Enhanced Pharmacokinetic Bioanalysis of Antibody-drug Conjugates using Hybrid Immunoaffinity Capture and Microflow LC-MS/MS.

The increasing complexity and diversity of antibody-drug conjugates (ADCs) have led to a need for comprehensive and informative bioanalytical methods to enhance pharmacokinetic (PK) understanding. This study aimed to evaluate the feasibility of a hybrid immunoaffinity (IA) capture microflow LC-MS/MS (μLC-MS/MS) method for ADC analysis, utilizing a minimal sample volume for PK assessments in a preclinical study. A robust workflow was established for the quantitative analysis of ADCs by the implementation of solid-phase extraction (SPE) and semi-automation in µLC-MS/MS.

Proteomic Analysis of Antibiotic Susceptibility in Enterobacteriaceae.

Mass spectrometry is a sensitive and specific analytical technique that is capable of providing qualitative and quantitative data to resolve the protein elements of biochemical pathways that are altered by antibiotics. Here we present methods to study antibiotic susceptibility by changes in protein abundance, as exemplified by Klebsiella pneumoniae, a Gram-negative pathogen that colonizes mucosal surfaces of the human gastrointestinal and respiratory tracts. Cultured bacteria are exposed to antibiotics, the total proteomes of collected cell pellets are converted to complex peptide mixtures by filter-aided sample preparation (FASP), and the peptides are further processed by an optimized desalting procedure.

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the Proteome.

Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A β-lactamase+ invasive strain of that was isolated from a lethal outbreak within laboratory colonies of monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic.

Type 1 Diabetes: Urinary Proteomics and Protein Network Analysis Support Perturbation of Lysosomal Function.

While insulin replacement therapy restores the health and prevents the onset of diabetic complications (DC) for many decades, some T1D patients have elevated hemoglobin A1c values suggesting poor glycemic control, a risk factor of DC. We surveyed the stool microbiome and urinary proteome of a cohort of 220 adolescents and children, half of which had lived with T1D for an average of 7 years and half of which were healthy siblings. Phylogenetic analysis of the 16S rRNA gene did not reveal significant differences in gut microbial alpha-diversity comparing the two cohorts.

Antibiotic-dependent perturbations of extended spectrum beta-lactamase producing Klebsiella pneumoniae proteome.

Extended spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-KP) causes life-threatening infections in susceptible and immuno-compromised individuals. Because of the emergence of multidrug resistance and tolerance, it is crucial to better understand the mechanisms by which ESBL-KP can adapt to antibiotic stress. The aim of this study was to provide an overview of the global proteome changes occurring in ESBL-KP in response to sub-lethal concentrations of the antibiotics doxycycline (DC, bacteriostatic) and streptomycin (SM, bactericidal), which both impair ribosomal synthesis of bacterial proteins.

Quantitative Differences in the Urinary Proteome of Siblings Discordant for Type 1 Diabetes Include Lysosomal Enzymes.

Individuals with type 1 diabetes (T1D) often have higher than normal blood glucose levels, causing advanced glycation end product formation and inflammation and increasing the risk of vascular complications years or decades later. To examine the urinary proteome in juveniles with T1D for signatures indicative of inflammatory consequences of hyperglycemia, we profiled the proteome of 40 T1D patients with an average of 6.3 years after disease onset and normal or elevated HbA1C levels, in comparison with a cohort of 41 healthy siblings.

Proteomes of pathogenic Escherichia coli/Shigella group surveyed in their host environments.

Proteomic studies on Shigella dysenteriae, Shigella flexneri, enterohemorrhagic Escherichia coli and uropathogenic E. coli (UPEC) are reviewed. UPEC causes infections in the urogenital tract, whereas the other species colonize and, to varying degrees, invade the intestinal tract.

Urine sample preparation in 96-well filter plates for quantitative clinical proteomics.

Urine is an important, noninvasively collected body fluid source for the diagnosis and prognosis of human diseases. Liquid chromatography mass spectrometry (LC-MS) based shotgun proteomics has evolved as a sensitive and informative technique to discover candidate disease biomarkers from urine specimens. Filter-aided sample preparation (FASP) generates peptide samples from protein mixtures of cell lysate or body fluid origin.

Analysis of the proteome of intracellular Shigella flexneri reveals pathways important for intracellular growth.

Global proteomic analysis was performed with Shigella flexneri strain 2457T in association with three distinct growth environments: S. flexneri growing in broth (in vitro), S. flexneri growing within epithelial cell cytoplasm (intracellular), and S.

Proteomic View of Interactions of Shiga Toxin-Producing Escherichia coli with the Intestinal Environment in Gnotobiotic Piglets.

Background: Shiga toxin (Stx)-producing Escherichia coli cause severe intestinal infections involving colonization of epithelial Peyer's patches and formation of attachment/effacement (A/E) lesions. These lesions trigger leukocyte infiltration followed by inflammation and intestinal hemorrhage. Systems biology, which explores the crosstalk of Stx-producing Escherichia coli with the in vivo host environment, may elucidate novel molecular pathogenesis aspects.

Integrated next-generation sequencing of 16S rDNA and metaproteomics differentiate the healthy urine microbiome from asymptomatic bacteriuria in neuropathic bladder associated with spinal cord injury.

Background: Clinical dogma is that healthy urine is sterile and the presence of bacteria with an inflammatory response is indicative of urinary tract infection (UTI). Asymptomatic bacteriuria (ABU) represents the state in which bacteria are present but the inflammatory response is negligible. Differentiating ABU from UTI is diagnostically challenging, but critical because overtreatment of ABU can perpetuate antimicrobial resistance while undertreatment of UTI can result in increased morbidity and mortality.

Development stage-specific proteomic profiling uncovers small, lineage specific proteins most abundant in the Aspergillus Fumigatus conidial proteome.

Background: The pathogenic mold Aspergillus fumigatus is the most frequent infectious cause of death in severely immunocompromised individuals such as leukemia and bone marrow transplant patients. Germination of inhaled conidia (asexual spores) in the host is critical for the initiation of infection, but little is known about the underlying mechanisms of this process.

Results: To gain insights into early germination events and facilitate the identification of potential stage-specific biomarkers and vaccine candidates, we have used quantitative shotgun proteomics to elucidate patterns of protein abundance changes during early fungal development.

Characterizing the Escherichia coli O157:H7 proteome including protein associations with higher order assemblies.

Background: The recent outbreak of severe infections with Shiga toxin (Stx) producing Escherichia coli (STEC) serotype O104:H4 highlights the need to understand horizontal gene transfer among E. coli strains, identify novel virulence factors and elucidate their pathogenesis. Quantitative shotgun proteomics can contribute to such objectives, allowing insights into the part of the genome translated into proteins and the connectivity of biochemical pathways and higher order assemblies of proteins at the subcellular level.

Using chemical derivatization and mass spectrometric analysis to characterize the post-translationally modified Staphylococcus aureus surface protein G.

The Staphylococcus aureus surface protein G (SasG) is an important mediator of biofilm formation in virulent S. aureus strains. A detailed analysis of its primary sequence has not been reported to date.

Integral and peripheral association of proteins and protein complexes with Yersinia pestis inner and outer membranes.

Yersinia pestis proteins were sequentially extracted from crude membranes with a high salt buffer (2.5 M NaBr), an alkaline solution (180 mM Na2CO3, pH 11.3) and membrane denaturants (8 M urea, 2 M thiourea and 1% amidosulfobetaine-14).

Limited proteolysis analysis of the ribosome is affected by subunit association.

Our understanding of the structural organization of ribosome assembly intermediates, in particular those intermediates that result from misfolding leading to their eventual degradation within the cell, is limited because of the lack of methods available to characterize assembly intermediate structures. Because conventional structural approaches, such as NMR, X-ray crystallography, and cryo-EM, are not ideally suited to characterize the structural organization of these flexible and sometimes heterogeneous assembly intermediates, we have set out to develop an approach combining limited proteolysis with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that might be applicable to ribonucleoprotein complexes as large as the ribosome. This study focuses on the limited proteolysis behavior of appropriately assembled ribosome subunits.

Retinol saturase promotes adipogenesis and is downregulated in obesity.

Adipocyte differentiation is controlled by many transcription factors, but few known downstream targets of these factors are necessary for adipogenesis. Here we report that retinol saturase (RetSat), which is an enzyme implicated in the generation of dihydroretinoid metabolites, is induced during adipogenesis and is directly regulated by the transcription factor peroxisome proliferator activated receptor gamma (PPARgamma). Ablation of RetSat dramatically inhibited adipogenesis but, surprisingly, this block was not overcome by the putative product of RetSat enzymatic activity.

Widespread Occurrence of Non-Enzymatic Deamidations of Asparagine Residues in Yersinia pestis Proteins Resulting from Alkaline pH Membrane Extraction Conditions.

Extraction of crude membrane fractions with alkaline solutions, such as 100-200 mM Na(2)CO(3) (pH ~11), is often used to solubilize peripheral membrane proteins. Integral membrane proteins are largely retained in membrane pellets. We applied this method to the fractionation of membrane proteins of the plague bacterium Yersinia pestis.

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography.

An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights.

Cell proliferation inhibition and alterations in retinol esterification induced by phytanic acid and docosahexaenoic acid.

We investigated the effects of two natural dietary retinoid X receptor (RXR) ligands, phytanic acid (PA) and docosahexaenoic acid (DHA), on proliferation and on the metabolism of retinol (vitamin A) in both cultured normal human prostate epithelial cells (PrECs) and PC-3 prostate carcinoma cells. PA and DHA inhibited the proliferation of the parental PC-3 cells and PC-3 cells engineered to overexpress human lecithin:retinol acyltransferase (LRAT) in both the absence and presence of retinol. A synthetic RXR-specific ligand also inhibited PC-3 cell proliferation, whereas all-trans retinoic acid (ATRA) did not.

Structure elucidation of retinoids in biological samples using postsource decay laser desorption/ionization mass spectrometry after high-performance liquid chromatography separation.

Retinoids [retinol (vitamin A) and its metabolites] function in the visual cycle, embryonic development, cellular differentiation, and tissue homeostasis. Notwithstanding pivotal roles of retinoids in mammals, the limited number of commercially available retinoid standards is a major roadblock to identifying and studying retinoids in biological samples. Therefore, a need exists for improved methods to identify retinoid metabolites.

Extending ribosomal protein identifications to unsequenced bacterial strains using matrix-assisted laser desorption/ionization mass spectrometry.

A protocol has been developed that allows protein identifications using available DNA-based or protein sequences from a reference strain of a bacterial species to be extended to bacterial strains for which no prior DNA-based or protein sequence information exists. The protocol is predicated on careful isolation of a specific sub-cellular group of proteins. In this study, ribosomal proteins were chosen due to their high relative abundance and similarity in copy number per cell.

Thermus thermophilus L11 methyltransferase, PrmA, is dispensable for growth and preferentially modifies free ribosomal protein L11 prior to ribosome assembly.

The ribosomal protein L11 in bacteria is posttranslationally trimethylated at multiple amino acid positions by the L11 methyltransferase PrmA, the product of the prmA gene. The role of L11 methylation in ribosome function or assembly has yet to be determined, although the deletion of Escherichia coli prmA has no apparent phenotype. We have constructed a mutant of the extreme thermophile Thermus thermophilus in which the prmA gene has been disrupted with the htk gene encoding a heat-stable kanamycin adenyltransferase.

Investigation of methods suitable for the matrix-assisted laser desorption/ionization mass spectrometric analysis of proteins from ribonucleoprotein complexes.

A variety of protein isolation and purification techniques for ribonucleoprotein (RNP) complexes were investigated for their compatibility with downstream analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Ribosomal proteins from Escherichia coli 70S ribosomes were obtained using methods such as phenol extraction and precipitation by organic solvents or acids. Under optimal conditions, more than 90% of the expected ribosomal proteins were detected in a single MALDI-MS experiment.