Mass Profiling Research Articles

Spatial lipidomics reveals myelin defects and pro-tumor macrophage infiltration in MPNST adjacent nerves

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas arising from peripheral nerves, accounting for 3-5% of soft tissue sarcomas. MPNSTs often recur locally, leading to poor survival. Achieving tumor-free surgical margins is essential to prevent recurrence, but current methods for determining tumor margins are limited, highlighting the need for improved biomarkers. In this study we investigated the degree to which MPNST extends into nerves adjacent to tumors. Alterations to the lipidome of MPNST and adjacent peripheral nerves were assessed using spatial lipidomics. Tissue samples from 5 MPNST patients were analyzed, revealing alterations of the lipid profile extending into the peripheral nerves beyond what was expected based on macroscopic and histological observations. Integration of spatial lipidomics and high-resolution accurate mass profiling identified distinct lipid profiles associated with healthy nerves, connective tissue, and tumors. Notably, histologically normal nerves exhibited myelin degradation and infiltration of pro-tumoral M2 macrophages, particularly near the tumor. Furthermore, aberrant osmium staining patterns and loss of H3K27me3 staining in absence of atypia were observed in a case with tumor recurrence. This exploratory study thereby highlights the changes occurring in the nerves affected by MPNST beyond what is visible on H&E, and provides leads for further biomarker studies, including aberrant osmium staining, to assess resection margins in MPNST

TBL38 atypical homogalacturonan-acetylesterase activity and cell wall microdomain localization in Arabidopsis seed mucilage secretory cells

Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity invitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.

Abstract 5976: R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC)

Abstract Triple Negative Breast Cancer (TNBC) is a highly aggressive breast cancer subtype, disproportionately affecting young and African American populations. TNBC constitutes up to 15% of breast cancers that lack effective targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex, a pivotal chromatin remodeling entity, plays a crucial role in cancer development and progression. Specifically, the NuRD complex associates with SALL4 in cancer cells, contributing to the silencing of tumor-suppressor genes like PTEN. In this study, we discover R9, a novel small-molecule nucleotide analog, a first-in-class NuRD inhibitor. Utilizing computational and cellular methods, R9 demonstrated selective engagement with RBBP4, a key NuRD complex subunit. Cellular target engagement technology has been utilized to verify direct interaction of R9 with NuRD complex as well as to guide medicinal chemistry efforts. Mass spectrometry thermal proteome profiling (TPP) affirmed RBBP4 as the primary target of R9 with minimal off-target effects. In vitro studies reveal R9 displaying robust toxicity in TNBC cells, while sparing normal tissue derived cells. Encouragingly, R9 effectively impeded TNBC tumors growth in mice without inducing toxicity. Mechanistically, we demonstrate R9 induces DNA damage in sensitive TNBC cells, and the sensitivity correlates to DNA damage-vulnerable cancer cells, indicating DNA damage is a major effector of NuRD complex inhibition in TNBCs. We further demonstrate that R9 induces DNA damage through a c-MYC dependent manner. Ongoing studies aim to elucidate key transcription factors involved in NuRD complex inhibition and the role of MYC and DNA damage in response to NuRD complex inhibition. This research positions R9 as a promising candidate for targeted therapy in TNBC, offering potential insights into the intricate molecular pathways involved in TNBC tumor progression. Citation Format: Kun Zhang, Sheeba Jacob, Mikhail Dozmorov, Ivan Babic, Elmar Nurmemmedov, Anthony C. Faber. R9: A novel NuRD complex inhibitor for targeted therapy in triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5976.

Abstract 1988: Mass spectrometry profiling identifies ENO2 as a driver of lenvatinib resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the predominant type of primary liver cancer, and lenvatinib is a multi-kinase inhibitor approved by the FDA as a first-line drug for advanced HCC since 2018. Lenvatinib demonstrated antitumor activity with a higher objective response rate, longer progression-free survival, and time to progression in a non-inferiority trial compared with sorafenib. However, the clinical benefits of lenvatinib remain limited as acquired resistance to lenvatinib during HCC treatment is inevitable. This highlights the need to understand the molecular mechanisms of lenvatinib resistance and to explore new therapeutic approaches to overcome this resistance. Tumor lineage plasticity, a recognized hallmark of cancer, is a phenomenon in which tumor cells dedifferentiate into a more stemness state to attain cellular plasticity, enabling them to evade targeted therapeutic interventions. However, the mechanism by which cellular plasticity contributes to lenvatinib resistance remains elusive. In this study, we employed proteomic mass spectrometry to investigate altered proteins in two pairs of parental lenvatinib-sensitive and their corresponding trained lenvatinib-resistant HCC cell lines. This analysis revealed that upregulation of enolase 2 (ENO2), a glycolytic enzyme that catalyzes the dehydration and conversion of 2-phosphoglycerate to phosphoenolpyruvate, is a critical contributor. ENO2 upregulation is frequently observed in HCC and is strongly correlated with aggressive clinical features, including poor survival, advanced tumor stage, and poor differentiation status. We further showed that ENO2 upregulation in HCC is positively correlated with stemness and hepatic progenitor marker signatures, as well as hypoxia, and subsequently demonstrated that ENO2 is regulated by hypoxia/HIF-1α, with hypoxia response element sequences located in the ENO2 promoter region. Research is underway to investigate the functional role and underlying molecular mechanisms associated with ENO2-driven lenvatinib resistance and cellular plasticity in HCC. Citation Format: Sukbum Kim, Jia-Jian Loh, Huajian Yu, Ki-Fong Man, Kai-Yu Ng, Terence Kin-Wah Lee, Clive Yik-Sham Chung, Stephanie Ma. Mass spectrometry profiling identifies ENO2 as a driver of lenvatinib resistance in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1988.

Abstract 6055: COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation

Abstract Histone deacetylase 3 (HDAC3) is overexpressed in cancer cells and functions as transcription repression complex with nuclear receptor corepressor (NCOR) to inhibit gene transcription. The pan-HDAC inhibitors have been approved for T-cell lymphoma with evident side effects. The selective HDAC3 inhibitors needs to be developed. Proteolysis-targeting chimera (PROTAC) approach is taken to design HDAC3 degraders with unsolved bioavailability obstacle. We screened small molecules with HDAC3 degradation ability and found two small molecules derived from glycyrrhetinic acid, methyl 2-cyano-3-oxo-18β-olean-1,9(11), 12-trien-30-oate (COOTO, 10e) and methyl 2-cyano-3,12-dioxo-18β-olean-1,9(11)-dien-30-oate (CDODO-Me, 10d), selectively decreased HDAC3 protein with increased acetylation of Histone 3. We explored the mechanism of HDAC3 degradation and found that 10e interrupted the interaction of HDAC3 with NCOR and induced degradation of both HDAC3 and NCOR protein. Biotin labeled 10e shows binding to both HDAC3 and NCOR directly as well as to E3 ligases SIAH2 and ITCH. Mass spectrometry analysis profiling reveals covalent interactions of 10e with multiple cysteine sites of SIAH2 in the binding regions for NCOR and HDAC3. Silencing SIAH2 blocks degradation of HDAC3. N-Acetylcysteine and GSH block the binding of 10e to HDAC3, NCOR and SIAH2. In addition, 10e decreased the levels of c-FLIP protein and increased the levels of NOXA protein leading to apoptosis induction. These data suggest that 10e functions as a molecular glue to attract SIAH2 to HDAC3 and NCOR to cause degradation through covalent binding. Citation Format: Ping Gong, Min Huang, Xin Li, Haoling Wang, Samuel Waxman, Linxiang Zhao, Yongkui Jing. COOTO-Me (10e) acts as a molecular glue to target NCOR/HDAC3 degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6055.

Pectolytic enzyme treatment partially degrades an arabinogalactan protein–rhamnogalacturonan I–xyloglucan co-polymer in red wine as characterised using epitope mass profiling

Polysaccharides are important metabolites in red wine but are challenging to separate from yeast-derived mannoproteins, and costly to identify using sialylation and gas chromatography. Affinity and gel permeation chromatography was combined with immunodetection of various specific plant cell wall polysaccharide epitopes to understand this complex mixture of extracted cell wall polymers. A survey of Cabernet Sauvignon wines produced with and without pectolytic enzymes suggested that enzyme-treated wines have fewer polysaccharides in the 85–105 kDa and 1050–6000 kDa ranges. When yeast-derived mannoproteins are excluded (15.3% of total), pectolytic enzyme treatment was shown to alter the molecular weight wine polysaccharide composition between the 100–1000 kDa ranges. Furthermore, ELISA data suggested that many of the soluble polysaccharides in the 300–1000 kDa range are an arabinogalactan protein–rhamnogalacturonan I–xyloglucan co-polymer. This is the first report to use ELISA to identify changes in specific polysaccharide classes during enzyme preparation used in wine maceration.

Parasite and Pesticide Impacts on the Bumblebee (Bombus terrestris) Haemolymph Proteome

Pesticides pose a potential threat to bee health, especially in combination with other stressors, such as parasites. However, pesticide risk assessment tests pesticides in isolation from other stresses, i.e., on otherwise healthy bees. Through molecular analysis, the specific impacts of a pesticide or its interaction with another stressor can be elucidated. Molecular mass profiling by MALDI BeeTyping® was used on bee haemolymph to explore the signature of pesticidal and parasitic stressor impacts. This approach was complemented by bottom-up proteomics to investigate the modulation of the haemoproteome. We tested acute oral doses of three pesticides-glyphosate, Amistar and sulfoxaflor-on the bumblebee Bombus terrestris, alongside the gut parasite Crithidia bombi. We found no impact of any pesticide on parasite intensity and no impact of sulfoxaflor or glyphosate on survival or weight change. Amistar caused weight loss and 19-41% mortality. Haemoproteome analysis showed various protein dysregulations. The major pathways dysregulated were those involved in insect defences and immune responses, with Amistar having the strongest impact on these dysregulated pathways. Our results show that even when no response can be seen at a whole organism level, MALDI BeeTyping® can detect effects. Mass spectrometry analysis of bee haemolymph provides a pertinent tool to evaluate stressor impacts on bee health, even at the level of individuals.

Ephedra alata Subsp. Alenda as a Novel Source of Bioactive Phytochemicals: Characterization Based on the Mass Spectrometry and Profiling of Antioxidant and Anti-Inflammatory Properties.

The aim of the present study was to examine, for the first time, the phytochemical content of Ephedra alata pulp extract (EAP) and explore its antioxidant and anti-inflammatory capacities. High-performance liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometry (HPLC-ESI-QTOF/MS) was used for phytochemical analysis and three in vitro antioxidant assays together with three in vitro anti-inflammatory tests were used for the assessment of biological activity. The HPLC-ESI-QTOF/MS analysis revealed the presence of 42 metabolites, including flavonoids, sphingolipides, fatty acids, ephedrine derivatives, and amino acid derivatives. In vitro findings revealed that EAP has interesting 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide, and ferrous ion chelating capacities (IC50 values were 0.57 mg/mL, 0.55 mg/mL, and 0.51 mg/mL for DPPH, superoxide radical, and ferrous ion, respectively). Furthermore, EAP showed a noticeable anti-inflammatory ability by inhibiting the two cyclooxygenase isoforms, COX-1 and COX-2 (IC50 of 59.1 and 58.8 µg/mL for COX-1 and COX-2, respectively), preventing protein denaturation (IC50 = 0.51 mg/mL), and protecting membrane stabilization (IC50 = 0.53 mg/mL). The results highlighted the use of Ephedra alata pulp as a potential source of natural compounds with therapeutic effects for the management of inflammatory disorders.

Genetic mapping of maize metabolites using high-throughput mass profiling

Plant metabolites are the basis of human nutrition and have biological relevance in ecology. Farmers selected plants with favorable characteristics since prehistoric times and improved the cultivars, but without knowledge of underlying mechanisms. Understanding the genetic basis of metabolite production can facilitate the successful breeding of plants with augmented nutritional value. To identify genetic factors related to the metabolic composition in maize, we generated mass profiles of 198 recombinant inbred lines (RILs) and their parents (B73 and Mo17) using direct-injection electrospray ionization mass spectrometry (DLI-ESI MS). Mass profiling allowed the correct clustering of samples according to genotype. We quantified 71 mass features from grains and 236 mass features from leaf extracts. For the corresponding ions, we identified tissue-specific metabolic ‘Quantitative Trait Loci’ (mQTLs) distributed across the maize genome. These genetic regions could regulate multiple metabolite biosynthesis pathways. Our findings demonstrate that DLI-ESI MS has sufficient analytical resolution to map mQTLs. These identified genetic loci will be helpful in metabolite-focused maize breeding. Mass profiling is a powerful tool for detecting mQTLs in maize and enables the high-throughput screening of loci responsible for metabolite biosynthesis.

Mass spectrometry profiling of single bacterial cells reveals metabolic regulation during antibiotics induced bacterial filamentation

Bacterial antimicrobial resistance (AMR) is a severe threat to global health and development. Under the stimulation of antibiotics, bacterial cells can undergo filamentation and generate daughter cells with stronger AMR. The current research on bacterial AMR mechanism is mainly conducted with a population of cells. However, bacterial cells exhibit heteroresistance, making the study at population level not reliable. Herein, we developed single bacterial cell metabolic profiling by mass spectrometry (MS) to study bacterial AMR at single-cell level. By utilizing a microprobe controlled by a microoperation platform, single filamentous extended spectrum beta-lactamase (ESBL) producing Escherichia coli (ESBL-E. coli) cells generated by ceftriaxone sodium stimulation can be extracted and spray-ionized for MS analysis. Heterogeneous among ESBL-E. coli cells under the same antibiotic stimulus condition was observed from mass spectra as well as cell morphology. The metabolic profiles by MS of different individual cells can be clustered into subgroups well in accordance with bacterial cell length. Metabolic pathways including arginine and proline metabolism, as well as cysteine and methionine metabolism were disclosed to play an important role in the bacterial SOS-associated filamentation against antibiotics. The microprobe electrospray ionization-MS-based single bacterial cell analysis method is promising in the study of various bacterial AMR mechanism and can reveal the heterogeneity of bacterial AMR from-cell-to-cell.

Charting the Proteoform Landscape of Serum Proteins in Individual Donors by High-Resolution Native Mass Spectrometry.

Most proteins in serum are glycosylated, with several annotated as biomarkers and thus diagnostically important and of interest for their role in disease. Most methods for analyzing serum glycoproteins employ either glycan release or glycopeptide centric mass spectrometry-based approaches, which provide excellent tools for analyzing known glycans but neglect previously undefined or unknown glycosylation and/or other co-occurring modifications. High-resolution native mass spectrometry is a relatively new technique for the analysis of intact glycoproteins, providing a “what you see is what you get” mass profile of a protein, allowing the qualitative and quantitative observation of all modifications present. So far, a disadvantage of this approach has been that it centers mostly on just one specific serum glycoprotein at the time. To address this issue, we introduce an ion-exchange chromatography-based fractionation method capable of isolating and analyzing, in parallel, over 20 serum (glyco)proteins, covering a mass range between 30 and 190 kDa, from 150 μL of serum. Although generating data in parallel for all these 20 proteins, we focus the discussion on the very complex proteoform profiles of four selected proteins, i.e., α-1-antitrypsin, ceruloplasmin, hemopexin, and complement protein C3. Our analyses provide an insight into the extensive proteoform landscape of serum proteins in individual donors, caused by the occurrence of various N- and O-glycans, protein cysteinylation, and co-occurring genetic variants. Moreover, native mass intact mass profiling also provided an edge over alternative approaches revealing the presence of apo- and holo-forms of ceruloplasmin and the endogenous proteolytic processing in plasma of among others complement protein C3. We also applied our approach to a small cohort of serum samples from healthy and diseased individuals. In these, we qualitatively and quantitatively monitored the changes in proteoform profiles of ceruloplasmin and revealed a substantial increase in fucosylation and glycan occupancy in patients with late-stage hepatocellular carcinoma and pancreatic cancer as compared to healthy donor samples.

Axions as a model of Dark Matter

The true nature of dark matter is an extremely important and fundamental problem in the study of astrophysics, particle physics, cosmology and many other areas within the study of physics. This paper presents experimental evidence for the existence of dark matter through discussing the experimental results of mass profiling a galaxy and gravitational lensing. The fundamental properties of dark matter are then discussed, and evidence for these properties is presented. This allows further discussion of one of the most promising models of dark matter - the axion. The purpose of this paper is to present the evidence for the axion model, describe the nature of the theoretical axion particle, and to highlight the effects this model would have on other theories in physics such as solving the Strong CP Problem in the theory of quantum chromodynamics.

Correlation of body composition and aerobic capacity with heart rate variability in Indian elite soccer players

Objective: This study aimed to investigate the relationship between Heart Rate Variability (HRV) indexes with body composition and aerobic capacity (VO2max) in Indian Elite soccer players. Methods: 115 male soccer players were recruited and all the subjects were measured for age: 16.8 ± 3.11; height: 167.85 ± 8.27; weight: 57.05 ± 10.66; BMI: 20.16 ± 3.02; body fat %, lean body mass and anthropometry profiling. The aerobic performance was measured using a 20-meter shuttle run test (20 MSRT) for VO2max. Heart Rate Variability (HRV) was recorded (RMSSD, pNN50%, LF, HF, LH/HF ratio) at a resting state (~5 minutes) using the Heartware Shimmer ECG device. The Pearson correlation coefficient for % BF and VO2max with heart rate variability was calculated. Result: The result revealed a highly significant negative correlation between VO2max with Body fat% (4-site skinfold) at (p<0.01; r = -.280), while Body fat %( 4-fold) showed strong significant positive correlation with BMI (p<0.01; r= .631). Resting heart rate showed strong significant negative correlation with SDNN and positive correlation with Body fat % (4-fold) with their r- values at -.318 and .258 respectively (p<0.01).

Mass Spectrometry-Based Proteome Profiling of Extracellular Vesicles Derived from the Cerebrospinal Fluid of Adult Rhesus Monkeys Exposed to Cocaine throughout Gestation.

Cocaine use disorder has been reported to cause transgenerational effects. However, due to the lack of standardized biomarkers, the effects of cocaine use during pregnancy on postnatal development and long-term neurobiological and behavioral outcomes have not been investigated thoroughly. Therefore, in this study, we examined extracellular vesicles (EVs) in adult (~12 years old) female and male rhesus monkeys prenatally exposed to cocaine (n = 11) and controls (n = 9). EVs were isolated from the cerebrospinal fluid (CSF) and characterized for the surface expression of specific tetraspanins, concentration (particles/mL), size distribution, and cargo proteins by mass spectrometry (MS). Transmission electron microscopy following immunogold labeling for tetraspanins (CD63, CD9, and CD81) confirmed the successful isolation of EVs. Nanoparticle tracking analyses showed that the majority of the particles were <200 nm in size, suggesting an enrichment for small EVs (sEV). Interestingly, the prenatally cocaine-exposed group showed ~54% less EV concentration in CSF compared to the control group. For each group, MS analyses identified a number of proteins loaded in CSF-EVs, many of which are commonly listed in the ExoCarta database. Ingenuity pathway analysis (IPA) demonstrated the association of cargo EV proteins with canonical pathways, diseases and disorders, upstream regulators, and top enriched network. Lastly, significantly altered proteins between groups were similarly characterized by IPA, suggesting that prenatal cocaine exposure could be potentially associated with long-term neuroinflammation and risk for neurodegenerative diseases. Overall, these results indicate that CSF-EVs could potentially serve as biomarkers to assess the transgenerational adverse effects due to prenatal cocaine exposure.

Magnetic bioassembly platforms towards the generation of extracellular vesicles from human salivary gland functional organoids for epithelial repair.

Salivary glands (SG) are exocrine organs with secretory units commonly injured by radiotherapy. Bio-engineered organoids and extracellular vesicles (EV) are currently under investigation as potential strategies for SG repair. Herein, three-dimensional (3D) cultures of SG functional organoids (SGo) and human dental pulp stem cells (hDPSC) were generated by magnetic 3D bioassembly (M3DB) platforms. Fibroblast growth factor 10 (FGF10) was used to enrich the SGo in secretory epithelial units. After 11 culture days via M3DB, SGo displayed SG-specific acinar epithelial units with functional properties upon neurostimulation. To consistently develop 3D hDPSC in vitro, 3 culture days were sufficient to maintain hDPSC undifferentiated genotype and phenotype for EV generation. EV isolation was performed via sequential centrifugation of the conditioned media of hDPSC and SGo cultures. EV were characterized by nanoparticle tracking analysis, electron microscopy and immunoblotting. EV were in the exosome range for hDPSC (diameter: 88.03 ± 15.60 nm) and for SGo (123.15 ± 63.06 nm). Upon ex vivo administration, exosomes derived from SGo significantly stimulated epithelial growth (up to 60%), mitosis, epithelial progenitors and neuronal growth in injured SG; however, such biological effects were less distinctive with the ones derived from hDPSC. Next, these exosome biological effects were investigated by proteomic arrays. Mass spectrometry profiling of SGo exosomes predicted that cellular growth, development and signaling was due to known and undocumented molecular targets downstream of FGF10. Semaphorins were identified as one of the novel targets requiring further investigations. Thus, M3DB platforms can generate exosomes with potential to ameliorate SG epithelial damage.

Salt-Mediated Organic Solvent Precipitation for Enhanced Recovery of Peptides Generated by Pepsin Digestion.

Conventional solvent-based precipitation makes it challenging to obtain a high recovery of low mass peptides. However, we previously demonstrated that the inclusion of salt ions, specifically ZnSO4, together with high concentrations of acetone, maximizes the recovery of peptides generated from trypsin digestion. We herein generalized this protocol to the rapid (5 min) precipitation of pepsin-digested peptides recovered from acidic matrices. The precipitation protocol extended to other organic solvents (acetonitrile), with high recovery from dilute peptide samples permitting preconcentration and purification. Mass spectrometry profiling of pepsin-generated peptides demonstrated that the protocol captured peptides as small as 800 u, although with a preferential bias towards recovering larger and more hydrophobic peptides. The precipitation protocol was applied to rapidly quench, concentrate, and purify pepsin-digested samples ahead of MS. Complex mixtures of yeast and plasma proteome extracts were successfully precipitated following digestion, with over 95% of MS-identified peptides observed in the pellet fraction. The full precipitation workflow—including the digestion step—can be completed in under 10 min, with direct MS analysis of the recovered peptide pellets showing exceptional protein sequence coverage.

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-μHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-μHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Mass spectrometry profiling and quantitation of changes in circulating hormones secreted over time in Cancer borealis hemolymph due to feeding behavior.

The crustacean stomatogastric ganglion (STG) is a valuable model for understanding circuit dynamics in neuroscience as it contains a small number of neurons, all easily distinguishable and most of which contribute to two complementary feeding-related neural circuits. These circuits are modulated by numerous neuropeptides, with many gaining access to the STG as hemolymph-transported hormones. Previous work characterized neuropeptides in the hemolymph of the crab Cancer borealis but was limited by low peptide abundance in the presence of a complex biological matrix and the propensity for rapid peptide degradation. To improve their detection, a data-independent acquisition (DIA) mass spectrometry (MS) method was implemented. This approach improved the number of neuropeptides detected by approximately twofold and showed greater reproducibility between experimental and biological replicates. This method was then used to profile neuropeptides at different stages of the feeding process, including hemolymph from crabs that were unfed, or 0min, 15min, 1h, and 2h post-feeding. The results show differences both in the presence and relative abundance of neuropeptides at the various time points. Additionally, 96 putative neuropeptide sequences were identified with de novo sequencing, indicating there may be more key modulators within this system than is currently known. These results suggest that a distinct cohort of neuropeptides provides modulation to the STG at different times in the feeding process, providing groundwork for targeted follow-up electrophysiological studies to better understand the functional role of circulating hormones in the neural basis of feeding behavior.

What's in a mass?

This short essay pretends to make the reader reflect on the concept of biological mass and on the added value that the determination of this molecular property of a protein brings to the interpretation of evolutionary and translational snake venomics research. Starting from the premise that the amino acid sequence is the most distinctive primary molecular characteristics of any protein, the thesis underlying the first part of this essay is that the isotopic distribution of a protein's molecular mass serves to unambiguously differentiate it from any other of an organism's proteome. In the second part of the essay, we discuss examples of collaborative projects among our laboratories, where mass profiling of snake venom PLA2 across conspecific populations played a key role revealing dispersal routes that determined the current phylogeographic pattern of the species.

The Xenopus spindle is as dense as the surrounding cytoplasm.

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle's material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle's mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle's emergent physical properties-essential to advance predictive frameworks of spindle assembly and function.