancer progression. MYC is known to promote oncogenic transcription by binding to active promoters. In addition, MYC has also been shown to invade distal enhancers when expressed at oncogenic levels, but this enhancer binding has been proposed to have low gene-regulatory potential. Here, we demonstrate that MYC enhancer binding directly promotes cancer type-specific gene programs predictive of poor patient prognosis. MYC induces transcription of enhancer RNA through recruitment of RNAPII, rather than regulating RNAPII pause-release as is the case at promoters. This is mediated by MYC-induced H3K9 demethylation by KDM3A and acetylation by GCN5, leading to enhancer-specific BRD4 recruitment through its bromodomains, which facilitates RNAPII recruitment. Thus, we propose that MYC drives prognostic cancer type-specific gene programs by promoting RNAPII recruitment to enhancers through induction of an epigenetic switch.

genomics↗

bioRxiv2022

Top-Down Ion Mobility Separations of Isomeric Proteoforms

Continuing advances in proteomics highlight the ubiquity and biological importance of proteoforms - the proteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs ("localization variants") commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The variants with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.

biochemistry↗

bioRxiv2021

FLASHIda: Intelligent data acquisition for top-down proteomics that doubles proteoform level identification count

Top-down proteomics (TDP) has gained a lot of interest in biomedical application for detailed analysis and structural characterization of proteoforms. Data-dependent acquisition (DDA) of intact proteins is non-trivial due to the diversity and complex signal of proteoforms. Dedicated acquisition methods thus have the potential to greatly improve TDP. We present FLASHIda, an intelligent online data acquisition algorithm for TDP that ensures the real-time selection of high-quality precursors of diverse proteoforms. FLASHIda combines fast charge deconvolution algorithms and machine learning-based quality assessment for optimal precursor selection. In analysis in E. coli lysates, FLASHIda increased the number of unique proteoform level identifications from 800 to 1,500, or generated a near-identical number of identifications in [1/3] of instrument time when compared to standard DDA mode. Furthermore, FLASHIda enabled sensitive mapping of post translational modifications and detection of chemical adducts. As an extension module to the instrument, FLASHIda can be readily adopted for TDP studies of complex samples to enhance proteoform identification rates.

bioinformatics↗

bioRxiv2021

Distinct and diverse chromatin-proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

Temporal molecular changes in ageing mammalian organs are of relevance to disease etiology because many age-related diseases are linked to changes in the transcriptional and epigenetic machinery that regulate gene expression. We performed quantitative proteome analysis of chromatin-enriched protein extracts to investigate the dynamics of the chromatin-proteomes of the mouse brain, heart, lung, kidney, liver, and spleen at 3, 5, 10, and 15 months of age. Each organ exhibited a distinct chromatin-proteome and sets of unique proteins. The brain and spleen chromatin-proteomes were the most extensive, diverse, and heterogenous among the six organs. The spleen chromatin proteome appeared static during the lifespan, presenting a young phenotype that reflects the permanent alertness state and important role of this organ in physiological defense and immunity. We identified a total of 5928 proteins, including 2472 nuclear or chromatin associated proteins across the six mouse organs. Up to 3125 proteins were quantified in each organ demonstrating distinct and organ-specific temporal protein expression timelines and regulation at the post-translational level. Bioinformatics meta- analysis of these chromatin proteomes revealed distinct physiological and ageing- related features for each organ. Our results demonstrate the efficiency of organelle specific proteomics for in vivo studies of a model organism and consolidate the hypothesis that chromatin-associated proteins are involved in distinct and specific physiological functions in ageing organs. HIGHLIGHTSO_LIQuantitative chromatin-proteome analysis during mouse lifespan; C_LIO_LIChromatin analysis in vitro and in vivo mouse models; C_LIO_LIDistinct chromatin proteomes of six organs during mouse lifespan; C_LIO_LICorrelations between ageing and chromatin regulation in mammalian lifespan. C_LI

cell biology↗

bioRxiv2020

Zirconium(IV)-IMAC for phosphopeptide enrichment in phosphoproteomics

Phosphopeptide enrichment is an essential step in large-scale, quantitative phosphoproteomics studies by mass spectrometry. Several phosphopeptide affinity enrichment techniques exist, such as Immobilized Metal ion Affinity Chromatography (IMAC) and Metal Oxide Affinity Chromatography (MOAC). We compared Zirconium (IV) IMAC (Zr-IMAC) magnetic microparticles to more commonly used Titanium (IV) IMAC (Ti-IMAC) and TiO2 magnetic microparticles for phosphopeptide enrichment from simple and complex protein samples prior phosphopeptide sequencing and characterization by mass spectrometry (LC-MS/MS). We optimized sample-loading conditions to increase phosphopeptide recovery for Zr-IMAC, Ti-IMAC and TiO2 based workflows. The performance of Zr-IMAC was enhanced by 19-22% to recover up to 5173 phosphopeptides from 200 {micro}g of protein extract from HepG2/C3A cells, making Zr-IMAC the preferred method for phosphopeptide enrichment in this study. Ti-IMAC and TiO2 performance were also optimized to improve phosphopeptide numbers by 28% and 35%, respectively. Furthermore, Zr-IMAC based phosphoproteomics in the magnetic microsphere format identified 23% more phosphopeptides than HPLC-based Fe(III)-IMAC for same sample amount (200 {micro}g), thereby adding 37% more uniquely identified phosphopeptides. We conclude that Zr-IMAC improves phosphoproteome coverage and recommend that this affinity enrichment method should be more widely used in biological and biomedical studies of cell signalling and in the search for disease-biomarkers.

biochemistry↗

bioRxiv2020

Histo-molecular differentiation of renal cancer subtypes by imaging mass spectrometry and rapid proteome profiling

Pathology differentiation of renal cancer types is challenging due to tissue similarities or overlapping histological features of various tumor (sub)types. As assessment is often manually conducted outcomes can be prone to human error and therefore require high-level expertise and experience. Mass spectrometry can provide detailed histo-molecular information on tissue and is becoming increasingly popular in clinical settings. Spatially resolving technologies such as mass spectrometry imaging and quantitative microproteomics profiling in combination with machine learning approaches provide promising tools for automated tumor classification of clinical tissue sections. In this proof of concept study we used MALDI-MS imaging (MSI) and rapid LC-MS/MS-based microproteomics technologies (15 min/sample) to analyze formalin-fixed paraffin embedded (FFPE) tissue sections and classify renal oncocytoma (RO, n=11), clear cell renal cell carcinoma (ccRCC, n=12) and chromophobe renal cell carcinoma (ChRCC, n=5). Both methods were able to distinguish ccRCC, RO and ChRCC in cross-validation experiments. MSI correctly classified 87% of the patients whereas the rapid LC-MS/MS-based microproteomics approach correctly classified 100% of the patients. This strategy involving MSI and rapid proteome profiling by LC-MS/MS reveals molecular features of tumor sections and enables cancer subtype classification. Mass spectrometry provides a promising complementary approach to current pathological technologies for precise digitized diagnosis of diseases.

biochemistry↗