Evaluating Solid Phase Adsorption Toxin Tracking (SPATT) for passive monitoring of per- and polyfluoroalkyl substances (PFAS) with Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS).

Detection and monitoring of per- and polyfluoroalkyl substances (PFAS) in aquatic environments has become an increasingly higher priority of regulatory agencies as public concern for human intake of these chemicals continues to grow. While many methods utilize active sampling strategies ("grab samples") for precise PFAS quantitation, here we evaluate the efficacy of low-cost passive sampling devices (Solid Phase Adsorption Toxin Tracking, or SPATTs) for spatial and temporal PFAS assessment of aquatic systems. For this study, passive samplers were initially deployed in North Carolina along the Cape Fear River during the summer and fall of 2016 and 2017. These were originally intended for the detection of microcystins and monitoring potentially harmful algal blooms, though this period also coincided with occurrences of PFAS discharge from a local fluorochemical manufacturer into the river. Additional samplers were then deployed in 2022 to evaluate changes in PFAS fingerprint and abundances. Assessment of PFAS showed legacy compounds were observed across almost all sampling sites over all 3 years (PFHxS, PFOS, PFHxA, etc.), while emerging replacement PFAS (e.g., Nafion byproducts) were predominantly localized downstream from the manufacturer. Furthermore, samplers deployed downstream from the manufacturer in 2022 noted sharp decreases in observed signal for replacement PFAS in comparison to samplers deployed in 2016 and 2017, indicating mitigation and remediation efforts in the area were able to reduce localized fluorochemical contamination.

Zero-profile implant system versus novel plate systems after ACDF for comparison of sagittal balance parameters and clinical efficacy analysis.

BACKGROUND: The zero-profile implant system (Zero-P) and conventional plates have been widely used in anterior cervical discectomy and fusion (ACDF) to treat cervical spondylosis. The purpose of this study was to compare the effects of the application of Zero-P and new conventional plates (ZEVO, Skyline) in ACDF on the sagittal imaging parameters of cervical spondylosis patients and to analyze their clinical efficacy. METHODS: We conducted a retrospective study on 119 cervical spondylosis patients from January 2018 to December 2021, comparing outcomes between those receiving the Zero-P device (n = 63) and those receiving a novel conventional plate (n = 56, including 46 ZEVO and 10 Skyline plates) through ACDF. Cervical sagittal alignment was assessed pre- and postoperatively via lateral radiographs. The Japanese Orthopedic Association (JOA), Neck Disability Index (NDI), and visual analog scale (VAS) scores were recorded at baseline, after surgery, and at the 2-year follow-up to evaluate patient recovery and intervention success. RESULTS: There were significant differences in the postoperative C0-C2 Cobb angle and postoperative sagittal segmental angle (SSA) between patients in the novel conventional plate group and those in the Zero-P group (P < 0.05). Postoperatively, there were significant changes in the C2‒C7 Cobb angle, C0‒C2 Cobb angle, SSA, and average surgical disc height (ASDH) compared to the preoperative values in both patient groups (P < 0.05). Dysphagia in the immediate postoperative period was lower in the Zero-P group than in the new conventional plate group (0% in the Zero-P group, 7.14% in the novel conventional plate group, P = 0.046), and the symptoms disappeared within 2 years in both groups. There was no statistically significant difference between the two groups in terms of complications of adjacent spondylolisthesis (ASD) at 2 years postoperatively (3.17% in the Zero-P group, 8.93% in the novel conventional plate group; P = 0.252). According to the subgroup analysis, there were significant differences in the postoperative C2‒C7 Cobb angle, C0‒C2 Cobb angle, T1 slope, and ASDH between the ZEVO group and the Skyline group (P < 0.05). Compared with the preoperative scores, the JOA, NDI, and VAS scores of all groups significantly improved at the 2-year follow-up (P < 0.01). According to the subgroup analysis, the immediate postoperative NDI and VAS scores of the ZEVO group were significantly better than those of the Skyline group (P < 0.05). CONCLUSION: In ACDF, both novel conventional plates and Zero-P can improve sagittal parameters and related scale scores. Compared to the Zero-P plate, the novel conventional plate has a greater advantage in correcting the curvature of the surgical segment, but the Zero-P plate is less likely to produce postoperative dysphagia.

Minimal impact of beam projection angle deviations in skyline (Laurin) view and the efficacy of the anterior border of proximal tibia as a guiding landmark.

OBJECTIVE: Obtaining an optimal knee skyline view is challenging due to inaccuracies in beam projection angles (BPAs) and soft tissue obscuring bony landmarks. This study aimed to assess the impact of BPA deviations on patellofemoral index measurements and assessed the anterior border of the proximal tibia as an anatomic landmark for guiding BPAs. MATERIALS AND METHODS: This retrospective study consisted of three parts. The first was a simulation study using 52 CT scans of knees with a 20° flexion contracture to replicate the skyline (Laurin) view. Digitally reconstructed radiographs simulated neutral, 5° downward, and 5° upward tilt BPAs. Five patellofemoral indices (sulcus angle, congruence angle, patellar tilt angle, lateral facet angle, and bisect ratio) were measured and compared. The second part was a proof of concept study on 162 knees to examine patellar indices differences across these BPAs. Lastly, the alignment of the anterior border of the proximal tibia with the BPA tangential to the patellar articular surface was tested from the CT scans. RESULTS: No significant differences in patellofemoral indices were found across various BPAs in both the simulation and proof of concept studies (all p > 0.05). The angle between the anterior border of the proximal tibia and the patellar articular surface was 1.5 ± 5.3°, a statistically significant (p = 0.037) yet clinically acceptable deviation. CONCLUSION: Patellofemoral indices in skyline view remained consistent regardless of BPA deviations. The anterior border of the proximal tibia proved to be an effective landmark for accurate beam projection.

Jump dispersal drives the relationship between micro- and macroevolutionary dynamics in the Sicydiinae (Gobiiformes: Oxudercidae) of Sundaland and Wallacea.

Insular biodiversity hotspots of Southeast Asia are remarkable for their biodiverse faunas. With a marine larval phase lasting up to several months, the freshwater fish subfamily Sicydiinae has colonized most islands of these hotspots. However, Sicydiinae diversity is still poorly understood in Southeast Asia. With the objective to estimate intraspecific genetic diversity and infer past demography, we conducted the molecular inventory of Sicydiinae species in Sundaland and Wallacea using 652 bp of the mitochondrial cytochrome oxidase I gene, species delimitation methods and Bayesian Skyline plot reconstructions. In total, 24 Molecular Operational Taxonomic Units are delimited among the 603 sequences belonging to 27 species and five genera. Two cases of discordance between morphology and mitochondrial sequence are observed suggesting ongoing speciation and/or introgression in two genera. Multiple new occurrences are reported, either for a single biodiversity hotspot or both, some of which corresponding to observations of a few individuals far from the range distribution of their conspecifics. Among the ten species or species group whose intraspecific diversity was examined, high levels of genetic diversity and past population expansion are revealed by Tajima's D tests and Bayesian Skyline Plot reconstructions. Together these results indicate that long-distance dispersal is common and suggest that most endemic species originated through founder events followed by population expansion. Patterns of sexual dimorphism and males' coloration among diverging species pair seem to point to sexual selection as an important mechanism contributing to speciation in the Sicydiinae of Sundaland and Wallacea.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

A P2P multi-path routing algorithm based on Skyline operator for data aggregation in IoMT environments.

The integration of Internet of Things (IoT) technologies, particularly the Internet of Medical Things (IoMT), with wireless sensor networks (WSNs) has revolutionized the healthcare industry. However, despite the undeniable benefits of WSNs, their limited communication capabilities and network congestion have emerged as critical challenges in the context of healthcare applications. This research addresses these challenges through a dynamic and on-demand route-finding protocol called P2P-IoMT, based on LOADng for point-to-point routing in IoMT. To reduce congestion, dynamic composite routing metrics allow nodes to select the optimal parent based on the application requirements during the routing discovery phase. Nodes running the proposed routing protocol use the multi-criteria decision-making Skyline technique for parent selection. Experimental evaluation results show that P2P-IoMT protocol outperforms its best rivals in the literature in terms of residual network energy and packet delivery ratio. The network lifetime is extended by 4% while achieving a comparable packet delivery ratio and communication delay compared to LRRE. These performances are offered on top of the dynamic path selection and configurable route metrics capabilities of P2P-IoMT.

Skyline view of the patella does not increase fracture detection: A non-inferiority diagnostic study.

PURPOSE: Patella fractures are frequent injuries in the adult population. Initial study is made by plain radiographs and the standard set includes the skyline view of patella. Recommendation for use of this projection is variable among the experts, without data that support its performance in the diagnosis of patella fractures. The main purpose of this study was to determine the sensitivity of the antero-posterior and lateral view of the knee, without skyline view, in the diagnosis of patella fracture. METHODS: A retrospective non-inferiority diagnostic study was designed with all the knee trauma adult patients of an Emergency Department in a single center in five years. A random sample of all the consecutive patella fracture cases were taken to elaborate the case group. The control group was matched by sex and age. Two blinded orthopedic surgeons reviewed the cases and control radiographs and defined the presence of fracture, with or without skyline view, with a wash-out time of three weeks between the two evaluations. Non-inferiority was defined a priori at 90% of minimum sensitivity. RESULTS: 140 patients were evaluated (70 cases and 70 controls). Sensitivity of the set without skyline view was 92.86% (CI 95% 0.90 - 0.96) and the standard set was 97.86% (CI 95% 0.96 - 0.99), without significant differences (p = 0.1) CONCLUSION: Sensitivity of the plain radiograph set without skyline view is non-inferior to the standard set. The routine use of skyline view when suspecting patella fractures is questionable.

Genetic signatures of lineage fusion closely resemble population decline.

Accurate interpretation of the genetic signatures of past demographic events is crucial for reconstructing evolutionary history. Lineage fusion (complete merging, resulting in a single panmictic population) is a special case of secondary contact that is seldom considered. Here, the circumstances under which lineage fusion can be distinguished from population size constancy, growth, bottleneck, and decline were investigated. Multi-locus haplotype data were simulated under models of lineage fusion with different divergence versus sampling lag times (D:L ratios). These pseudo-observed datasets also differed in their allocation of a fixed amount of sequencing resources (number of sampled alleles, haplotype length, number of loci). Distinguishability of lineage fusion versus each of 10 untrue non-fusion scenarios was quantified based on six summary statistics (neutrality tests). Some datasets were also analyzed using extended Bayesian skyline plots. Results showed that signatures of lineage fusion very closely resemble those of decline-high distinguishability was generally limited to the most favorable scenario (D:L = 9), using the most sensitive summary statistics (F S and Z nS), coupled with the optimal sequencing resource allocation (maximizing number of loci). Also, extended Bayesian skyline plots often erroneously inferred population decline. Awareness of the potential for lineage fusion to carry the hallmarks of population decline is critical.

Skyline Fossilized Birth-Death Model is Robust to Violations of Sampling Assumptions in Total-Evidence Dating.

Several total-evidence dating studies under the fossilized birth-death (FBD) model have produced very old age estimates, which are not supported by the fossil record. This phenomenon has been termed "deep root attraction (DRA)." For two specific data sets, involving divergence time estimation for the early radiations of ants, bees, and wasps (Hymenoptera) and of placental mammals (Eutheria), it has been shown that the DRA effect can be greatly reduced by accommodating the fact that extant species in these trees have been sampled to maximize diversity, so-called diversified sampling. Unfortunately, current methods to accommodate diversified sampling only consider the extreme case where it is possible to identify a cut-off time such that all splits occurring before this time are represented in the sampled tree but none of the younger splits. In reality, the sampling bias is rarely this extreme and may be difficult to model properly. Similar modeling challenges apply to the sampling of the fossil record. This raises the question of whether it is possible to find dating methods that are more robust to sampling biases. Here, we show that the skyline FBD (SFBD) process, where the diversification and fossil-sampling rates can vary over time in a piecewise fashion, provides age estimates that are more robust to inadequacies in the modeling of the sampling process and less sensitive to DRA effects. In the SFBD model we consider, rates in different time intervals are either considered to be independent and identically distributed or assumed to be autocorrelated following an Ornstein-Uhlenbeck (OU) process. Through simulations and reanalyses of Hymenoptera and Eutheria data, we show that both variants of the SFBD model unify age estimates under random and diversified sampling assumptions. The SFBD model can resolve DRA by absorbing the deviations from the sampling assumptions into the inferred dynamics of the diversification process over time. Although this means that the inferred diversification dynamics must be interpreted with caution, taking sampling biases into account, we conclude that the SFBD model represents the most robust approach currently available for addressing DRA in total-evidence dating.

Targeted proteomics data interpretation with DeepMRM.

Targeted proteomics is widely utilized in clinical proteomics; however, researchers often devote substantial time to manual data interpretation, which hinders the transferability, reproducibility, and scalability of this approach. We introduce DeepMRM, a software package based on deep learning algorithms for object detection developed to minimize manual intervention in targeted proteomics data analysis. DeepMRM was evaluated on internal and public datasets, demonstrating superior accuracy compared with the community standard tool Skyline. To promote widespread adoption, we have incorporated a stand-alone graphical user interface for DeepMRM and integrated its algorithm into the Skyline software package as an external tool.

A Comparative Analysis of Data Analysis Tools for Data-Independent Acquisition Mass Spectrometry.

Data-independent acquisition (DIA) mass spectrometry-based proteomics generates reproducible proteome data. The complex processing of the DIA data has led to the development of multiple data analysis tools. In this study, we assessed the performance of five tools (OpenSWATH, EncyclopeDIA, Skyline, DIA-NN, and Spectronaut) using six DIA datasets obtained from TripleTOF, Orbitrap, and TimsTOF Pro instruments. By comparing identification and quantification metrics and examining shared and unique cross-tool identifications, we evaluated both library-based and library-free approaches. Our findings indicate that library-free approaches outperformed library-based methods when the spectral library had limited comprehensiveness. However, our results also suggest that constructing a comprehensive library still offers benefits for most DIA analyses. This study provides comprehensive guidance for DIA data analysis tools, benefiting both experienced and novice users of DIA-mass spectrometry technology.

Eco-evolutionary factors that influence its demographic oscillations in Prochilodus costatus (Actinopterygii: Characiformes) populations evidenced through a genetic spatial-temporal evaluation.

The human activity impact on wild animal populations is indicated by eco-evolutionary and demographic processes, along with their survival and capacity to evolve; consequently, such data can contribute toward enhancing genetic-based conservation programs. In this context, knowledge on the life-history and the eco-evolutionary processes is required to understand extant patterns of population structure in Prochilodus costatus a Neotropical migratory fish that has been threatened due to loss and fragmentation of its natural habitat since 1960s promoted by the expansion of hydroelectric power plant construction programs. This study evaluated the eco-evolutionary parameters that cause oscillations in the demography and structure of P. costatus populations. An integrated approach was used, including temporal and spatial sampling, next-generation sequencing of eight microsatellite loci, multivariate genetic analysis, and demographic life-history reconstruction. The results provided evidence of the complex interplay of ecological-evolutionary and human-interference events on the life history of this species in the upper basin. In particular, spawning wave behavior might have ecological triggers resulting in an overlapping of distinct genetic generations, and arising distinct migratory and nonmigratory genetic patterns living in the same area. An abrupt decrease in the effective population size of the P. costatus populations in the recent past (1960-80) was likely driven by environment fragmentation promoted by the construction of the Três Marias hydropower dam. The low allelic diversity that resulted from this event is still detected today; thus, active stocking programs are not effective at expanding the genetic diversity of this species in the river basin. Finally, this study highlights the importance of using mixed methods to understand spatial and temporal variation in genetic structure for effective mitigation and conservation programs for threatened species that are directly affected by human actions.

Strategies for mapping protein hydrolysate profiles and pharmacokinetics based on non-targeted proteomics combining skyline-aided quantitative techniques.

Numerous works have been focused on the bioactivities of protein hydrolysates (PHs) and their application in food or drug formulations, but their composition and pharmacokinetics have never been addressed due to their complex constitutes, short half-life, extremely low concentrations and lack of authentic standards. The present study aims to develop systematic analytical strategy and technical platform with optimized sample preparation, separation and detection protocols for PHs. Lineal peptides (LPs), extraction of the spleen of healthy pigs or calves, were used as cases. First, solvents with polarity gradients were used to globally extract peptides of LP from biological matrix. Non-targeted proteomics based on a high-resolution MS system was used to establish a reliable qualitative analysis workflow for PHs. Based on the developed approach, 247 unique peptides were identified using NanoLC-Orbitrap-MS/MS, and then further verified on the MicroLC-Q-TOF/MS system. In the quantitative analysis workflow, Skyline software was used to predict and optimize the LC-MS/MS detection parameters of LPs followed by investigating the linearity and precision of the developed analytical assay. Note worthily, we innovatively prepared calibration curves by sequential dilution of LP solution to overcome the bottleneck of lacking authentic standards and complex PH composition. All the peptides exhibited good linearity and precision in biological matrix. The established qualitative and quantitative assays were successfully applied to study the distribution characteristics of LPs in mice, and would be conductive to systematically map the profile and pharmacokinetics of peptides in various PHs in vivo and in vitro.

In-Network Processing of Skyline Join Queries in Wireless Sensor Networks Using Synopses of Skyline Attribute Value Ranges.

We investigate the in-network processing of a skyline join query in wireless sensor networks (WSNs). While much research was conducted on processing skyline queries in WSNs, skyline join queries were dealt with only in traditional centralized or distributed database environments. However, such techniques cannot be applied to WSNs. Carrying out join filtering, as well as skyline filtering using them in WSNs, is infeasible due to limited memory in senor nodes and to excessive energy consumption in wireless communications. In this paper, we propose a protocol to process a skyline join query in WSNs energy efficiently with only a small amount of memory in each sensor node. It uses a synopsis of skyline attribute value ranges, which is a very compact data structure. The range synopsis is used both in the search of anchor points for skyline filtering and in 2-way semijoins for join filtering. We describe the structure of a range synopsis and present our protocol. To optimize our protocol, we solve some optimization problems. Through implementation and a set of detailed simulations, we show the effectiveness of our protocol. The range synopsis is confirmed to be compact enough for our protocol to work well with the limited memory and energy in each sensor node. For the correlated and random distributions, our protocol significantly outperforms other possible protocols, confirming the effectiveness of an in-network skyline as well as the join filtering capabilities of our protocol.

Genetic evidence for widespread population size expansion in North American boreal birds prior to the Last Glacial Maximum.

Pleistocene climate cycles are well documented to have shaped contemporary species distributions and genetic diversity. Northward range expansions in response to deglaciation following the Last Glacial Maximum (LGM; approximately 21 000 years ago) are surmised to have led to population size expansions in terrestrial taxa and changes in seasonal migratory behaviour. Recent findings, however, suggest that some northern temperate populations may have been more stable than expected through the LGM. We modelled the demographic history of 19 co-distributed boreal-breeding North American bird species from full mitochondrial gene sets and species-specific molecular rates. We used these demographic reconstructions to test how species with different migratory strategies were affected by glacial cycles. Our results suggest that effective population sizes increased in response to Pleistocene deglaciation earlier than the LGM, whereas genetic diversity was maintained throughout the LGM despite shifts in geographical range. We conclude that glacial cycles prior to the LGM have most strongly shaped contemporary genetic diversity in these species. We did not find a relationship between historic population dynamics and migratory strategy, contributing to growing evidence that major switches in migratory strategy during the LGM are unnecessary to explain contemporary migratory patterns.

TurnoveR: A Skyline External Tool for Analysis of Protein Turnover in Metabolic Labeling Studies.

In spite of its central role in biology and disease, protein turnover is a largely understudied aspect of most proteomic studies due to the complexity of computational workflows that analyze in vivo turnover rates. To address this need, we developed a new computational tool, TurnoveR, to accurately calculate protein turnover rates from mass spectrometric analysis of metabolic labeling experiments in Skyline, a free and open-source proteomics software platform. TurnoveR is a straightforward graphical interface that enables seamless integration of protein turnover analysis into a traditional proteomics workflow in Skyline, allowing users to take advantage of the advanced and flexible data visualization and curation features built into the software. The computational pipeline of TurnoveR performs critical steps to determine protein turnover rates, including isotopologue demultiplexing, precursor-pool correction, statistical analysis, and generation of data reports and visualizations. This workflow is compatible with many mass spectrometric platforms and recapitulates turnover rates and differential changes in turnover rates between treatment groups calculated in previous studies. We expect that the addition of TurnoveR to the widely used Skyline proteomics software will facilitate wider utilization of protein turnover analysis in highly relevant biological models, including aging, neurodegeneration, and skeletal muscle atrophy.

Distributed messaging and light streaming system for combating pandemics: A case study on spatial analysis of COVID-19 Geo-tagged Twitter dataset.

Real-time data processing and distributed messaging are problems that have been worked on for a long time. As the amount of spatial data being produced has increased, coupled with increasingly complex software solutions being developed, there is a need for platforms that address these needs. In this paper, we present a distributed and light streaming system for combating pandemics and give a case study on spatial analysis of the COVID-19 geo-tagged Twitter dataset. In this system, three of the major components are the translation of tweets matching with user-defined bounding boxes, name entity recognition in tweets, and skyline queries. Apache Pulsar addresses all these components in this paper. With the proposed system, end-users have the capability of getting COVID-19 related information within foreign regions, filtering/searching location, organization, person, and miscellaneous based tweets, and performing skyline based queries. The evaluation of the proposed system is done based on certain characteristics and performance metrics. The study differs greatly from other studies in terms of using distributed computing and big data technologies on spatial data to combat COVID-19. It is concluded that Pulsar is designed to handle large amounts of long-term on disk persistence.

Sensitive Detection of Pharmaceutical Drugs and Metabolites in Serum Using Data-Independent Acquisition Mass Spectrometry and Open-Access Data Acquisition Tools.

Data-independent acquisition (DIA) based strategies have been explored in recent years for improving quantitative analysis of metabolites. However, the data analysis is challenging for DIA methods as the resulting spectra are highly multiplexed. Thus, the DIA mode requires advanced software analysis to facilitate the data deconvolution process. We proposed a pipeline for quantitative profiling of pharmaceutical drugs and serum metabolites in DIA mode after comparing the results obtained from full-scan, Data-dependent acquisition (DDA) and DIA modes. using open-access software. Pharmaceutical drugs (10) were pooled in healthy human serum and analysed by LC-ESI-QTOF-MS. MS1 full-scan and Data-dependent (MS2) results were used for identification using MS-DIAL software while deconvolution of MS1/MS2 spectra in DIA mode was achieved by using Skyline software. The results of acquisition methods for quantitative analysis validated the remarkable analytical performance of the constructed workflow, proving it to be a sensitive and reproducible pipeline for biological complex fluids.

Application of Parallel Reaction Monitoring in 15N Labeled Samples for Quantification.

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope 15N to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, 15N labeling quantification has several challenges. Less identifications are often observed in the heavy-labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here, we demonstrate the application of parallel reaction monitoring (PRM) to 15N labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS.

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.