Background: Proteoforms are the critical functional species that drive biological processes. However, conventional proteogenomic (“shotgun”) analyses discard relevant proteoform-level information through bulk protein digestion. This study presents a proof-of-principle integrative Top-Down Proteomics workflow designed to identify and characterise proteoforms across the breadth of a proteome. Methods: This workflow combined two-dimensional electrophoresis, a custom grid-cutting lattice for reproducible gel sectioning, in-gel digestion, liquid chromatography–tandem mass spectrometry, and open-search algorithms. Gel-grid locations, isoelectric points, and molecular weights were integrated with mass-spectrometry results to further characterise identified proteoforms. Results: The approach enabled identification of a broad range of proteoforms and their inherent post-translational modifications, including phosphorylation, acetylation, methylation, and glycosylation. Notably, substantial numbers of proteoforms were identified in visually “blank regions” of replicate two-dimensional gels. This eliminated bias associated with assessing only visually detectable protein spots and substantially expanded proteome-level proteoform detection and characterisation. Conclusions: This integrative workflow provides a reproducible and comprehensive strategy for addressing the often-overlooked complexity of proteomes at the proteoform level. By extending analysis beyond visually detectable gel spots, it enables deeper characterisation of proteoform diversity and associated post-translational modifications.
Drought and heat stresses are among the most important environmental constraints limiting maize (Zea mays L.) productivity worldwide. Over the past two decades, advances in mass spectrometry-based proteomics have generated extensive datasets describing proteins with altered abundance in maize tissues exposed to water deficit and elevated temperatures. These studies have identified numerous stress-responsive proteins involved in photosynthesis, energy metabolism, antioxidant defense, proteostasis, signaling pathways, and cellular homeostasis. In parallel, phosphoproteomic investigations have revealed extensive remodeling of phosphorylation networks that regulate stress perception, signal transduction, and adaptive responses. However, most available studies remain focused on protein-level observations and provide limited insight into the molecular diversity underlying stress adaptation. This review synthesizes current knowledge on the proteomic and phosphoproteomic responding of maize to drought and heat stresses through the emerging perspective of proteoform biology. We discuss how phosphorylation, oxidative modifications, proteolytic processing, alternative splicing, and genetic variation contribute to proteoform generation and expand the functional complexity of the maize stress proteome. Particular emphasis is placed on the integration of quantitative proteomics, phosphoproteomics, and proteogenomics as complementary approaches for characterizing stress-responsive molecular networks. We further evaluate current methodological limitations, including the predominance of bottom-up workflows, the underrepresentation of combined-stress studies and reproductive tissues, and the limited application of proteoform-resolved analytical strategies. We propose that future advances in maize stress biology will require a transition from protein-centered analyses toward proteoform-centered investigations capable of resolving functionally distinct molecular species. The integration of top-down proteomics, phosphoproteomics, proteogenomics, and systems-level approaches is expected to provide a more comprehensive understanding of stress adaptation mechanisms and facilitate the identification of molecular determinants of climate resilience. Such efforts may ultimately support the development of maize cultivars better adapted to increasingly challenging environmental conditions.
Periodontal regeneration requires the reconstruction of root cementum, periodontal ligament, and alveolar bone, which are frequently disrupted by periodontitis, trauma, and other inflammatory conditions. Although periodontal ligament stem cells (PDLSCs) have been considered promising for periodontal tissue engineering because of their multipotency, self-renewal capacity, and immunomodulatory activity, direct cell-based therapy still faces various limitations, including donor variability, culture-related changes, delivery difficulties, and safety concerns. Exosomes and small extracellular vesicles derived from PDLSCs have therefore attracted attention as potential cell-free mediators of PDLSC paracrine activity. PDLSC-derived exosomes carry bioactive molecules, including proteins, lipids, messenger RNAs, and microRNAs, and can influence recipient cell behavior in periodontal and bone regenerative environments. In vitro and preclinical animal studies indicate that PDLSC-derived exosomes are involved in osteogenesis, periodontal attachment repair, cementogenesis, angiogenesis, mechanotransduction, cell proliferation/survival, and immune regulation. Their biological effects are closely related to the condition of their parental PDLSCs, their cargo, and the exosome isolation and characterization methods used. In this review, we summarize recent findings on the isolation strategies used, biological functions of, and therapeutic potential of PDLSC-derived exosomes, with particular focus on protein cargo, proteomic signatures, proteoform-related interpretation, and microRNA-mediated regulation. We also discuss current challenges in exosome isolation, characterization, delivery, stability, and reporting standards for future periodontal regenerative applications. The findings discussed throughout are preclinical, and their translation to clinical settings remains to be explored.
Background: Chronic stress contributes to the development of cardiometabolic, malignant, and other chronic diseases through cumulative multisystem physiological dysregulation, conceptualized as allostatic load (AL). However, traditional AL relies on heterogeneous clinical biomarkers that are frequently unavailable or inconsistently collected in large cohorts, limiting reproducibility and translational utility. We developed and validated ProAL50, a proteomics-based measure of AL derived from 50 circulating proteins. Methods: Using high-dimensional plasma proteomic data from the UK Biobank, we constructed ProAL50 via penalized regression and stability selection and externally validated its construct in the Coronary Artery Risk Development in Young Adults (CARDIA) Study. Results: Among 37,089 UK Biobank participants, ProAL50 correlated strongly with traditional AL in the held-out test set (r = 0.72) and showed moderate external construct validity in CARDIA (r = 0.62). ProAL50 closely mirrored traditional AL in its associations with sociodemographic characteristics, lifestyle behaviors, physical and mental health, inflammation, and biological aging, supporting construct validity. In prospective analyses, ProAL50 showed associations with incident chronic disease, including type 2 diabetes, ischemic heart disease, chronic lung disease, chronic liver disease, chronic kidney disease, overall cancer, and all-cause and cause-specific mortality that were broadly comparable to traditional AL. Representative associations included incident type 2 diabetes (HR per SD = 2.72, 95% CI 2.61–2.84) and chronic liver disease (HR = 2.12, 95% CI 1.96–2.29). Conclusions: These findings support ProAL50 as a biologically informative, protein-based surrogate of AL for research use that complements traditional AL, pending further external validation and assay standardization.
Background: Koalas are a vulnerable marsupial species with unique reproductive traits. Efforts to develop assisted breeding technologies have been hindered by a limited understanding of maternal recognition of pregnancy and physiological changes induced by the foeto-placental unit. Differences in the reproductive physiology of pregnant and non-pregnant koalas were examined to investigate the possibility of maternal recognition and identify potential pregnancy and/or embryonic loss biomarkers. Methods: Koalas were separated into three groups: pregnant (n = 4 cycles from three females), mated but non-parturient (n = 4 cycles from three females), and gonadotropin-releasing hormone (GnRH) agonist-treated females (n = 7). Plasma was collected on day of mating/GnRH injection (D0) and on multiple subsequent days. Progesterone concentrations were measured by enzyme immunoassay, and plasma proteomes were analysed using filter-aided sample preparation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), employing sequential window acquisition of all theoretical fragment ion spectra. Results: Ovulation induction mechanisms influenced peri-ovulatory progesterone secretion (pregnant 40.7 ± 3.3 ng/mL vs. GnRH-treated 15.7 ± 1.8 ng/mL), with no significant differences in progesterone that occurred later in the luteal phase. LC-MS/MS identified 158 proteins, representing the first koala plasma proteome. Leucine-rich alpha-2-glycoprotein (LRG1) was significantly elevated at D2 in pregnant females compared to GnRH-treated females, and pregnant D9 and D19. In pregnant females, fibronectin (FN1) was significantly more abundant at D19 compared to D9 but not significantly different between treatments. Conclusions: These preliminary findings provide foundational data for further investigation into maternal recognition and pregnancy/embryonic loss in koalas.
