Rapid quantification of insulin degludec by immunopurification combined with liquid chromatography high-resolution mass spectrometry.

Insulin degludec is an ultra-long-acting insulin analogue that is increasingly being used in diabetes due to its favourable efficacy and safety profile. Thus, there is an increasing demand for a reliable and specific analytical method to quantify insulin degludec for research, pharmaceutical industry and clinical applications. We developed and validated an automated, high-throughput method for quantification of insulin degludec in human blood samples across the expected clinical range combining immunopurification with high-resolution mass spectrometry.

Development and validation of a targeted affinity-enrichment and LC-MS/MS proteomics approach for the therapeutic monitoring of adalimumab.

Background: The anti-tumor necrosis factor alpha (TNFα) therapeutic monoclonal antibodies (mAbs), such as adalimumab, are widely used in the treatment of rheumatoid arthritis, inflammatory bowel diseases, and other auto-immune diseases. The administration of adalimumab can elicit the immune responses from some patients, resulting in the formation of anti-drug antibodies (ADAbs). The ADAbs can diminish the therapeutic effects of adalimumab by neutralizing the TNFα binding site or increasing its clearance from circulation.

Top-down mass spectrometric immunoassay for human insulin and its therapeutic analogs.

Unlabelled: Measurement of insulin and its therapeutic analogs is important in diabetes, hypoglycemia, sports anti-doping and toxicology. Commercial insulin immunoassays fail to detect commonly prescribed insulin analogs. Because of their unique sequences and masses, these analogs are readily measured and distinguished with mass spectrometric (MS) assays.

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.

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.

Detection of endogenous B-type natriuretic peptide at very low concentrations in patients with heart failure.

Background: The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32.

MS-based phenotypic characterization of a human blood protein from urinary waste products.

The urine proteome (urineome) has become an ideal biological fluid for biomarker study and detection due to its rich protein content as well as its ease and abundance in collection. Protein variation in human plasma has been linked to the presence of disease states in humans; however, it stands to reason that the same is true of the equally complex urineome. In this manuscript we present the combination of two proteomics technologies, mass spectrometric immunoassay and a bioreactive probe, for the detection and characterization of the protein variants of transthyretin (TTR) found in human urine.

Volumetric mass spectrometry protein arrays.

Affinity mass spectrometry is a proteomics approach for selectively isolating target proteins from complex biological fluids for mass spectrometric analysis. When executed in high throughput mode through affinity pipets, the resulting volumetric mass spectrometry arrays enable rapid protein assaying from hundreds of samples. Furthermore, in combination with postcapture proteolytic degradation, this top-down proteomics approach can reveal structural features (i.

High-throughput affinity mass spectrometry.

Affinity mass spectrometry (AMS) is a proteomics approach for selectively isolating target protein(s) from complex biological fluids for mass spectrometric analysis. The resulting high-content mass spectrometry (MS) data show the unique MS protein signatures (wild-type, posttranslationally modified, as well as genetically modified forms of the protein target) that are present within a biological sample. Information regarding such protein diversity is normally lost in classical proteomic or immunoassay analyses.

Population proteomics: the concept, attributes, and potential for cancer biomarker research.

This review outlines the concept of population proteomics and its implication in the discovery and validation of cancer-specific protein modulations. Population proteomics is an applied subdiscipline of proteomics engaging in the investigation of human proteins across and within populations to define and better understand protein diversity. Population proteomics focuses on interrogation of specific proteins from large number of individuals, utilizing top-down, targeted affinity mass spectrometry approaches to probe protein modifications.

Development of recombinant-based mass spectrometric immunoassay with application to resistin expression profiling.

This report addresses the need for additional assays for human resistin (hRES) by developing a rational progression of the mass spectrometric immunoassay to incorporate recombinant proteins. The recombinant-based hRES mass spectrometric immunoassay (RES-MSIA) was initially developed for the qualitative analysis of the human resistin homodimer from normal (healthy) plasma samples. The method involved selective extraction and detection of both endogenous and recombinant resistant proteins.

Multiplexed DNA sequencing-by-synthesis.

We report a new DNA sequencing-by-synthesis method in which the sequences of DNA templates, hybridized to a surface-immobilized array of DNA primers, are determined by sensing the number of nucleotides by which the primers in each array spot are extended in sequential DNA polymerase-catalyzed nucleotide incorporation reactions, each with a single fluorescein-labeled deoxyribonucleoside triphosphate (dNTP) species. The fluorescein label is destroyed after each readout by a photostimulated reaction with diphenyliodonium chloride. A DNA polymerase with enhanced ability to incorporate, and to extend beyond, modified nucleotides is used.

High-throughput MS-based protein phenotyping: application to haptoglobin.

A high-throughput affinity capture and reduction approach was developed for phenotype and post-translational modification analysis of a complexed globular protein, haptoglobin (Hp), directly from human plasma. Hp was selectively retrieved utilizing anti-Hp antibodies immobilized onto affinity pipette tips, eluted onto a formatted mass spectrometer target for reduction of Hp alpha-chains (Hpalpha1 and Hpalpha2) and subjected to subsequent MALDI-MS analysis. The affinity capture and reduction approach was originally developed from a pre-extraction reduction methodology that was optimized to an affinity capture post-reduction technique for intact Hp alpha-chain variant analysis, phenotype classification and ensuing post-translational variant detection.

Investigating diversity in human plasma proteins.

Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population.

Proteomic characterization of novel serum amyloid P component variants from human plasma and urine.

Serum amyloid P component (SAP) is a human plasma protein that has been widely studied for its influence on amyloid plaque formation and stabilization. SAP was characterized directly from human plasma and urine samples via novel affinity mass spectrometry-based proteomic technology that is able to readily discriminate between mass-altered protein variants. These analyses were able to identify several variants of SAP that have not been previously reported.

High-throughput comprehensive analysis of human plasma proteins: a step toward population proteomics.

A high-throughput (HT) comprehensive analysis approach was developed for assaying proteins directly from human plasma. Proteins were selectively retrieved, by utilizing antibodies immobilized within affinity pipet tips, and eluted onto enzymatically active mass spectrometer targets for subsequent digestion and structural characterization. Several parameters, including uniform parallel protein elution from 96 affinity pipet tips, proper buffering for on-target digestion, termination of the digestion, and MALDI matrix (re)introduction, were evaluated and optimized.

Transduction of biologically active motifs of the small heat shock-related protein HSP20 leads to relaxation of vascular smooth muscle.

Activation of cyclic nucleotide-dependent signaling pathways leads to phosphorylation of the small heat shock-related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose-dependent relaxation of coronary artery smooth muscle.

Comparative urine protein phenotyping using mass spectrometric immunoassay.

Reported here, human urine samples were analyzed for beta-2-microglobulin (beta2m), transthyretin (TTR), cystatin C, urine protein 1 (UP1), retinol binding protein (RBP), albumin, transferrin, and human neutrophil defensin peptides (HNP) using mass spectrometric immunoassay (MSIA). MSIA is a unique analytical technique, which allows for the generation of distinct protein profiles of specific target proteins from each subject, which may be subsequently used in comparative protein expression profiling between all subjects. Comparative profiling allows for the rapid identification of variations within individual protein expression profiles.

Detection of novel truncated forms of human serum amyloid A protein in human plasma.

Serum amyloid A protein (SAA) is a human plasma protein that has been recognized as potential biomarker of multiple ailments including myocardial infarction, inflammatory disease and amyloiosis. Presented here is the application of a novel immunoassay technique, termed mass spectrometric immunoassay for the detection and identification of SAA present in human plasma. Results demonstrate the ability to readily detect known SAA isotypes, and to identify novel truncated forms of SAA, in the plasma of healthy individuals and those suffering from acute and chronic inflammation.

Detection of bound and free IGF-1 and IGF-2 in human plasma via biomolecular interaction analysis mass spectrometry.

Insulin like growth factor (IGF)-1 and IGF-2 were assayed from human plasma via biomolecular interaction analysis mass spectrometry, utilizing antibodies as ligands for affinity retrieval. Detection of both targeted and non-targeted IGFs in the mass spectra indicated possible protein complex retrieval by the individual antibodies. A series of control experiments eliminated the possibility of analyte cross-walking between flow cells, significant antibodies cross-reactivity, and direct IGF interactions.

Novel mass spectrometric immunoassays for the rapid structural characterization of plasma apolipoproteins.

Novel mass spectrometric immunoassays (MSIAs) for the isolation and structural characterization of plasma apolipoprotein A-I (apoA-I), apoA-II, and apoE have been developed. The assays combine selective isolation of apolipoprotein species via affinity capture with mass-specific detection using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In application, plasma (from 50 microl of whole blood drawn from individuals, using finger lancet) was addressed with affinity pipette tips derivatized with antibodies toward the specific apolipoprotein.

Comparative phenotypic analyses of human plasma and urinary retinol binding protein using mass spectrometric immunoassay.

Mass spectrometric immunoassay (MSIA) is a proteomics technology that combines the selectivity of affinity capture with the sensitivity and resolution of mass spectrometric detection. This unique approach allows for intact protein identification therefore is readily capable of discriminating between protein variants, i.e.

High-throughput protein characterization using mass spectrometric immunoassay.

A high-throughput mass spectrometric immunoassay system for the analysis of proteins directly from plasma is reported. A 96-well format robotic workstation was used to prepare antibody-derivatized affinity pipette tips for subsequent use in the extraction of specific proteins from plasma and deposition onto 96-well format matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) targets. Samples from multiple individuals were screened with regard to the plasma protein transthyretin (TTR), followed by analysis of the same plasma samples for the transthyretin-associated transport protein, retinol-binding protein (RBP).