Affinity ligands such as antibodies are widely used in (bio)medical research for purifying proteins from complex biological samples. These ligands are generally immobilized onto solid supports which facilitate the separation of a captured protein from the sample matrix. Adsorptive microtiter plates are commonly used as solid supports prior to immunochemical detection (e.g., immunoassays) but hardly ever prior to liquid chromatography-mass spectrometry (LC-MS-)-based detection. Here, we describe the use of adsorptive microtiter plates for protein enrichment prior to LC-MS detection, and we discuss opportunities and challenges of corresponding workflows, based on examples of targeted (i.e., soluble receptor for advanced glycation end-products (sRAGE) in human serum) and discovery-based workflows (i.e., transcription factor p65 (NF-κB) in lysed murine RAW 264.7 macrophages and peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP5) in lysed human A549 alveolar basal epithelial cells). Thereby, we aim to highlight the potential usefulness of adsorptive microtiter plates in affinity purification workflows prior to LC-MS detection, which could increase their usage in mass spectrometry-based protein research.

Metastasis is a major obstacle in the therapeutic intervention of melanoma, and several GTP-binding proteins were found to play important roles in regulating cancer metastasis. To assess systematically the regulatory roles of these proteins in melanoma metastasis, we employed a targeted chemoproteomic method, which relies on the application of stable isotope-labeled desthiobiotin-GTP acyl phosphate probes in conjunction with scheduled multiple-reaction monitoring (MRM), for profiling quantitatively the GTP-binding proteins. Following probe labeling, tryptic digestion, and affinity pull-down of desthiobiotin-conjugated peptides, differences in expression levels of GTP-binding proteins in two matched pairs of primary/metastatic melanoma cell lines were measured using liquid chromatography-MRM analysis. We also showed that among the top upregulated proteins in metastatic melanoma cells, AK4 promotes the migration and invasion of melanoma cells; overexpression of AK4 in primary melanoma cells leads to augmented migration and invasion, and reciprocally, knockdown of AK4 in metastatic melanoma cells results in repressed invasiveness. In summary, we examined the relative expression levels of GTP-binding proteins in two pairs of primary/metastatic melanoma cell lines. Our results confirmed some previously reported regulators of melanoma metastasis and revealed a potential role of AK4 in promoting melanoma metastasis.

While substantial efforts have been made to optimize and standardize fecal metabolomics for studies in adults, the development of a standard protocol to analyze infant feces is, however, still lagging behind. Here, we present the development of a hands-on and robust protocol for proton 1H NMR spectroscopy of infant feces. The influence of extraction solvent, dilution ratio, homogenization method, filtration, and duration of centrifugation on the biochemical composition of infant feces was carefully evaluated using visual inspection of 1H NMR spectra in combination with multivariate statistical modeling. The optimal metabolomics protocol was subsequently applied on feces from seven infants collected at 8 weeks, 4, and 9 months of age. Interindividual variation was exceeding the variation induced by different fecal sample preparation methods, except for filtration. We recommend extracting fecal samples using water with a dilution ratio of 1:5 feces-to-water to homogenize using bead beating and to remove particulates using centrifugation. Samples collected from infants aged 8 weeks and 4 months showed elevated concentrations of milk oligosaccharide derivatives and lactic acid, whereas short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) were higher in the 9 month samples. The established protocol enables hands-on and robust analyses of the infant gut metabolome. The wide-ranging application of this protocol will facilitate interlaboratory comparison of infants' metabolic profiles and finally aid in a better understanding of infant gut health.

Gene products can affect the concentrations of small molecules (aka "metabolites"), and conversely, some metabolites can modulate the concentrations of gene transcripts. While many specific instances of this interplay have been revealed, a global approach to systematically uncover human gene-metabolite interactions is still lacking. We performed a metabolome- and transcriptome-wide association study to identify genes influencing the human metabolome using untargeted metabolome features, extracted from 1H nuclear magnetic resonance spectroscopy (NMR) of urine samples, and gene expression levels, quantified from RNA-Seq of lymphoblastoid cell lines (LCL) from 555 healthy individuals. We identified 20 study-wide significant associations corresponding to 15 genes, of which 5 associations (with 2 genes) were confirmed with follow-up NMR data. Using metabomatching, we identified the metabolites corresponding to metabolome features associated with the genes, namely, N-acetylated compounds with ALMS1 and trimethylamine (TMA) with HPS1. Finally, Mendelian randomization analysis supported a potential causal link between the expression of genes in both the ALMS1- and HPS1-loci and their associated metabolite concentrations. In the case of HPS1, we additionally observed that TMA concentration likely exhibits a reverse causal effect on HPS1 expression levels, indicating a negative feedback loop. Our study highlights how the integration of metabolomics, gene expression, and genetic data can pinpoint causal genes modulating metabolite concentrations.

DNA sequences of high guanine (G) content have the potential to form G quadruplex (G4) structures. A more complete understanding about the biological functions of G4 DNA requires the investigation about how these structures are recognized by proteins. Here, we conducted exhaustive quantitative proteomic experiments to profile the interaction proteomes of G4 structures by employing different sequences of G4 DNA derived from the human telomere and the promoters of c-MYC and c-KIT genes. Our results led to the identification of a number of candidate G4-interacting proteins, many of which were discovered here for the first time. These included three proteins that can bind to all three DNA G4 structures and 78 other proteins that can bind selectively to one or two of the three DNA G4 structure(s). We also validated that GRSF1 can bind directly and selectively toward G4 structure derived from the c-MYC promoter. Our quantitative proteomic screening also led to the identification of a number of candidate "antireader" proteins of G4 DNA. Together, we uncovered a number of cellular proteins that exhibit general and selective recognitions of G4 folding patterns, which underscore the complexity of G4 DNA in biology and the importance of understanding fully the G4-interaction proteome.

An important component of ionizing radiation (IR) exposure after a radiological incident may include low-dose rate (LDR) exposures either externally or internally, such as from 137Cs deposition. In this study, a novel irradiation system, VAriable Dose-rate External 137Cs irradiatoR (VADER), was used to expose male and female mice to a variable LDR irradiation over a 30 d time span to simulate fall-out-type exposures in addition to biofluid collection from a reference dose rate (0.8 Gy/min). Radiation markers were identified by untargeted metabolomics and random forests. Mice exposed to LDR exposures were successfully identified from control groups based on their urine and serum metabolite profiles. In addition to metabolites commonly perturbed after IR exposure, we identified and validated a novel metabolite (hexosamine-valine-isoleucine-OH) that increased up to 150-fold after LDR and 80-fold after conventional exposures in urine. A multiplex panel consisting of hexosamine-valine-isoleucine-OH with other urinary metabolites (N6,N6,N6-trimethyllysine, carnitine, 1-methylnicotinamide, and α-ketoglutaric acid) achieved robust classification performance using receiver operating characteristic curve analysis, irrespective of the dose rate or sex. These results show that in terms of biodosimetry, dysregulated energy metabolism is associated with IR exposure for both LDR and conventional IR exposures. These mass spectrometry data have been deposited to the NIH data repository via Metabolomics Workbench with study IDs ST001790, ST001791, ST001792, ST001793, and ST001806.

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.