Analytical Differences in Intraoperative Parathyroid Hormone Assays.

Background: We compared the rates of intraoperative parathyroid hormone (PTH) decline using the Siemens Immulite Turbo PTH and Roche Elecsys short turnaround time PTH assays in 95 consecutive surgical patients to investigate analytical and turnaround time (TAT) differences between the tests performed in the operating room (OR) vs the central clinical chemistry laboratory (CCL).

Methods: Serial blood samples from 95 patients undergoing parathyroidectomy were collected and measured using the 2 immunoassays. Specimens from the first 15 patients were measured simultaneously in the OR and CCL and used for the TAT study.

Opposing activities of oncogenic MIR17HG and tumor suppressive MIR100HG clusters and their gene targets regulate replicative senescence in human adult stem cells.

Growing evidence suggests that many diseases of aging, including diseases associated with robust changes and adipose deports, may be caused by resident adult stem cell exhaustion due to the process called cellular senescence. Understanding how microRNA pathways can regulate cellular senescence is crucial for the development of novel diagnostic and therapeutic strategies to combat these pathologies. Herein, using integrated transcriptomic and semi-quantitative proteomic analysis, we provide a system level view of the regulation of human adipose-derived stem cell senescence by a subset of mature microRNAs (termed senescence-associated-microRNAs) produced by biogenesis of oncogenic and tumor-suppressive clusters.

Assessment of a method to characterize antibody selectivity and specificity for use in immunoprecipitation.

Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins.

The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16(INK4A) and p14(ARF) by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex.

A semi-automated mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) reveals novel relationships between circulating PCSK9 and metabolic phenotypes in patient cohorts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels.

Proteomic signatures of serum albumin-bound proteins from stroke patients with and without endovascular closure of PFO are significantly different and suggest a novel mechanism for cholesterol efflux.

Background: The anatomy of PFO suggests that it can allow thrombi and potentially harmful circulatory factors to travel directly from the venous to the arterial circulation - altering circulatory phenotype. Our previous publication using high-resolution LC-MS/MS to profile protein and peptide expression patterns in plasma showed that albumin was relatively increased in donor samples from PFO-related than other types of ischemic strokes. Since albumin binds a host of molecules and acts as a carrier for lipoproteins, small molecules and drugs, we decided to investigate the albumin-bound proteins (in a similar sample cohort) in an effort to unravel biological changes and potentially discover biomarkers related to PFO-related stroke and PFO endovascular closure.

The Escherichia coli O157:H7 cattle immunoproteome includes outer membrane protein A (OmpA), a modulator of adherence to bovine rectoanal junction squamous epithelial (RSE) cells.

Building on previous studies, we defined the repertoire of proteins comprising the immunoproteome (IP) of Escherichia coli O157:H7 (O157) cultured in DMEM supplemented with norepinephrine (O157 IP), a β-adrenergic hormone that regulates E. coli O157 gene expression in the gastrointestinal tract, using a variation of a novel proteomics-based platform proteome mining tool for antigen discovery, called "proteomics-based expression library screening" (PELS; Kudva et al., 2006).

Comparative proteomic analysis reveals activation of mucosal innate immune signaling pathways during cholera.

Vibrio cholerae O1 is a major cause of acute watery diarrhea in over 50 countries. Evidence suggests that V. cholerae O1 may activate inflammatory pathways, and a recent study of a Bangladeshi population showed that variants in innate immune genes play a role in mediating susceptibility to cholera.

Delineating monoclonal antibody specificity by mass spectrometry.

Unlabelled: Generation of monoclonal antibody (mAb) libraries against antigens in complex matrices can prove a valuable analytical tool. However, delineating the specificity of newly generated antibodies is the limiting step of the procedure. Here, we propose a strategy for mAb production by injecting mice with complex biological fluid and mAb characterization by coupling immunoaffinity techniques with Mass spectrometry (immuno-MS).

Mass spectrometry assays of plasma biomarkers to predict radiographic progression of knee osteoarthritis.

Introduction: Biomarkers to identify osteoarthritis (OA) patients at risk for disease progression are needed. As part of a proteomic analysis of knee synovial fluid from normal and OA patients, differentially expressed proteins were identified that could represent potential biomarkers for OA. This study aimed to use mass spectrometry assays to identify representative peptides from several proteins in synovial fluid and peripheral blood, and assess their levels as biomarkers of OA progression.

Hybrid data acquisition and processing strategies with increased throughput and selectivity: pSMART analysis for global qualitative and quantitative analysis.

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis.

LC-MS candidate reference methods for the harmonisation of parathyroid hormone (PTH) measurement: a review of recent developments and future considerations.

The analysis of intact parathyroid hormone (PTH) (PTH1-84) is useful in the diagnosis of hyper- and hypocalcaemia, hyperparathyroidism, and in the prevention of bone mineral disorders in renal patients. The analysis is complicated by the presence of PTH fragments, which may accumulate in renal failure and cross-react in immunoassays, including the most recent third-generation immunoassays. Large variability exists between different commercially available assays.

The role of apolipoprotein E in neurodegeneration and cardiovascular disease.

Apolipoprotein E (ApoE) is an abundant plasma protein that interacts with low density lipoprotein receptors and other proteins, participating in the transport of cholesterol and lipids. Research has revealed many other roles for this multifunctional protein. ApoE is polymorphic and exists in three major isoforms: ApoE2, ApoE3 (the most common isoform) and ApoE4, which differ by only one amino acid, at positions 112 and 158.

An automated, high-throughput method for targeted quantification of intact insulin and its therapeutic analogs in human serum or plasma coupling mass spectrometric immunoassay with high resolution and accurate mass detection (MSIA-HR/AM).

The detection and quantification of insulin and its therapeutic analogs is important for medical, sports doping, and forensic applications. Synthetic variants contain slight sequence variations to affect bioavailability. To reduce sample handling bias, a universal extraction method is required for simultaneous extraction of endogenous and variant insulins with subsequent targeted quantification by LC-MS.

Parallel workflow for high-throughput (>1,000 samples/day) quantitative analysis of human insulin-like growth factor 1 using mass spectrometric immunoassay.

Insulin-like growth factor 1 (IGF1) is an important biomarker for the management of growth hormone disorders. Recently there has been rising interest in deploying mass spectrometric (MS) methods of detection for measuring IGF1. However, widespread clinical adoption of any MS-based IGF1 assay will require increased throughput and speed to justify the costs of analyses, and robust industrial platforms that are reproducible across laboratories.

Targeted selected reaction monitoring mass spectrometric immunoassay for insulin-like growth factor 1.

Insulin-like growth factor 1 (IGF1) is an important biomarker of human growth disorders that is routinely analyzed in clinical laboratories. Mass spectrometry-based workflows offer a viable alternative to standard IGF1 immunoassays, which utilize various pre-analytical preparation strategies. In this work we developed an assay that incorporates a novel sample preparation method for dissociating IGF1 from its binding proteins.

Rapid development of sensitive, high-throughput, quantitative and highly selective mass spectrometric targeted immunoassays for clinically important proteins in human plasma and serum.

Objectives: The aim of this study was to develop high-throughput, quantitative and highly selective mass spectrometric, targeted immunoassays for clinically important proteins in human plasma or serum.

Design And Methods: The described method coupled mass spectrometric immunoassay (MSIA), a previously developed technique for immunoenrichment on a monolithic microcolumn activated with an anti-protein antibody and fixed in a pipette tip, to selected reaction monitoring (SRM) detection and accurate quantification of targeted peptides, including clinically relevant sequence or truncated variants.

Results: In this report, we demonstrate the rapid development of MSIA-SRM assays for sixteen different target proteins spanning seven different clinically important areas (including neurological, Alzheimer's, cardiovascular, endocrine function, cancer and other diseases) and ranging in concentration from pg/mL to mg/mL.

Depletion of nuclear histone H2A variants is associated with chronic DNA damage signaling upon drug-evoked senescence of human somatic cells.

Cellular senescence is associated with global chromatin changes, altered gene expression, and activation of chronic DNA damage signaling. These events ultimately lead to morphological and physiological transformations in primary cells. In this study, we show that chronic DNA damage signals caused by genotoxic stress impact the expression of histones H2A family members and lead to their depletion in the nuclei of senescent human fibroblasts.

Heart-brain signaling in patent foramen ovale-related stroke: differential plasma proteomic expression patterns revealed with a 2-pass liquid chromatography-tandem mass spectrometry discovery workflow.

Patent foramen ovale (PFO) is highly prevalent and associated with more than 150,000 strokes per year. Traditionally, it is thought that PFOs facilitate strokes by allowing venous clots to travel directly to the brain. However, only a small portion of PFO stroke patients have a known tendency to form blood clots, and the optimal treatment for this multiorgan disease is unclear.

Comparison of immune responses to the O-specific polysaccharide and lipopolysaccharide of Vibrio cholerae O1 in Bangladeshi adult patients with cholera.

Immunity against Vibrio cholerae O1 is serogroup specific, and serogrouping is defined by the O-specific polysaccharide (OSP) part of lipopolysaccharide (LPS). Despite this, human immune responses to V. cholerae OSP have not previously been characterized.

Protein interactions with piALU RNA indicates putative participation of retroRNA in the cell cycle, DNA repair and chromatin assembly.

Recent analyses suggest that transposable element-derived transcripts are processed to yield a variety of small RNA species that play critical functional roles in gene regulation and chromatin organization as well as genome stability and maintenance. Here we report a mass spectrometry analysis of an RNA-affinity complex isolation using a piRNA homologous sequence derived from Alu retrotransposal RNA. Our data point to potential roles for piALU RNAs in DNA repair, cell cycle and chromatin regulations.

Proteins other than the locus of enterocyte effacement-encoded proteins contribute to Escherichia coli O157:H7 adherence to bovine rectoanal junction stratified squamous epithelial cells.

Background: In this study, we present evidence that proteins encoded by the Locus of Enterocyte Effacement (LEE), considered critical for Escherichia coli O157 (O157) adherence to follicle-associated epithelial (FAE) cells at the bovine recto-anal junction (RAJ), do not appear to contribute to O157 adherence to squamous epithelial (RSE) cells also constituting this primary site of O157 colonization in cattle.

Results: Antisera targeting intimin-γ, the primary O157 adhesin, and other essential LEE proteins failed to block O157 adherence to RSE cells, when this pathogen was grown in DMEM, a culture medium that enhances expression of LEE proteins. In addition, RSE adherence of a DMEM-grown-O157 mutant lacking the intimin protein was comparable to that seen with its wild-type parent O157 strain grown in the same media.

Interlaboratory reproducibility of selective reaction monitoring assays using multiple upfront analyte enrichment strategies.

Over the past few years, mass spectrometry has emerged as a technology to complement and potentially replace standard immunoassays in routine clinical core laboratories. Application of mass spectrometry to protein and peptide measurement can provide advantages including high sensitivity, the ability to multiplex analytes, and high specificity at the amino acid sequence level. In our previous study, we demonstrated excellent reproducibility of mass spectrometry-selective reaction monitoring (MS-SRM) assays when applying standardized standard operating procedures (SOPs) to measure synthetic peptides in a complex sample, as lack of reproducibility has been a frequent criticism leveled at the use of mass spectrometers in the clinical laboratory compared to immunoassays.