Journal of Proteome Research

The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has impacted public health globally. As the glycosylation of viral envelope glycoproteins is strongly associated with their immunogenicity, intensive studies have been conducted on the glycans of the glycoprotein of SARS-CoV-2, the spike (S) protein. Here, we conducted intensive glycoproteomic analyses of the SARS-CoV-2 S protein of ancestral and γ-variant strains using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarrays (LMA)...

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins...

We studied the effect of the column temperature on the selectivity of reversed-phase peptide separation in bottom-up proteomics. The number of peptide identifications from 2 h liquid chromatography with tandem mass spectrometry (LC-MS/MS) acquisitions reaches a plateau at 45-55 °C, driven simultaneously by improved separation efficiency, a gradual decrease in peptide retention, and possible on-column degradation of peptides at elevated temperatures. Performing 2D LC-MS/MS acquisitions at 25, 35, 45, and 55 °C resulted in the identification of ∼100,000 and ∼120,000 unique peptides for nonmodified and tandem mass tags (TMT)-labeled samples, respectively...

RNA-protein interactions within cellular signaling pathways have significant modulatory effects on RNA binding proteins' (RBPs') effector functions. During the innate immune response, specific RNA-protein interactions have been reported as a regulatory layer of post-transcriptional control. We investigated changes in the RNA-bound proteome of immortalized mouse macrophages (IMM) following treatment with lipopolysaccharide (LPS). Stable isotope labeling by amino acids in cell culture (SILAC) of cells followed by unbiased purification of RNP complexes at two time points after LPS stimulation and bottom-up proteomic analysis by LC-MS/MS resulted in a set of significantly affected RBPs...

Metabolomics is an emerging and powerful bioanalytical method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance, as platelet counts and function may vary substantially in individuals. A multiomics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well ( n = 461, R 2 = 0...

Delayed diagnosis of patients with sepsis or septic shock is associated with increased mortality and morbidity. UPLC-MS and NMR spectroscopy were used to measure panels of lipoproteins, lipids, biogenic amines, amino acids, and tryptophan pathway metabolites in blood plasma samples collected from 152 patients within 48 h of admission into the Intensive Care Unit (ICU) where 62 patients had no sepsis, 71 patients had sepsis, and 19 patients had septic shock. Patients with sepsis or septic shock had higher concentrations of neopterin and lower levels of HDL cholesterol and phospholipid particles in comparison to nonsepsis patients...

Disruption of the glycosylation machinery is a common feature in many types of cancer, and colorectal cancer (CRC) is no exception. Core fucosylation is mediated by the enzyme fucosyltransferase 8 (FucT-8), which catalyzes the addition of α1,6-l-fucose to the innermost GlcNAc residue of N -glycans. We and others have documented the involvement of FucT-8 and core-fucosylated proteins in CRC progression, in which we addressed core fucosylation in the syngeneic CRC model formed by SW480 and SW620 tumor cell lines from the perspective of alterations in their N -glycosylation profile and protein expression as an effect of the knockdown of the FUT8 gene that encodes FucT-8...

Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized cofractionation mass spectrometry (CF-MS) to map protein complexes within the postmortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions and then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates...

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion...

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Our prior investigations have evidenced that bone marrow mesenchymal stem cell (BMSC) therapy can significantly improve the outcomes of rheumatoid arthritis (RA). This study aims to conduct a comprehensive analysis of the proteomics between BMSCs and BMSCs-Exos, and to further elucidate the potential therapeutic effect of BMSCs-Exos on RA, so as to establish a theoretical framework for the prevention and therapy of BMSCs-Exos on RA. The 4D label-free LC-MS/MS technique was used for comparative proteomic analysis of BMSCs and BMSCs-Exos...

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts...

Colitis has a multifactorial pathogenesis with a strong cross-talk among microbiota, hypoxia, and tissue metabolism. Here, we aimed to characterize the molecular signature of the disease in symptomatic and presymptomatic stages of the inflammatory process at the tissue and fecal level. The study is based on two different murine models for colitis, and HR-MAS NMR on "intact" colon tissues and LC-MS/MS on colon tissue extracts were used to derive untargeted metabolomics and proteomics information, respectively...

We introduce single cell Proteoform imaging Mass Spectrometry ( scPiMS ), which realizes the benefit of direct solvent extraction and MS detection of intact proteins from single cells dropcast onto glass slides. Sampling and detection of whole proteoforms by individual ion mass spectrometry enable a scalable approach to single cell proteomics. This new scPiMS platform addresses the throughput bottleneck in single cell proteomics and boosts the cell processing rate by several fold while accessing protein composition with higher coverage...

Rescoring of peptide-spectrum matches (PSMs) has emerged as a standard procedure for the analysis of tandem mass spectrometry data. This emphasizes the need for software maintenance and continuous improvement for such algorithms. We introduce MS2 Rescore 3.0, a versatile, modular, and user-friendly platform designed to increase peptide identifications. Researchers can install MS2 Rescore across various platforms with minimal effort and benefit from a graphical user interface, a modular Python API, and extensive documentation...

MDR3 (multidrug resistance 3) deficiency in humans (MDR2 in mice) causes progressive familial intrahepatic cholestasis type 3 (PFIC3). PFIC3 is a lethal disease characterized by an early onset of intrahepatic cholestasis progressing to liver cirrhosis, a preneoplastic condition, putting individuals at risk of hepatocellular carcinoma (HCC). Hepatocyte-like organoids from MDR2-deficient mice (MDR2KO) were used in this work to study the molecular alterations caused by the deficiency of this transporter. Proteomic analysis by mass spectrometry allowed characterization of 279 proteins that were differentially expressed in MDR2KO compared with wild-type organoids...

To ensure biological validity in metabolic phenotyping, findings must be replicated in independent sample sets. Targeted workflows have long been heralded as ideal platforms for such validation due to their robust quantitative capability. We evaluated the capability of liquid chromatography-mass spectrometry (LC-MS) assays targeting organic acids and bile acids to validate metabolic phenotypes of SARS-CoV-2 infection. Two independent sample sets were collected: (1) Australia: plasma, SARS-CoV-2 positive ( n = 20), noninfected healthy controls ( n = 22) and COVID-19 disease-like symptoms but negative for SARS-CoV-2 infection ( n = 22)...

Organisms respond to dietary and environmental challenges by altering the molecular composition of their glycerolipids and glycerophospholipids (GPLs), which may favorably adjust the physicochemical properties of lipid membranes. However, how lipidome changes affect the membrane proteome and, eventually, the physiology of specific organs is an open question. We addressed this issue in Drosophila melanogaster , which is not able to synthesize sterols and polyunsaturated fatty acids but can acquire them from food...

Breast cancer is the second leading cause of cancer-related death among women and a major source of brain metastases. Despite the increasing incidence of brain metastasis from breast cancer, the underlying mechanisms remain poorly understood. Altered glycosylation is known to play a role in various diseases including cancer metastasis. However, profiling studies of O -glycans and their isomers in breast cancer brain metastasis (BCBM) are scarce. This study analyzed the expression of O -glycans and their isomers in human breast cancer cell lines (MDA-MB-231, MDA-MB-361, HTB131, and HTB22), a brain cancer cell line (CRL-1620), and a brain metastatic breast cancer cell line (MDA-MB-231BR) using nanoLC-MS/MS, identifying 27 O -glycan compositions...

C18ORF25 was recently shown to be phosphorylated at S67 by AMP-activated protein kinase (AMPK) in the skeletal muscle, following acute exercise in humans. Phosphorylation was shown to improve the ex vivo skeletal muscle contractile function in mice, but our understanding of the molecular mechanisms is incomplete. Here, we profiled the interactome of C18ORF25 in mouse myotubes using affinity purification coupled to mass spectrometry. This analysis included an investigation of AMPK-dependent and S67-dependent protein/protein interactions...