Spatial tissue proteomics reveals distinct landscapes of heterogeneity in cutaneous papillomavirus-induced keratinocyte carcinomas.

Infection with certain cutaneous human papillomaviruses (HPV), in conjunction with chronic ultraviolet (UV) exposure, are the major cofactors of non-melanoma skin cancer (NMSC), the most frequent cancer type worldwide. Cutaneous squamous cell carcinomas (SCCs) as well as tumors in general represent three-dimensional entities determined by both temporal and spatial constraints. Whole tissue proteomics is a straightforward approach to understand tumorigenesis in better detail, but studies focusing on different progression states toward a dedifferentiated SCC phenotype on a spatial level are rare. Here, we applied an innovative proteomic workflow on formalin-fixed, paraffin-embedded (FFPE) epithelial tumors derived from the preclinical animal model Mastomys coucha. This rodent is naturally infected with its genuine cutaneous papillomavirus and closely mimics skin carcinogenesis in the context of cutaneous HPV infections in humans. We deciphered cellular networks by comparing diverse epithelial tissues with respect to their differentiation level and infection status. Our study reveals novel regulatory proteins and pathways associated with virus-induced tumor initiation and progression of SCCs. This approach provides the basis to better comprehend the multistep process of skin carcinogenesis.

Updated and enhanced pig cardiac transcriptome based on long-read RNA sequencing and proteomics.

Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel therapeutic interventions. In particular, the pig has emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Currently, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and are provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.

Automated sample preparation with SP3 for low-input clinical proteomics.

High-throughput and streamlined workflows are essential in clinical proteomics for standardized processing of samples from a variety of sources, including fresh-frozen tissue, FFPE tissue, or blood. To reach this goal, we have implemented single-pot solid-phase-enhanced sample preparation (SP3) on a liquid handling robot for automated processing (autoSP3) of tissue lysates in a 96-well format. AutoSP3 performs unbiased protein purification and digestion, and delivers peptides that can be directly analyzed by LCMS, thereby significantly reducing hands-on time, reducing variability in protein quantification, and improving longitudinal reproducibility. We demonstrate the distinguishing ability of autoSP3 to process low-input samples, reproducibly quantifying 500-1,000 proteins from 100 to 1,000 cells. Furthermore, we applied this approach to a cohort of clinical FFPE pulmonary adenocarcinoma (ADC) samples and recapitulated their separation into known histological growth patterns. Finally, we integrated autoSP3 with AFA ultrasonication for the automated end-to-end sample preparation and LCMS analysis of 96 intact tissue samples. Collectively, this constitutes a generic, scalable, and cost-effective workflow with minimal manual intervention, enabling reproducible tissue proteomics in a broad range of clinical and non-clinical applications.

Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging.

Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

Effects of tubing degradation and pump position on extracorporeal circulation performance.

Extracorporeal circulation (ECC) is an integral part of cardiac surgery. Yet, the contact with artificial surfaces, mechanical stress, shear, and suction forces within the ECC pose a pronounced risk for damaging the corpuscular components of the blood. These suction forces may be reduced by a repositioning of the roller pumps employed below the reservoir. Furthermore, the repeated compression of the involved silicone tubing is likely to cause degradation. We present our observations regarding changes in the ECC performance following this degradation process. In vitro experiments were performed in a standard as well as a simplified ECC setup and included measurements of pressure, effective flow, and tubing restoring force over a time frame of 12 hours with two different pump positions utilizing transit time flow measurement. Suction forces within the tubing could be significantly reduced by shifting the pump position below the reservoir. Regardless of the setup, the tubing showed significant loss of restoring force as well as effective flow over time. A shift of the pump position within the ECC setup can be recommended to prevent blood damaging negative pressures. Further research is necessary to evaluate the exact cause of the reduction of restoring force overtime as well as its risks for the patients. Finally, our results underline the importance of transit time flow measurements to collect reliable flow data.

Red Blood Cell Stiffness and Adhesion Are Species-Specific Properties Strongly Affected by Temperature and Medium Changes in Single Cell Force Spectroscopy.

Besides human red blood cells (RBC), a standard model used in AFM-single cell force spectroscopy (SCFS), little is known about apparent Young's modulus (Ea) or adhesion of animal RBCs displaying distinct cellular features. To close this knowledge gap, we probed chicken, horse, camel, and human fetal RBCs and compared data with human adults serving as a repository for future studies. Additionally, we assessed how measurements are affected under physiological conditions (species-specific temperature in autologous plasma vs. 25 °C in aqueous NaCl solution). In all RBC types, Ea decreased with increasing temperature irrespective of the suspension medium. In mammalian RBCs, adhesion increased with elevated temperatures and scaled with reported membrane sialic acid concentrations. In chicken only adhesion decreased with higher temperature, which we attribute to the lower AE-1 concentration allowing more membrane undulations. Ea decreased further in plasma at every test temperature, and adhesion was completely abolished, pointing to functional cell enlargement by adsorption of plasma components. This halo elevated RBC size by several hundreds of nanometers, blunted the thermal input, and will affect the coupling of RBCs with the flowing plasma. The study evidences the presence of a RBC surface layer and discusses the tremendous effects when RBCs are probed at physiological conditions.

Systematic Evaluation of Protein Reduction and Alkylation Reveals Massive Unspecific Side Effects by Iodine-containing Reagents.

Reduction and alkylation of cysteine residues is part of virtually any proteomics workflow. Despite its frequent use, up to date no systematic investigation of the impact of different conditions on the outcome of proteomics studies has been performed. In this study, we compared common reduction reagents (dithiothreitol, tris-(2-carboxyethyl)-phosphine, and ß-mercaptoethanol) and alkylation reagents (iodoacetamide, iodoacetic acid, acrylamide, and chloroacetamide). Using in-gel digests as well as SAX fractionated in-solution digests of cytosolic fractions of HeLa cells, we evaluated 13 different reduction and alkylation conditions resulting in considerably varying identification rates. We observed strong differences in offsite alkylation reactions at 7 amino acids as well as at the peptide N terminus, identifying single and double adducts of all reagents. Using dimethyl labeling, mass tolerant searches, and synthetic peptide experiments, we identified alkylation of methionine residues by iodine-containing alkylation reagents as one of the major factors for the differences. We observed differences of more than 9-fold in numbers of identified methionine-containing peptide spectral matches for in-gel digested samples between iodine- and noniodine-containing alkylation reagents. This was because of formation of carbamidomethylated and carboxymethylated methionine side chains and a resulting prominent neutral loss during ESI ionization or in MS/MS fragmentation, strongly decreasing identification rates of methionine-containing peptides. We achieved best results with acrylamide as alkylation reagent, whereas the highest numbers of peptide spectral matches were obtained when reducing with dithiothreitol and ß-mercaptoethanol for the in-solution and the in-gel digested samples, respectively.

Spermidine-Induced Attraction of Like-Charged Surfaces Is Correlated with the pH-Dependent Spermidine Charge: Force Spectroscopy Characterization.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

Impact of reduced tillage on greenhouse gas emissions and soil carbon stocks in an organic grass-clover ley - winter wheat cropping sequence.

Organic reduced tillage aims to combine the environmental benefits of organic farming and conservation tillage to increase sustainability and soil quality. In temperate climates, there is currently no knowledge about its impact on greenhouse gas emissions and only little information about soil organic carbon (SOC) stocks in these management systems. We therefore monitored nitrous oxide (N2O) and methane (CH4) fluxes besides SOC stocks for two years in a grass-clover ley - winter wheat - cover crop sequence. The monitoring was undertaken in an organically managed long-term tillage trial on a clay rich soil in Switzerland. Reduced tillage (RT) was compared with ploughing (conventional tillage, CT) in interaction with two fertilisation systems, cattle slurry alone (SL) versus cattle manure compost and slurry (MC). Median N2O and CH4 flux rates were 13 µg N2O-N m-2 h-1 and -2 µg CH4C m-2 h-1, respectively, with no treatment effects. N2O fluxes correlated positively with nitrate contents, soil temperature, water filled pore space and dissolved organic carbon and negatively with ammonium contents in soil. Pulse emissions after tillage operations and slurry application dominated cumulative gas emissions. N2O emissions after tillage operations correlated with SOC contents and collinearly to microbial biomass. There was no tillage system impact on cumulative N2O emissions in the grass-clover (0.8-0.9 kg N2O-N ha-1, 369 days) and winter wheat (2.1-3.0 kg N2O-N ha-1, 296 days) cropping seasons, with a tendency towards higher emissions in MC than SL in winter wheat. Including a tillage induced peak after wheat harvest, a full two year data set showed increased cumulative N2O emissions in RT than CT and in MC than SL. There was no clear treatment influence on cumulative CH4 uptake. Topsoil SOC accumulation (0-0.1 m) was still ongoing. SOC stocks were more stratified in RT than CT and in MC than SL. Total SOC stocks (0-0.5 m) were higher in RT than CT in SL and similar in MC. Maximum relative SOC stock difference accounted for +8.1 Mg C ha-1 in RT-MC compared to CT-SL after 13 years which dominated over the relative increase in greenhouse gas emissions. Under these site conditions, organic reduced tillage and manure compost application seems to be a viable greenhouse gas mitigation strategy as long as SOC is sequestered.

Single-cell force spectroscopy of bacteria enabled by naturally derived proteins.

Bringing the study of bacterial adhesion down to a single-cell level is critical for understanding the molecular mechanisms involved in initial bacterial attachment. We have developed a simple and versatile method for making single-cell bacterial probes to study the adhesion of single bacterial cells by atomic force microscopy (AFM). A single-cell probe was made by picking up a bacterial cell from a glass surface using a tipless AFM cantilever coated with a commercial cell adhesive Cell-Tak. The method was applied to four different bacterial strains, and single-cell adhesion was measured on three surfaces (fresh glass, hydrophilic glass, and mica). Attachment to the cantilever was stable during the AFM force measurements that were conducted for 2 h, and viability was confirmed by Live/Dead fluorescence staining at the end of each experiment. The adhesion force and final rupture length were dependent on bacterial strains, surfaces properties, and contact time. The single-cell probe offers control of cell immobilization and thus holds advantages over the commonly used multicell probes with which random immobilization is obtained by submerging the cantilever in a bacterial suspension. The reported method provides a general platform for investigating single-cell interactions of bacteria with different surfaces and other cells by AFM force spectroscopy, thus improving our understanding of the mechanisms of bacterial attachment.

Effectiveness of bio-effectors on maize, wheat and tomato performance and phosphorus acquisition from greenhouse to field scales in Europe and Israel: a meta-analysis.

Biostimulants (Bio-effectors, BEs) comprise plant growth-promoting microorganisms and active natural substances that promote plant nutrient-acquisition, stress resilience, growth, crop quality and yield. Unfortunately, the effectiveness of BEs, particularly under field conditions, appears highly variable and poorly quantified. Using random model meta-analyses tools, we summarize the effects of 107 BE treatments on the performance of major crops, mainly conducted within the EU-funded project BIOFECTOR with a focus on phosphorus (P) nutrition, over five years. Our analyses comprised 94 controlled pot and 47 field experiments under different geoclimatic conditions, with variable stress levels across European countries and Israel. The results show an average growth/yield increase by 9.3% (n=945), with substantial differences between crops (tomato > maize > wheat) and growth conditions (controlled nursery + field (Seed germination and nursery under controlled conditions and young plants transplanted to the field) > controlled > field). Average crop growth responses were independent of BE type, P fertilizer type, soil pH and plant-available soil P (water-P, Olsen-P or Calcium acetate lactate-P). BE effectiveness profited from manure and other organic fertilizers, increasing soil pH and presence of abiotic stresses (cold, drought/heat or salinity). Systematic meta-studies based on published literature commonly face the inherent problem of publication bias where the most suspected form is the selective publication of statistically significant results. In this meta-analysis, however, the results obtained from all experiments within the project are included. Therefore, it is free of publication bias. In contrast to reviews of published literature, our unique study design is based on a common standardized protocol which applies to all experiments conducted within the project to reduce sources of variability. Based on data of crop growth, yield and P acquisition, we conclude that application of BEs can save fertilizer resources in the future, but the efficiency of BE application depends on cropping systems and environments.

Characterization of novel nitrite-based nitric oxide generating delivery systems for topical dermal application.

Topical application of nitric oxide (NO) has been shown to exert beneficial effects in the therapy of chronic wounds, impaired microcirculation, and skin infections. Nitrite acidified by ascorbic acid has been widely used in many studies as NO-donor system, unfortunately with inflammatory and toxic effects on the treated skin due to unregulated excessive NO generation, low pH and possible toxic side products. Here we describe an essentially modified nitrite based NO generating system that avoid the mentioned unwanted side effects on human skin by using a pH-stable acetate/acetic acid buffer with a skin neutral pH of 5.5 and sodium ascorbate. In order to overcome the shortcoming of lower NO yields due to the higher pH-value and low nitrite concentrations, we have determined additionally the influence of copper ions. To investigate the influence of different NO release and penetration kinetics on NO-induced toxicity, we have developed a fibroblast assay using cell culture plates with gas permeable bottoms. The results show clearly that the donor system can achieve a sustained NO generation without generating high peaks. Furthermore, the presence of Cu(2+) ions enhances manifold NO generation of pH/ascorbate-induced nitrite decomposition, a mechanism comprising the reduction of Cu(2+) ions to Cu(1+) by ascorbate. Finally, we have found that apart from the NO dose the NO release kinetics had a significant influence of cell toxicity. Thus, application of comparable NO amounts within a time interval of 600s led to the development of variable cell toxicities, which predominantly depended on the NO concentration values generated in the first 200s. In summary, we here describe a novel nitrite-based NO-donor system that can provide well defined NO concentrations at skin neutral pH-values for side effect poor topical dermal application, i.e. in the therapy of chronic wounds and impaired microcirculation.

An integrated workflow for quantitative analysis of the newly synthesized proteome.

The analysis of proteins that are newly synthesized upon a cellular perturbation can provide detailed insight into the proteomic response that is elicited by specific cues. This can be investigated by pulse-labeling of cells with clickable and stable-isotope-coded amino acids for the enrichment and mass spectrometric characterization of newly synthesized proteins (NSPs), however convoluted protocols prohibit their routine application. Here we report the optimization of multiple steps in sample preparation, mass spectrometry and data analysis, and we integrate them into a semi-automated workflow for the quantitative analysis of the newly synthesized proteome (QuaNPA). Reduced input requirements and data-independent acquisition (DIA) enable the analysis of triple-SILAC-labeled NSP samples, with enhanced throughput while featuring high quantitative accuracy. We apply QuaNPA to investigate the time-resolved cellular response to interferon-gamma (IFNg), observing rapid induction of targets 2 h after IFNg treatment. QuaNPA provides a powerful approach for large-scale investigation of NSPs to gain insight into complex cellular processes.

Automated Sample Preparation for Mass Spectrometry-Based Clinical Proteomics.

Mass spectrometry (MS)-based proteomics is a rapidly maturing discipline, thus gaining momentum for routine molecular profiling of clinical specimens to improve disease classification, diagnostics, and therapy development. Yet, hurdles need to be overcome to enhance reproducibility in preanalytical sample processing, especially in large, quantity-limited sample cohorts. Therefore, automated sonication and single-pot solid-phase-enhanced sample preparation (autoSP3) was developed as a streamlined workflow that integrates all tasks from tissue lysis and protein extraction, protein cleanup, and proteolysis. It enables the concurrent processing of 96 clinical samples of any type (fresh-frozen or FFPE tissue, liquid biopsies, or cells) on an automated liquid handling platform, which can be directly interfaced to LC-MS for proteome analysis of clinical specimens with high sensitivity, high reproducibility, and short turn-around times.

Soil phosphorus availability and fractionation in response to different phosphorus sources in alkaline and acid soils: a short-term incubation study.

Using agricultural wastes as an alternative phosphorus (P) source has great prospects to improve soil P status. A 70-day incubation experiment was carried out to investigate the effects of superphosphate (SSP), poultry manure (PM), cattle manure (CM), maize straw (MS), and cattle bone meal (CB) with the same total P input on soil P availability and fractions in typical acidic (red soil) and alkaline (fluvo-aquic soil) soils. The results showed that in both fluvo-aquic and red soils, CM out-performed other P sources in improving soil P availability. Changes in soil Olsen-P (ΔOlsen-P) were greater in fluvo-aquic soils with SSP, PM and CM additions than in red soils. Among the different P sources used, only CM has increased the labile soil P fractions to levels similar to that with SSP. Compared with SSP, more monoester P and inositol hexakisphosphate were detected in soils amended with PM and CM. A structural equation model (SEM) analysis suggested that soil pH had a direct positive effect on the labile P fractions in the acidic red soil amended with different P sources. In summary, CM is a superior P source for increasing plant available soil P, with considerable practical implications for P recycling.

Mechanical properties and chemical stability of alginate-based anisotropic capillary hydrogels.

Anisotropic capillary hydrogels are formed by ionotropic gel formation of an aqueous sodium alginate solution during unidirectional diffusion and complexation with divalent cations. The type of cation used for gel formation dictates the size of the resulting capillary structure which might facilitate applications as biomaterial scaffolds in tissue engineering and regenerative medicine. Until now, such capillary hydrogel structures have not been characterized regarding their mechanical properties, and we hypothesise that both, the macroscopic capillary structure and the concentration of a chemical crosslinking agent, influence the mechanical properties and the stability of the hydrogels against degradation and dissolution in physiological environment. We prepared anisotropic gels with capillary sizes ranging between 12 and 100 µm using electrolyte solutions containing Ba2+, Sr2+, Zn2+ or Ni2+ cations. They were chemically crosslinked with hexamethylene diisocyanate in concentrations varying between 1 and 100 mmol L-1. Hydrogel properties were determined by swelling experiments, oscillatory rheometry and microindentation analysis and monitored during incubation in phosphate-buffered solution at 37 °C for up to three months. We found, that the mechanical strength generally decreases with increasing capillary diameter. The higher the concentration of the crosslinking agent, the higher is the mechanical strength. The resistance of the alginate hydrogels against degradation is positively correlated with the concentration of the crosslinking agent. Furthermore, microindentation experiments revealed a microscopic anisotropy of the mechanical properties resulting from a perpendicular orientation of the polymer fibres relative to the capillary axis.

A Global Network Meta-Analysis of the Promotion of Crop Growth, Yield, and Quality by Bioeffectors.

Bioeffector (BE) application is emerging as a strategy for achieving sustainable agricultural practices worldwide. However, the effect of BE on crop growth and quality is still controversial and there is still no adequate impact assessment that determines factors on the efficiency of BE application. Therefore, we carried out a network metaanalysis on the effect of BEs using 1,791 global observations from 186 studies to summarize influencing factors and the impact of BEs on crop growth, quality, and nutrient contents. The results show that BEs did not only improve plant growth by around 25% and yield by 30%, but also enhanced crop quality, e.g., protein (55% increase) and soluble solids content (75% increase) as well as aboveground nitrogen (N) and phosphate (P) content by 28 and 40%, respectively. The comparisons among BE types demonstrated that especially non-microbial products, such as extracts and humic/amino acids, have the potential to increase biomass growth by 40-60% and aboveground P content by 54-110%. The soil pH strongly influenced the efficiency of the applied BE with the highest effects in acidic soils. Our results showed that BEs are most suitable for promoting the quality of legumes and increasing the yield of fruits, herbs, and legumes. We illustrate that it is crucial to optimize the application of BEs with respect to the right application time and technique (e.g., placement, foliar). Our results provide an important basis for future research on the mechanisms underlying crop improvement by the application of BEs and on the development of new BE products.

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens.

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.

Pre-analytical processing of plasma and serum samples for combined proteome and metabolome analysis.

Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

IceR improves proteome coverage and data completeness in global and single-cell proteomics.

Label-free proteomics by data-dependent acquisition enables the unbiased quantification of thousands of proteins, however it notoriously suffers from high rates of missing values, thus prohibiting consistent protein quantification across large sample cohorts. To solve this, we here present IceR (Ion current extraction Re-quantification), an efficient and user-friendly quantification workflow that combines high identification rates of data-dependent acquisition with low missing value rates similar to data-independent acquisition. Specifically, IceR uses ion current information for a hybrid peptide identification propagation approach with superior quantification precision, accuracy, reliability and data completeness compared to other quantitative workflows. Applied to plasma and single-cell proteomics data, IceR enhanced the number of reliably quantified proteins, improved discriminability between single-cell populations, and allowed reconstruction of a developmental trajectory. IceR will be useful to improve performance of large scale global as well as low-input proteomics applications, facilitated by its availability as an easy-to-use R-package.