Sex-partitioning of the <i>Plasmodium falciparum</i> stage V gametocyte proteome provides insight into <i>falciparum</i>-specific cell biology

One of the critical gaps in malaria transmission biology and surveillance is our lack of knowledge about Plasmodium falciparum gametocyte biology, especially sexual dimorphic development and how sex ratios that may influence transmission from the human to the mosquito. Dissecting this process has been hampered by the lack of sex-specific protein markers for the circulating, mature stage V gametocytes. The current evidence suggests a high degree of conservation in gametocyte gene complement across Plasmodium, and therefore presumably for sex-specific genes as well. To better our understanding of gametocyte development and subsequent infectiousness to mosquitoes, we undertook a Systematic Subtractive Bioinformatic analysis (filtering) approach to identify sex-specific P. falciparum NF54 protein markers based on a comparison with the Dd2 strain, which is defective in producing males, and with syntenic male and female proteins from the reanalyzed and updated P. berghei (related rodent malaria parasite) gametocyte proteomes. This produced a short list of 174 male- and 258 female-enriched P. falciparum stage V proteins, some of which appear to be under strong diversifying selection, suggesting ongoing adaptation to mosquito vector species. We generated antibodies against three putative female-specific gametocyte stage V proteins in P. falciparum and confirmed either conserved sex-specificity or the lack of cross-species sex-partitioning. Finally, our study provides not only an additional resource for mass spectrometry-derived evidence for gametocyte proteins but also lays down the foundation for rational screening and development of novel sex-partitioned protein biomarkers and transmission-blocking vaccine candidates

Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

Computational prediction and in vivo protein coupling experiments identify candidate plant G-protein coupled receptors in Arabidopsis, rice and poplar

Sampling and Analytical Strategies for Biomarker Discovery Using Mass Spectrometry

There is an often unspoken truth behind the course of scientific investigation that involves not what is necessarily academically worthy of study, but rather what is scientifically worthy in the eyes of funding agencies. The perception of worthy research is, as cost is driven in the simplest sense in economics, often driven by demand. Presently, the demand for novel diagnostic and therapeutic protein biomarkers that possess high sensitivity and specificity is placing major impact on the field of proteomics. The focal discovery technology that is being relied on is mass spectrometry (MS), whereas the challenge of biomarker discovery often lies not in the application of MS but in the underlying proteome sampling and bioinformatic processing strategies. Although biomarker discovery research has been historically technology-driven, it is clear from the meager success in generating validated biomarkers that increasing attention must be placed at the pre-analytic stage, such as sample retrieval and preparation. As diseases vary, so do the combinations of sampling and sample analyses necessary to discover novel biomarkers. In this review, we highlight different strategies used toward biomarker discovery and discuss them in terms of their reliance on technology and methodology

Deep Proteomics of Mouse Skeletal Muscle Enables Quantitation of Protein Isoforms, Metabolic Pathways, and Transcription Factors

Skeletal muscle constitutes 40% of individual body mass and plays vital roles in locomotion and whole-body metabolism. Proteomics of skeletal muscle is challenging because of highly abundant contractile proteins that interfere with detection of regulatory proteins. Using a state-of-the art MS workflow and a strategy to map identifications from the C2C12 cell line model to tissues, we identified a total of 10,218 proteins, including skeletal muscle specific transcription factors like myod1 and myogenin and circadian clock proteins. We obtain absolute abundances for proteins expressed in a muscle cell line and skeletal muscle, which should serve as a valuable resource. Quantitation of protein isoforms of glucose uptake signaling pathways and in glucose and lipid metabolic pathways provides a detailed metabolic map of the cell line compared with tissue. This revealed unexpectedly complex regulation of AMP-activated protein kinase and insulin signaling in muscle tissue at the level of enzyme isoforms

Histological and top-down proteomic analyses of the visual pathway in the cuprizone demyelination model

A change in visual perception is a frequent early symptom of multiple sclerosis (MS), the pathoaetiology of which remains unclear. Following a slow demyelination process caused by 12 weeks of low-dose (0.1%) cuprizone (CPZ) consumption, histology and proteomics were used to investigate components of the visual pathway in young adult mice. Histological investigation did not identify demyelination or gliosis in the optic tracts, pretectal nuclei, superior colliculi, lateral geniculate nuclei or visual cortices. However, top-down proteomic assessment of the optic nerve/tract revealed a significant change in the abundance of 34 spots in high-resolution two-dimensional (2D) gels. Subsequent liquid chromatography-tandem mass spectrometry (LC-TMS) analysis identified alterations in 75 proteoforms. Literature mining revealed the relevance of these proteoforms in terms of proteins previously implicated in animal models, eye diseases and human MS. Importantly, 24 proteoforms were not previously described in any animal models of MS, eye diseases or MS itself. Bioinformatic analysis indicated involvement of these proteoforms in cytoskeleton organization, metabolic dysregulation, protein aggregation and axonal support. Collectively, these results indicate that continuous CPZ-feeding, which evokes a slow demyelination, results in proteomic changes that precede any clear histological changes in the visual pathway and that these proteoforms may be potential early markers of degenerative demyelinating conditions

Tackling the biological meaning of the human olfactory bulb dyshomeostatic proteome across neurological disorders: an integrative bioinformatic approach

Olfactory dysfunction is considered an early prodromal marker of many neurodegenerative diseases. Neuropathological changes and aberrant protein aggregates occur in the olfactory bulb (OB), triggering a tangled cascade of molecular events that is not completely understood across neurological disorders. This study aims to analyze commonalities and differences in the olfactory protein homeostasis across neurological backgrounds with different spectrums of smell dysfunction. For that, an integrative analysis was performed using OB proteomics datasets derived from subjects with Alzheimer’s disease (AD), Parkinson´s disease (PD), mixed dementia (mixD), dementia with Lewy bodies (DLB), frontotemporal lobar degeneration (FTLD-TDP43), progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) with respect to OB proteome data from neurologically intact controls. A total of 80% of the differential expressed protein products were potentially disease-specific whereas the remaining 20% were commonly altered across two, three or four neurological phenotypes. A multi-level bioinformatic characterization revealed a subset of potential disease-specific transcription factors responsible for the downstream effects detected at the proteome level as well as specific densely connected protein complexes targeted by several neurological phenotypes. Interestingly, common or unique pathways and biofunctions were also identified, providing novel mechanistic clues about each neurological disease at olfactory level. The analysis of olfactory epithelium, olfactory tract and primary olfactory cortical proteotypes in a multi-disease format will functionally complement the OB dyshomeostasis, increasing our knowledge about the neurodegenerative process across the olfactory axis.This work was funded by grants from the Spanish Ministry of Science, Innovation and Universities (Ref. PID2019-110356RB-I00/ AEI / 10.13039/501100011033 to JF-I and ES) and the Department of Economic and Business Development of the Government of Navarra (Ref. 0011-14112020-000028 to ES).The Proteomics Platform of Navarrabiomed, member of Proteored (PRB3ISCIII), was supported by grant PT17/0019/009, of the PE I+D+I 2013-2016 funded by ISCIII and FEDER to JF. The Clinical Neuroproteomics Unit of Navarrabiomed is member of the Spanish Olfactory Network (ROE) (supported by grant RED2018-102662-T funded by Spanish Ministry of Science and Innovation) and the Global Consortium for Chemosensory Research (GCCR)

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools